Program and abstracts for the 2011 Meeting of the Society for Glycobiology

(1) Functions of Mucin Glycoproteins in Metastatic PancreaticCancer

M. Tony HollingsworthEppley Cancer Institute, Omaha, NE

Cell surface mucins configure the cell surface by presenting extendedprotein backbones that are heavily O-glycosylated. The glycopeptidestructures establish physicochemical properties at the cell surface thatenable and block the formation of biologically important molecularcomplexes. Some mucins, such as MUC1, associate with receptortyrosine kinases and other cell surface receptors, and engage in signaltransduction in order to communicate information regarding con-ditions at the cell surface to the nucleus. In that context, the MUC1cytoplasmic tail (MUC1CT) receives phosphorylation signals fromreceptor tyrosine kinases and serine/threonine kinases, which enablesits association with different signaling complexes that conduct thesesignals to the nucleus and perhaps other subcellular organelles. Wehave detected the MUC1CT at promoters of over 500 genes, inassociation with several different transcription factors, and haveshown that promoter occupancy can vary under different growthfactor conditions. However, the full biochemical nature of the nuclearforms of MUC1 and its function at these promoter regions remainundefined. I will present evidence that nuclear forms of theMUC1CT include extracellular and cytoplasmic tail domains. Inaddition, I will discuss evidence for a hypothesis that the MUC1CTpossesses a novel catalytic function that enables remodeling of thetranscription factor occupancy of promoters, and thereby engages inregulation of gene expression.

(2) What Do Glycans Do? - Finding the Rightful Placefor Carbohydrates in the Central Dogma of Life!

Gerald W. HartJohn Hopkins University School of Med, Baltimore, MD

All glycobiologists throughout their careers have often been asked bycolleagues the question: “So what exactly do glycans do anyway?”Of course, this is a ridiculous question born of ignorance. I usuallyreply, “lots of things, but do you know what alanine does?” Formany years, our field focused on the biosynthesis and structural com-plexities of glycans. However, in the past decades, great advanceshave taken place in our understanding of the biological roles of bothcomplex and simple glycans. These data are forcing our reluctant col-leagues in other fields to begin to realize that glycans might be actu-ally important to what they study! The data speaks for itself. In thisoverview lecture, I will chronicle the evolution of glycosciences froma field of mainly chemistry, structural biochemistry, and enzymologyto its current emphasis on relating structure to biological functionsand disease. Given current advances in glycomics, we are indeedentering a time when glycans will join other modifications of proteinsand lipids to become the major focus of research in the next decades.

(3) Functional GlycomicsJames C. Paulson

The Scripps Research Institute, La Jolla, CA

Over the last decade the field of glycomics has grown exponen-tially, spurred by the development of tools that facilitate the

bridging of glycomics to fields of proteomics and genomics. Amajor boost was the emergence of the Consortium for FunctionalGlycomics funded in 2002 by a ‘glue grant’ from the NationalInstitute of General Medical Sciences. With the development ofunique tools and databases that integrate diverse sets of biologicaldata, the CFG provided novel resources and a networking form fordiverse group of investigators whose research was impacted byglycans and their roles in biology. As the momentum for glyco-mics research continues to grow, increased emphasis needs to beplaced on the ‘bottlenecks’ that restrain progress. Critically neededare improved methods for analysis of glycan structures, bioinfor-matics tools that support the database deposition and mining ofglycomics data, and robust methods for synthesis glycan structuresrepresentative of the human glycome. Advances in these andrelated areas will accelerate the understanding of the functions ofglycans and glycan binding proteins that impact human heath anddisease. (Supported by NIH grant GM62116).

(4) O-Glycosylation of the Envelope Glycoproteinof the Human Immunodeficiency Virus

Elizabeth Stansell1, Kevin Canis2, I-Cheuh Huang1,Maria Panico2, Howard Morris2, Stuart Haslam2, Michael Farzan1,

Anne Dell2, Ronald Desrosiers11Harvard Medical School, Southborough, MA; 2Imperial College,

London, UK

The literature is unclear regarding the occurrence of O-linkedcarbohydrate on the HIV-1 Envelope glycoprotein; there arecertainly no reports defining putative sites of O-glycosylation norwhat the function might be. We have now defined a highly-conserved site of O-glycan attachment on HIV-1 gp120, the natureof the attached carbohydrate, and we have obtained evidence onthe function of the O-glycan. Our findings also indicate asimilarly-positioned site of O-glycan attachment on other familiesof enveloped viruses. These results define a previously unrecog-nized structure-function relationship that is conserved acrossmultiple families of enveloped viruses.

(5) Probing the 150-Cavity of Influenza AVirus Group-1Sialidases

Mark von ItzsteinGriffith University, Gold Coast Campus, Australia

The significant flexibility that has been structurally observed in thesialidase (neuraminidase, NA) catalytic domain of some influenzaA viruses has led to the synthesis of inhibitors that take advantageof this new architecture. We have used N-acetylneuraminic acid asthe framework to explore the so-called 150-cavity that has beenobserved in protein X-ray crystallographic studies of a number ofinfluenza A virus sialidases. In our present work we have beenable to restrict the flexibility of the 150-loop by installing appro-priate functional groups off the C-3 position of theN-acetylneuraminc acid template. More recently we have usedmolecular dynamic simulations to explore the flexibility of thisloop using available structural data of several influenza Avirus sia-lidases to better understand the extent of flexibility in thesesystems. This information would better inform the inhibitor

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discovery process. Aspects of our latest work in the design, syn-thesis and evaluation of influenza virus sialidase inhibitors will bediscussed.

(6) Specificity in the Host Range and Pathogenicityof Influenza Virus

Mikhail MatroscovichPhilipps-Universität Marburg, Germany

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(7) A Mimivirus Enzyme Provides Confirmation of CollagenGlycosylation Outside the Domains of Life

Kelvin B. Luther, Andreas J. Hülsmeier, Belinda Schegg,Thierry Hennet

University of Zürich, Zürich, Switzerland

While the O-galactosyltransferases responsible for initiation ofglycan synthesis in human collagen have only recently beendescribed, we now report that the giant virus Mimivirus expressesa collagen glycosyltransferase and viral collagens. The viral glyco-syltransferase and collagen proteins are expressed together duringinfection of the amoeba Ancanthamoeba polyphaga. Interestingly,the enzyme is bi-functional, possessing both collagen lysyl-hydroxylase activity and collagen glycosyltransferase activity. Wefind that the Mimivirus enzyme is substrate promiscuous, exhibit-ing lysyl-hydroxylase and collagen protein: O-glycosyltransferaseactivities toward both Mimivirus and human collagen substrates.While all other hydroxylysine modifying glycosyltransferasestransfer galactose, this enzyme adds glucose directly to hydroxyly-sine. We have purified functional Mimivirus enzyme from E. coli,and in addition, we are expressing the enzyme in concert witheither Mimivirus or human collagen substrates. This is the firstdescription of a collagen modifying enzyme outside metazoans,and the first report of a sugar other than galactose linked directlyto hydroxylysine. These results demonstrate that glycosylatedcollagen proteins exist outside the domains of life.

(8) Synthesis of Sialylated poly-LacNAc extended N- andO-Glycans and Their Evaluation as Receptors of Influenza

AVirus HemagglutininCorwin Nycholat1, Ryan McBride1, Damian Ekiert1, Rui Xu1,

Wenjie Peng1, Nahid Razi1, Michel Gilbert2, Warren Wakarchuk2,Ian A. Wilson1, James C. Paulson1

1The Scripps Research Institute, La Jolla, CA; 2National ResearchCouncil of Canada, Ottawa, ON

Poly-N-acetyl lactosamine (poly-LacNAc) chains are commonextensions of both glycolipids and glycoproteins, including both N-and O-glycans. Poly-LacNAc structures are commonly composedof type-2 chain (Galβ1-4GlcNAcβ1-3) tandem repeats. Their bio-synthesis is controlled by alternating reaction with β1-4 galactosyl-and β1-3 N-acetylglucosaminyl-transferases. Poly-LacNAc chainsare subject to further modifications serving as scaffolds for sialyla-tion, fucosylation and O-sulfation. Many important biological func-tions mediated by glycan binding proteins are thought to involveligands with poly-LacNAc structures. For example, extended sialo-side structures have received increased attention as receptors for

influenza Avirus hemagglutinin with suggestions they may be criti-cally important for viral adaption to humans (Chandrasekaran et.al., Nat. Biotech. 2008, 107). Recently, human and swine respirat-ory epithelial cells have been found to express a diverse range ofsialylated poly-LacNAc extended N-glycans. However, studies todate have employed linear glycans with LacNAc repeats that occuras terminal fragments of natural glycans. Here we report thesynthesis of sialylated poly-LacNAc on intact N- and O-glycancore structures. Key LacNAc extensions were attained byalternating reaction using the Helicobacter pylori JHP1032β1,3-N-acetylglucosaminyltransferase (β1,3GlcNAcT) (Sauerzapfeet. al., Glycoconj. J. 2009, 141) and the bacterial β1,4-galactosyl-transferase/UDP-4′-Gal-epimerase fusion protein (GalT-GalE).Divergent reaction of the Gal terminating intermediates using eitherrat ST3Gal-III or human ST6Gal-I allowed preparation of the corre-sponding α2,3 or α2,6 sialosides respectively. These glycans wereelaborated on a custom sialoside microarray for analysis of humanand avian influenza A virus HA specificity. (Supported in part byNIH grants AI058113 and GM62116)

(9) Repository of Recombinant Expression Constructsfor Mammalian Glycosylation Enzymes: Baculovirus Vectorsfor Glycosyltransferase and Glycoside Hydrolase Production

in Insect CellsGagandeep Gahlay1, Christoph Geisler1, Jared J. Aumiller1,

Kelley Moremen2, Jason Steel3, Joshua LaBaer3, Donald L. Jarvis11University of Wyoming, Laramie, WY; 2University of Georgia,

Athens, GA; 3Arizona State University, Tempe, AZ

A large set of glycosylation enzymes is responsible for the biosyn-thesis of complex carbohydrate structures, which have numerousroles in critical biological recognition in animal systems. Inmammals, �204 glycosyltransferase (GT) and �75 glycosidehydrolase (GH) family members assemble and degrade the diversearray of glycan structures attached to intracellular and secreted pro-teins and lipids. In order to enable structural and functional studieson these enzymes, we are assembling repositories of recombinantplasmids and baculovirus expression vectors encoding all knownhuman GT and GH coding sequences for production of these pro-teins in HEK293 and insect cells, respectively. Expression in theseeukaryotic hosts is expected to provide the correct context of cha-perones and post-translational modifications needed for the properfolding and enzymatic activity of these proteins. In each case, theoriginal GT vectors designed for this project encode catalyticdomains in which the native N-termini were replaced by a honey-bee mellitin signal peptide, 8X HIS and Strep II affinity purifi-cation tags, and the Tobacco Etch Virus (TEV) protease cleavagesite. Similarly, each original GH vector encodes the full-lengthprotein with a C-terminal TEV cleavage site followed by the 8XHIS and Strep II affinity tags. This poster will summarize theresults obtained to date in our efforts to isolate and test recombi-nant baculoviruses encoding the human GT and GH constructs.Our poster also will show the results obtained with selectedsubsets of GT constructs expressed using baculovirus vectors witha variety of different transcriptional promoters and/or purificationtags, in an ongoing effort to examine the influence of these key

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variables on protein production levels, secretion efficiencies, andnet productivity.Supported by Grant #3P41 RR-005351-20S1 from the National

Institutes of Health.

(10) Multiple Target Recognition Strategies of Glycan-BindingReceptors

Kurt Drickamer, Maureen TaylorImperial College London, London, United Kingdom

Identification of novel sugar-specific receptors in the humangenome provides one way to understand the importance ofglycan diversity. Glycan-binding proteins that contain C-typecarbohydrate-recognition domains have roles in pathogen recog-nition, leading to neutralisation, and in binding to endogenousglycans, resulting in cell adhesion, signalling and clearance of gly-coproteins from circulation. Analysis of the sugar-binding activi-ties and functions of novel receptors illustrates potential new rolesfor glycan-receptor interactions. SRCL, the endothelial scavengerreceptor C-type lectin, binds specifically to the Lewisx oligosac-charide. Glycoproteomic analysis of neutrophil ligands reveals thatmany are glycoproteins of the secondary granules. Identification ofthese ligands supports a mannose receptor-like model for SRCL asa distributed clearance system for proteins released at sites ofinflammation. A second receptor, prolectin, is found exclusivelyon a small subset of dividing B cells in germinal centres. It has anunusual cytoplasmic domain that contains multiple signallingdomains that are targets for phosphorylation by B-cell receptor-activated tyrosine kinases, resulting in interactions with intracellu-lar adapter molecules. The results suggest two models for prolectinfunction, either as a sugar-binding B cell co-receptor or as a celladhesion molecule involved in trafficking of B cells within germ-inal centres. Finally, LSECtin, a liver and lymph node sinusoidalendothelial cell receptor, binds very selectively to a terminal disac-charide on truncated complex glycans. This specificity allows it tomediate infection by binding such glycans on viral glycoproteins,but its biochemical and cell biological properties suggest that thenatural function of LSECtin may be in intracellular trafficking.

(11) Pathogen Subversion of Host Siglec ReceptorsVictor Nizet

UCSD School of Medicine, La Jolla, CA

The essence of innate immunity is the rapid detection and eliminationof infectious pathogens all the while avoiding self-reactivityand limiting inflammatory tissue damage. Human CD33-relatedSia-recognizing immunoglobulin (Ig) superfamily lectins(hCD33rSiglecs) are expressed prominently on the surface of leuko-cytes including neutrophils and macrophages. A number of key,including hSiglec-5 and hSiglec-9 possess tyrosine-based inhibitorymotifs (ITIMs) in their cytoplasmic tail. We hypothesize that berecognizing host ‘Sias’ as self, these inhibitory receptors limitunwanted neutrophil reactivity. We have begun to examine inter-actions between certain leading human bacterial pathogens andhCD33rSiglecs during the innate immune response. Group BStreptococcus (GBS) possesses a sialylated capsular polysaccharidethat engages neutrophil Siglec-9, reducing oxidative burst and

extracellular trap production in a Sia- and Siglec-9-dependentmanner. Thus, GBS can impair neutrophil defense functions bycoopting a host inhibitory receptor via sialoglycan molecularmimicry, a novel mechanism of bacterial immune evasion. Micedeficient in SiglecE exhibit enhanced innate immune responses andresistance to Siglec-binding GBS, since they lack this target receptorfor bacterial immune subversion. In further studies, we have discov-ered that GBS and other pathogens can also engage hCD33rSiglecsin a Sia-independent fashion, through specific bacterial cell surfaceproteins, once again blocking phagocytosis and bacterial killing.Another interesting phenomenon is observed with the sialidase-expressing pathogen Streptococcus pneumoniae (SPN), whichappears to elicit exaggerated inflammatory cytokine release frommonocytes and neutrophils by ‘uncapping’ cis-Sia engagement of thehCD33rSiglecs with self ligands. Finally, paired ‘activatory’ Siglecsexpressing ITAM motifs and capable of DAP12 recruitment/signal-ing, e.g. hSiglec-14 vis-a-vis Siglec-5, may provide and evolutionarycounterbalance to recognize and respond to Siglec-subverting patho-gens. Rapid evolution of hCD33rSiglecs and balanced polymorph-isms in the population may reflect an ongoing arms race betweenhost recognition and pathogen evasion, with implications forinfectious disease susceptibility and pathogenesis.

(12) Galectin-glycan Interactions Control Different ImmuneCompartments to Temper Inflammation-Induced

NeurodegenerationGabriel Rabinovich

IBYME-CONICET, Buenos Aires, Argentina

Inflammation-mediated neurodegeneration occurs in the acute andthe chronic/progressive phases of multiple sclerosis (MS) and itsanimal model experimental autoimmune encephalomyelitis (EAE).A number of strategies, targeting different immune compartments,have been proposed to ameliorate the severity of demyelinatingdiseases. These include down-modulation of pathogenic Th1 andTh17 cells, induction of tolerogenic dendritic cells (DCs), modu-lation of oligodendrocyte differentiation and de-activation of acti-vated M1-type microglia. Using in vitro and in vivo strategies, wehave studied the role of galectin-1, an endogenous lectin thatrecognizes galactose-beta1-4-N-acetylglucosamine (LacNAc), inthe control peripheral and CNS immune cells. Galectin-1-deficient(Lgals1-/-) mice exhibited increased EAE severity and pronouncedinflammation-induced neurodegeneration. While galectin-1-glycaninteractions selectively blunted Th1 and Th17, but not Th2- differ-entiated cells consistent with the differential ‘glycosylation signa-ture’ of these cells, interaction of this lectin with cell surfaceglycoproteins (mainly CD43) on DCs did not affect survival ofthese cells. However, galectin-1 signaled DCs to differentiate intotolerogenic DCs which in turn favored the expansion of FoxP3(-)regulatory T cells through mechanisms involving IL-27 and IL-10.Moreover, we recently identified galectin-1 as a key player in thecontrol of microglia activation and neurodegeneration. Throughselective interactions with M1-type, but not M2-type microglia,galectin-1 targets activation of p38MAPK- and CREB-dependentpathways and hierarchically controls downstream pro-inflammatorymediators such as iNOS, TNF and CCL2. Our findings identify


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essential roles of galectin-1-glycan interactions in tempering braininflammation and neurodegeneration through modulation of T cell,DC and microglia physiology with critical therapeutic implicationsin relapsing-remitting and secondary progressive MS.

(13) Carbohydrate Antigen Oxidation: Regulationand Disease-Associated Changes

Colleen Lewis, Brian CobbCase Western Reserve University, Cleveland, OH

Carbohydrate antigens (glycoantigens; GlyAgs) from commensalbacteria have been shown to associate with class II major histocom-patibility complex (MHCII) within professional antigen presentingcells. CD4+ T cells respond to these molecules in either apro-inflammatory or anti-inflammatory manner, depending on thecircumstances. Over the past several years, we have demonstratedthat GlyAgs require processing from high molecular weight (>100kDa) to low molecular weight species ranging in size from 3 to 8kDa in order to bind MHCII. The mechanism for this cleavage isdriven by nitric oxide oxidation within endo/lysosomal compart-ments. First, we wanted to determine how a non-specific cleavagereaction was regulated to prevent complete degradation of the anti-genic unit in cells and maintain the required 3 to 8 kDa fragments.Using confocal microscopy, we determined that active oxidationof the endocytosed antigen induces an acidic microenvironmentwithin GlyAg-carrying vesicles due to the release of protons duringthe reaction. This acidic pH inhibits further oxidation and preservesfragments large enough for appropriate loading onto MHCII mol-ecules. Second, we wanted to discover whether disease-associatedchanges in redox might alter GlyAg processing and downstream Tcell-dependent immune responses. We found that the oxidationpathway was highly dysregulated in mouse models of chronic gran-ulomatous disease (CGD) in which the NADPH oxidase complex isdefective. The lack of superoxide synthesis via NADPH oxidaseresulted in significantly higher concentrations of nitric oxide andthis lead to increased GlyAg processing efficiency, increasedpro-inflammatory T cell activation, and more severe inflammation.These findings reveal how carbohydrate oxidation is regulated incells and provide possible drug targets to slow pro-inflammatory Tcell signals triggered by GlyAg-expressing commensal bacteria.

(14) Modulation of Immune Responses by Targeting CD169/Siglec-1 with the Glycan Ligand

Norihito Kawasaki1, Corwin Nycholat1, Christoph Rademacher1,Weihsu Chen1, Jose Vela2, Igor Maricic3, Paul Crocker4,Vipin Kumar3, Mitchell Kronenberg2, James Paulson1

1The Scripps Research Institute, La Jolla, CA; 2La Jolla Institutefor Allergy and Immunology, San Diego, CA; 3Torrey Pines

Institute for Molecular Studies, San Diego, CA; 4University ofDundee, Dundee, UK

Immunotherapy directed towards enhancement of an immuneresponse has been proposed as an effective way to combat cancersand infections. Development of vaccine with enhanced delivery ofantigen to immune cells is envisioned as a key factor in potent induc-tion of immune responses. Recent studies have identified CD169/Sialoadhesin/Siglec-1 expressing macrophages as versatile antigen

presenting cells to orchestrate antigen specific immune responses,making CD169 a promising target to control immune responses.CD169 is widely recognized as a cell surface marker expressed onsubsets of macrophages in humans, mice and other mammals. Uponrecognition of its glycan ligand, N-acetylneuraminic acid (NeuAc)a2-3 linked to galactose residue of glycans, CD169 internalizes thesialylated molecules into the cell, demonstrating its role as an endo-cytic receptor on macrophages. We have developed a sialoside ligandof CD169, 9-N-thienochromenecarboxyl-NeuAca2-3Galb1-4GlcNAc (TCCNe uAc) glycan ligand. We observed thatTCCNeuAc liposomes were captured by macrophages in a CD169-dependent manner, internalized into the cell and delivered to lyso-somes. Using this targeted liposome, we incorporated antigens ofinterest to modulate immune responses via CD169 positive macro-phages. We found that intravenous injection of ovalubmin (OVA), amodel protein antigen, containing TCCNeuAc liposomes activatedOVA-specific CD8 + T cells in vivo. This activation of CD8 + T cellswas largely abrogated in CD169 KO mouse, suggesting theTCCNeuAc liposomes are captured by CD169 macrophages, interna-lized and antigens are subsequently processed and presented to CD8+ T cells. Alternatively, we made TCCNeuAc liposomes containingα-Galactosylceramide (a-GalCer) to activate natural killer T (NKT)cells, the glycolipid-antigen reactive T cells with immunoregulatoryfunction. By intravenous injection of the α-GalCer containingTCCNeuAc liposomes, we saw activation of NKT cells in vivo,which was completely CD169 dependent. In summary our datademonstrate that delivering antigens via CD169-targeted liposomesprovides an effective way to modulate immune responses in vivo.(NIH CA138891 and AI050143)

(15) FBG1 is a Ubiquitin Ligase that Preferentially RecognizesUnfolded Glycoproteins

Kevin Glenn1,3, Adam Mallinger2, Hsiang Wen31Veterans Affairs Medical Center, Iowa City, IA; 2Kansas CityUniversity of Medicine and Bioscience, Kansas City, MO;

3University of Iowa Hospital & Clinics, Iowa City, IA

Glycoproteins produced in the endoplasmic reticulum undergo aquality control mechanism, endoplasmic reticulum-associateddegradation (ERAD), to eliminate misfolded proteins. One markerused by the ER to identify misfolded glycoproteins is the presenceof a trimmed, high-mannose group. This high-mannose tag with theexposed chitobiose core serves as the signal for retrotranslocationinto the cytosol, and subsequent targeted destruction by the ubiqui-tin proteasome pathway. Uniquely among ubiquitin ligases, FBG1recognizes this exposed chitobiose core as a target for ubiquitina-tion. To date, assessing misfolding has only been possible at thelevel of individual proteins using a variety of techniques such as,circular dichroism, fluorescence/infrared spectroscopy, native PAGEgels, X-ray crystallography, and NMR. Here, we demonstrate that aunique ubiquitin ligase, FBG1, can be used as a general unfoldedglycoprotein sensor. Using glycan arrays we show that FBG1 recog-nizes the exposed chitobiose core and that it has an enhancedaffinity for an exposed chitobiose core and a trimmed highmannose group. We also demonstrate that FBG1 binds unfoldedglycoproteins by showing that FBG1 substrates increase under


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conditions that cause cellular misfolding such as heat stress, protea-some inhibition, or induction of ER stress. Additionally, usingspecific glycoproteins we show that FBG1 prefers genetically orchemically unfolded glycoprotein compared to the native isoforms.Furthermore we tested the ability of various sugars to competitivelyelute off the natural substrates and found that FBG1 substrates weremost effectively eluted by the core sugar, Man3GlcNAc2, the sameexposed chitobiose core found on misfolded glycoproteins. Usingaging fibroblasts we also demonstrate an increase in misfolded gly-coproteins within the cell on the cell surface during aging. We alsofound an increase in misfolded glycoprotein in aging mouse tissue,suggesting that the accumulation of misfolded glycoproteins is atleast a consequence of aging if not a cause.

(16) LNFPIII Type 2 Activation of Antigen Presenting CellsOccurs via Endocytosis and Clathrin/Dynamin Mediated

PathwayLeena Srivastava, Smanla Tundup, Donald Harn

University of Georgia, Athens, GA

LNFPIII is a biologically conserved pentasaccharide that contains theLewis X trisaccharide. LNFPIII is found in human milk and Lewis Xis expressed by parasitic helminthes such as Schistosomes. LNFPIIIconjugates induce anti-inflammatory and Th2-type responses in vivo,preventing or reversing inflammation induced diseases/conditionsincluding psoriasis and transplant organ rejection. We believe theability of LNFPIII conjugates to drive anti-inflammatory responses invivo, is by inducing alternative activation of antigen presenting cells(APCs). We report here that LNFPIII interacts with APCs and isinternalized through clathrin/dynamin dependent and caveoli inde-pendent endocytotic pathway. Disruption of actin filaments partiallyprevented the entry of LNFPIII into APCs. Furthermore, LNFPIIIco-localizes with both early and late endosomal markers and even-tually induces lysosomal maturation. Using specific inhibitors andsiRNA against clathrin we show that endocytosis is required forLNFPIII conjugates to drive dendritic cells to DC2-type. Overall, thisstudy demonstrates not only how LNFPIII is taken into APCs butsuggests a partial mechanism related to how LNFPIII induces anti-in-flammatory and Th2-type responses.

(17) Biochemical Reconstitution and Resolution of LipidFlippase Activities Required for Protein Glycosylation

in the ERAnant K. Menon

Weill Cornell Medical College, New York, NY

The oligosaccharide donor for protein N-glycosylation isGlc3Man9GlcNAc2-PP-dolichol. This lipid is required in the ERlumen. However, its biosynthesis is initiated on the cytoplasmicface of the ER necessitating transbilayer movement, or flip-flop,of the biosynthetic intermediate Man5GlcNAc2-PP-dolichol(M5-DLO), and the sugar donors Man-P-dolichol (MPD; requiredalso for GPI-anchoring, O-mannosylation and C-mannosylation inthe ER lumen) and Glc-P-dolichol across the ER membrane. Sinceflip-flop of these lipids is intrinsically unfavorable it is presumedto be facilitated by specific transport proteins, or flippases. Thepredicted flippases have yet to be identified. We now report

reconstitution of rapid, ATP-independent flipping of M5-DLO andMPD in proteoliposomes generated from detergent-solubilized ERmembrane proteins. We show that the M5-DLO and MPD flippaseactivities can be biochemically resolved from each other as well asfrom the ER glycerophospholipid flippase activity, and are highlyspecific with respect to the structure of the lipid that they transport.These results provide the first clear indication of at least threespecific flippase proteins in the ER and establish the basis for theireventual purification and identification.

(18) An Approach to Understand the Structure-FunctionRelationships of Glycan-Binding Proteins

Yoshiki YamaguchiStructural Glycobiology Team, RIKEN ASI, Wako-Shi, Japan

The biological effect of beta-glucan in relation to its tertiary struc-ture is one of the fundamental immunological issues. Althoughmany efforts have been made to reveal the structure-functionrelationship, there is still no consensus on the basic requirement ofthe biological activity of beta-glucan. This is in part due to insuffi-cient characterization of the tertiary structure of beta-glucan inassociation with its counter receptor.

We solved the crystal structures of the N-terminal beta1,3-glucanrecognition domain of betaGRP/GNBP3 (betaGRP-N) in complexwith glucose hexamer, laminarihexaose. In the crystals, three struc-tured laminarihexaoses simultaneously interact through six glucoseresidues (two from each chain) with one betaGRP-N. The spatialarrangement of the laminarihexaoses bound to betaGRP-N isalmost identical to that of a beta1,3-glucan triplex. Therefore, ourcrystallographic structures together with site-directed mutagenesisdata provide a structural basis for the unique recognition by suchreceptors of the triple-helix structure of beta1,3-glucan.

In typical carbohydrate-protein interaction, the actual region ofcontact between the carbohydrate and the proteins involves onlyone to three monosaccharide residues. As a consequence,carbohydrate-binding proteins tend to be of relatively low affinity.Many carbohydrate-binding proteins are oligomeric and achievehigh-affinity binding by making multiple interactions with multi-valent ligands, in order to mediate biologically relevant interaction.Another possible strategy to gain the affinity is to have an extendedcarbohydrate-binding site. betaGRP adopts the latter strategy. Aunique interaction mode is attained using an extensive bindingsurface of the betaGRP-N protein. The unexpectedly large contactarea of betaGRP-N reap a benefit of high-affinity binding to triplehelical beta-glucan using one carbohydrate recognition domain.

(19) Genetics and the Environment Converge to dysRegulateN-glycosylation in Multiple Sclerosis

Haik Mkhikian3, Ani Grigorian1, Carey Li1, Hung-Lin Chen1,Barbara Newton1, Raymond W. Zhou1, Christine Beeton1,Sevan Torossian1, Gevork Grikor Tatarian1, Sung-Uk Lee1,

Ken Lau2, Erin Walker2, Katherine A. Siminovitch2,K. George Chandy1, Zhaoxia Yu1, James W. Dennis2,

Michael Demetriou11UC Irvine, Irvine, CA; 2SLRI - Mt Sinai Hospital, Toronto, ON;

3University of California, Irvine, CA


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How environmental factors combine with genetic risk at the mol-ecular level to promote complex trait diseases such as MultipleSclerosis (MS) is largely unknown. In mice, N-glycan branchingby the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyper-activity, cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis,spontaneous inflammatory demyelination and neurodegeneration,the latter pathologies characteristic of MS. Here we show that mul-tiple MS risk modulators converge to alter N-glycosylation and/orCTLA-4 surface retention conditional on metabolism andVitamin D3, including genetic variants in interleukin-7 receptor-α(IL7RA*C), interleukin-2 receptor-α (IL2RA*T), MGAT1(IVAVT-T) and CTLA-4 (Thr17Ala). Down-regulation of Mgat1 byIL7RA*C and IL2RA*T is opposed by MGAT1 (IVAVT-T) andVitamin D3, optimizing branching and mitigating MS risk whencombined with enhanced CTLA-4 N-glycosylation by CTLA-4Thr17. Our data suggest a molecular mechanism in MS wherebymultiple environmental and genetic inputs lead to dysregulation ofa final common pathway, namely N-glycosylation.

(21) Binding of Smaller, but Not Larger, Hyaluronan toHyaluronan Receptor for Endocytosis (HARE) Activates NF-kB

Mediated Gene ExpressionMadhu S. Pandey, Bruce A. Baggenstoss, Jennifer L. Washburn,

Paul H. WeigelUniv of Oklahoma Health Sciences Center, Oklahoma City, OK

The Hyaluronic Acid Receptor for Endocytosis (HARE, akaStab-2) is the primary mammalian scavenger receptor for systemicclearance of 13 ligands, including hyaluronan (HA), heparin,chondroitin sulfates (A, C, D & E), dermatan sulfate, acetylatedLDL, and apoptotic cells. Two HARE isoforms, full-length315-HARE and a functional proteolyic 1416aa C-terminal product(190-HARE), are highly expressed in sinusoidal endothelial cellsof liver, lymph node and spleen, in oviduct, corneal and lens epi-thelium, and in heart valve mesenchymal cells. HARE contains a72aa cytoplasmic domain with four endocytic motifs. We pre-viously found that HARE-HA endocytosis initiates intracellularsignaling leading to ERK activation (Kyosseva et al, J. Biol.Chem. 283: 15047, 2008). Here we used a dual-luciferase NF-kBreporter gene expression assay to test purified fractions of low-endotoxin non-animal derived HA, with narrow size ranges andminimal overlap (by SEC-MALLS), for the ability to stimulatecell signaling and gene expression mediated by rat or human190-HARE in stable Flp-In 293 cell lines. Both HARE speciesmediated HA dose-dependent gene activation using smaller HApreparations with weight-average masses (Mw) of 80, 107 or 178kDa. Activation of gene expression occurred with apparent Kmvalues of 10 nM HA (107 kDa) and maximal responses at �20nM. In contrast, intermediate and larger HA (Mw = 436 or 780kDa) did not activate NF-kB as effectively. HA stimulation ofNF-kB mediated gene expression did not occur in cells expressing(i) empty vector, (ii) HARE mutants lacking the HA-binding Linkdomain or one of the four endocytic motifs, or (iii) in cells treatedwith HA-blocking antibody. The results indicate that HAREbinding to small (but not large) HA and all four cytoplasmicdomain endocytic motifs are required for intracellular signaling

leading to ERK activation and downstream NF-kB mediated geneexpression. (Supported by OCAST HR10-074).

(22) Structural and Functional Characterization of HumanProtein O-fucosyltransferase 2

Chun-I Chen, Jeremy J. Keusch, Dominique Klein,Jan Hofsteenge, Heinz Gut

Friedrich Miescher Institute, Basel, Switzerland

Protein O-fucosylation is a post-translational modification foundon epidermal growth factor-like (EGF) repeats and thrombospon-din type 1 repeats (TSR). The EGF modification was shown toregulate embryonic development and tissue renewal by controllingthe ligand specificity of Notch [1] but the role of proteinO-fucosyltransferase 2 (POFUT2) -dependent fucosylation ofTSRs is less well understood. Nevertheless, recent work on Pofut2knockout mice found that O-fucosylation of TSRs is essential forrestricting epithelial to mesenchymal transition, correct patteringof mesoderm and localization of endoderm, suggesting a criticalrole of POFUT2 in the early development of the mouse embryo[2]. More than 40 human proteins, located either on the membraneor being secreted, contain the TSR consensus sequence forPOFUT2-dependent fucosylation. Together, these proteins regulatea wide variety of biological processes such as anti-angiogenesis,growth factor release or innate immunity response. In order tobetter understand O-fucosylation on TSRs we carried out a com-plete structural and functional analysis of human POFUT2. Crystalstructures of native POFUT2 and of a sugar donor complex willbe discussed and correlated with enzyme activity measurements ofwild-type and mutant proteins using a HPLC mass-spectrometryassay. Our experimental findings give insight into the catalyticmechanism, the role of divalent cations in catalysis, and thespecial structural requirements of a protein glycosyltransferasemodifying a completely folded protein sugar acceptor that transi-ently forms a protein-protein interaction.

References[1] Kopan and Ilagan, Cell, 2009, 137, 216.[2] Du et al., Developmental Biology, 2010, 346, 25.

(23) New Discoveries in Bacterial N-Linked ProteinGlycosylation

Christine Szymanski1,21University of Alberta, Edmonton, Canada; 2Alberta Innovates

Centre for Carbohydrate Science, Edmonton, Canada

The foodborne pathogen, Campylobacter jejuni, is one of theleading causes of bacterial gastroenteritis worldwide and fre-quently associated with the post-infectious neuropathy, Guillain–Barré syndrome. C. jejuni was the first bacterium demonstrated topossess a general N-linked protein glycosylation (pgl) pathwaythat adds a conserved heptasaccharide (GalNAc-GalNAc-(Glc)GalNAc-GalNAc-GalNAc-diNAcBac) to the extended sequon D/E-X-N-X-S/T of more than 60 proteins. We recently demonstratedthat C. jejuni also produces large amounts of free oligosaccharides(fOS) derived from the N-glycan pathway. The fOS produced byC. jejuni are structurally identical to the N-linked glycan and are


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produced in response to changes in the osmotic environment ofthe organism.Comparative genomics of 31 Campylobacter species revealed that

pgl genes are present in all these strains and mass-spectrometry studiesshowed that they all produce fOS. These pgl pathway-derived glycanscan be divided into two structural groups. The thermotolerant campy-lobacters produce the C. jejuni heptasaccharide (group I) and includethe other two common gastrointestinal pathogens, C. coli and C. upsa-liensis. A subgroup of campylobacters within group I cluster with theniche-adapted species, C. lari. These species have all lost the glucosyl-transferase enzyme and therefore synthesize the hexasaccharide:GalNAc-GalNAcGalNAc-GalNAc-GalNAc-diNAcBac. The secondcluster (group II) includes species such as C. fetus, C. concisus,C. hyointestinalis, C. rectus and C. hominis. These non-thermotolerantcampylobacters can be divided into 8 subgroups based on the glycanstructures produced. Similar to previous studies with the C. jejuni hep-tasaccharides, group II glycans are immunogenic and surface exposed.Analysis of the glycoproteomes of C. fetus fetus, C. rectus andC. hominis also identified several glycoproteins that were modified atthe extended N-glycosylation sequon. However, in addition to synthe-sizing a diverse array of related glycans by the group II campylobac-ters, several strains are capable of producing more than one N-glycanstructure further compounding the diversity.

(24) Identifying and Exploiting O-Glycosylation Systemsfrom Bacterial Pathogens

Mario FeldmanUniversity of Alberta, Edmonton, Canada

Several bacterial species synthesize glycoproteins using oligosac-charyltransferases (OTases) that transfer glycans preassembledonto a lipid donor to proteins. The PglB protein is the OTase fromCampylobacter jejuni involved in the N-glycosylation of multipleproteins. Likewise, PglL from Neisseria meningitidis is the OTaseresponsible for O-glycosylation of multiple proteins in these bac-teria. These two OTases have been functionally expressed inEscherichia coli and exhibit relaxed glycan specificity, whichopened the door to the possibility of engineering recombinant gly-coproteins for biotechnological applications. Bacteria, in particularE. coli cells, constitute a perfect toolbox for glycoengineering, asthey tolerate the incorporation and manipulation of foreign bac-terial glycosylation pathways. In this talk I will discuss the mostpromising application for bacterial glycoproteins in the design andsynthesis of a new generation of conjugate vaccines and novelglycan-based diagnostics. I will also present recent work on thecharacterization of additional O-glycosylation systems in diversebacterial species, including important human pathogens likeAcinetobacter baumannii and Vibrio cholerae.

(25) The S-layer Glycome – Adding to the Sugar Coatof Bacteria

Christina SchafferUniv of Nat Resrcs & Life Sci, Österreich, Austria

S-layer glycoproteins are a distinct class of bacterial cell surfaceglycoconjugates, mostly decorating Gram-positive cells. They are

equipped with the intrinsic capability of self-assembling into 2Dlattices that display glycans with nanometer-scaled periodicity.Typically, S-layer glycans are heteropolysaccharidesO-glycosidically linked to multiple serine, threonine or tyrosineresidues on the S-layer proteins; they rely on a much widervariety of constituents, glycosidic linkage types, and structuresthan their eukaryotic counterparts. As the S-layer glycome ofseveral bacteria is unravelling, a picture of how S-layer glyco-proteins are biosynthesized is evolving. S-layer glycan biosyn-thesis pathways utilize different modules of the biosynthesisroutes of lipopolysaccharide O-antigens, albeit in new combi-nation. Production of S-layer O-glycans is initiated in the cyto-plasm by a WbaP homologue that transfers a Gal residue fromGal-1-P to a carrier lipid, followed by processive assembly ofthe glycan on this lipid-linked primer by frequently multifunc-tional glycosyltransferases, ABC transporter-mediated export ofthe assembly across the cytoplasmic membrane and subsequenten bloc transfer of the glycan to distinct O-glycosylation sitesof the S-layer protein, which would be exported upon signalpeptide cleavage, by an O-oligosaccharyltransferase. It isassumed that glycosylation proceeds co-secretionally, followedby surface self-assembly of the mature glycoprotein subunits.The mature S-layer glycoprotein lattice is anchored to the under-lying cell envelope via a species-specific cell wall saccharide.While S-layer glycans are generally assumed to participate indifferent biological cell-surface mediated phenomena andprovide a selection advantage to the bacterium in the naturalhabitat, for the Gram-negative pathogen Tannerella forsythia, wecould recently demonstrate that S-layer O-glycosylation rep-resents a general glycosylation system which is in linked topathogenicity. In the future, it will be exciting to fully exploitthe S-layer glycome for glycoengineering purposes and to linkit to the bacterial interactome.

This work is funded by the Austrian Science Fund FWF.

(26) O-antigen Synthesis in Enterohaemorrhagic Escherichiacoli Serotype O157: Characterization of a Novel

GlucosyltransferaseYin Gao1, Scott Strum1, Bin Liu2, John S. Schutzbach1,

Tatyana N. Druzhinina3, N. S. Utkina3, Vladimir I. Torgov3,Walter A. Szarek1, Lei Wang2, Inka Brockhausen1

1Queen’s University, Kingston, Canada; 2Nankai University,Tianjin, China; 3Russian Academy of Sciences, Moscow, Russia

The enterohaemorrhagic O157 strain of Escherichia coli displaysits O-antigen on the extracellular face of the outer membrane.The O157 antigen consists of repeating units with the sequence[-2-D-Rha4NAc-alpha1-3-L-Fuc-alpha1-4-D-Glc-beta1-3-D-GalN-Ac1-]. We recently characterized Glc-transferases from E.coliserotypes O152 and O56 that add Glc in beta1-3 linkage toGlcNAc-R. The wbdN gene found in the O157 antigen genecluster has little similarity to the Glc-transferase genes in O152and O56 and to other Glc-transferase genes. We showed herethat wbdN encodes a Glc-transferase that adds Glc to GalNAcas the second step in O-antigen repeat unit synthesis. ThewbdN gene was expressed in E. coli BL21 bacteria, purified


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and WbdN activity was characterized using the donor substrateUDP-Glc and the synthetic acceptor substrate GalNAc-alpha-PO3-PO3-(CH2)11-O-Phenyl. The disaccharide product was iso-lated by hydrophobic chromatography. EI-MS (negative ionmode) showed that one Glc residue was transferred by WbdN.WbdN has a DxD motif and requires Mn2+ ions for fullactivity. Mg2+ and Co2+ ions also activate the enzyme. WbdNactivity has a broad pH optimum and is specific for UDP-Glcas the donor substrate. GalNAc-alpha derivatives lacking thepyrophosphate group were inactive as acceptors. Our resultsillustrate that WbdN is a UDP-Glc: GalNAc-alpha-pyrophosphate-lipid glucosyltransferase that is different frompreviously studied Glc-transferases. Supported by an NSERCDiscovery grant (to I.B.).

(27) Structural Characterisation of Sulfolobus Glycoproteinsby Mass Spectrometry

Paul Hitchen1, Elham Peyfoon1, Benjamin Meyer2,Stuart Haslam1, Sonja-Verena Albers2, Anne Dell1

1Imperial College London, London, United Kingdom; 2MaxPlanck Institute for Terrestrial Microbiology, Marburg, Germany

A detailed structural understanding of archaeal glycoproteins andthe genes involved in their biosynthesis is important to our under-standing of the function and integrity cell surface structures. Weare using mass spectrometry as a tool for glycan structural determi-nation of archaeal glycoproteins and applying glycoproteomicstrategies and technologies that have evolved for the functionalanalysis of bacterial glycosylation and optimized on studies of theS-layer glycoprotein from the archaea Haloferax volcanii(Abu-Qarn et al., J Mol Biol (2007) 374:1224–36). Currently weare undertaking glycoproteomic studies on glycoproteins fromSulfolobus species, which phylogenetically are closer to theeukarya than other archaea. In contrast to the glycans found on theS-layer of H. volcanii, which like other previously characterizedarchaeal glycoproteins, contain linear glycan sequences whichbear no similarity to eukaryotic glycans, Sulfolobus S-layer glyco-proteins have been found to carry chitosbiose linked, mannosecontaining N-glycans. Initial mapping strategies looking atproteolytic digest products from the S-layer glycoprotein ofS. acidocaldarius yielded particularly rich data on the C-terminaldomain and identified a heterogeneous family of glycans, withthe largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS). Remarkably all the consensus sequencesthat have been mapped have been found to be glycosylated(Peyfoon et al., Archaea (2009) 29;754101–11). Given the matureprotein contains 31 predicted glycosylation sites, it is likely thatthere is a dense coat of glycans that blanket the cell surface ofS. acidocaldarius. Currently we are exploring the biosyntheticpathways involved in S-layer glycosylation and here we willpresent data from the functional analysis of the N-glycanbiosynthetic pathway. In complementary work, we are looking atS. solfataricus and S. islandicus, where we have discovered verysimilar glycan decorations and like S. acidocaldarius, it appearsthat the majority, if not all, of the consensus sequence sitesare occupied.

(28) Human Milk Oligosaccharides Affect Growth of InfantFecal Microbiota in vitro

Zhuoteng Yu, Ceng Chen, Bo Liu, David S. NewburgDavid S Newburg, Chestnut Hill, MA

The microbiota of the human gut is important for human health;colonization begins at birth. The influence of human milk consump-tion on colonization was tested by measuring utilization of humanmilk oligosaccharides (HMOS) by infant fecal bacteria (prebioticactivity). Prebiotics are indigestible glycans that stimulate growth ofmutualist symbiotic bacteria, suppress the growth of some patho-gens, and decrease gut pH through production of organic acids.First, effects of the entire human milk oligosaccharide fraction onthe entire population of the infant gut microbial community weretested. Infant feces supplemented with HMOS prior to 24 hours offermentation had significantly lower pH than in controls, and thepH was lower than in the groups supplemented with FOS. Lacticacid concentrations in the HMOS groups were significantly higherthan in controls by the end of fermentation. For each of the fecalsamples, irrespective of the donor source, HMOS could signifi-cantly increase the number of Bifidobacteria and Lactobacillus sp.For some donors, HMOS significantly decreased the number of E.coli (P < 0.05) and Clostridium perfringens (P < 0.05). To investi-gate which individual HMOS components were directly responsiblefor the growth of specific bacteria in the infant microbiota,2′-fucosyllactose (2’-FL), a principal HMOS component, was fed toindividual bacteria of the infant microbiota. 2’-FL and HMOS, atthe concentration found in human milk, significantly increased thegrowth of the Bifidobacteria sp. and Bacteroides sp., and the pHdecreased during fermentation. With higher concentrations of 2’-FLand HMOS, the growth increased and pH decreased accordingly. Incontrast, supplementing E. coli and Clostridium perfringens with2’-FL and HMOS did not significantly increase growth or decreasepH. Thus, HMOS and 2’-FL are prebiotics, stimulating growth ofBifidobacteria and Lactobacilli, and inhibiting that of pathogens ininfants, thereby supporting colonization of the infant humanintestine by desired mutualist symbiotic bacteria.

(29) Glycosylation and Its Function in Aspergillus fumigatusCheng Jin

Institute of Microbiology, CAS, Beijing, China

Aspergillus fumigatus is the predominant mold pathogen ofhuman, causing fatal invasive aspergillosis (IA) among the immu-nocompromised population. The crude mortality is 60-90% andremains around 29-42% even a treatment is given. A deep under-standing of A. fumigatus at molecular level will help to developthe efficient drug therapies to treat IA.

The fungal cell wall has a variety of biological functions, suchas maintaining cell shape and providing osmotic and physical pro-tections against an adverse environment, it has been recognizedfor a long time as essential and unique specific drug targets.A. fumigatus cell wall mainly consists of a covalently connectedpolysaccharide skeleton interlaced and coated with glycoproteinsand GPI proteins, which contain N- and O-glycans derivedprimarily from the process of glycosylation. Although these


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glycoproteins are involved in morphogenesis and cell wall organiz-ation, it is poorly understood how glycosylation affects the cellwall organization.During the past few years, the glycans on glycoproteins from

A. fumigatus have been characterization. Several genes, includingstt3, pmi1, srb1, cwh41, msdS, ams1, pmt1, pmt2, and pig-a,have been identified to be required for N-glycosylation,O-glycosylation, or GPI-anchoring in A. fumigatus. Their func-tions were analyzed by gene-disruption. Our results show that gly-cosylation is essential for cell wall biogenensis, morphogenesisand development of A. fumigatus. Although their physiologicalsignificances are vary, some of these genes would be potentialtargets for development of anti-fungal drugs. In addition, evi-dences we obtained show that A. fumigatus is an ideal model forunderstanding the basic roles and regulations of protein glycosyla-tion in devlopment of multi-cellular eukaryotes.

(30) Moving Malaria Glycobiology Forward: RecentApproaches to Addressing the Existing Gaps in Knowledge

viz. O-GlcNAc Modifications in Parasite DevelopmentRhoel David R. Dinglasan

Johns Hopkins Bloomberg School of Public Health,Baltimore, MD

The comprehensive role of glycosylation in malaria parasitebiology remains a major gap in knowledge. O-GlcNAc, a widelyconserved nuclear and cytoplasmic O-linked post-translationalmodification (PTM) of proteins across eukaryotes, is involved in aplethora of important cellular processes. However, this intracellularglycosylation has not been conclusively shown to be present in thePlasmodium parasite, the etiological agent of malaria. In fact, theconcept of O-GlcNAcylation in the malaria parasite remains con-troversial. The prevailing perception is that demonstration of theintracellular parasite-derived O-GlcNAc modification is con-founded by the contamination of intrinsic host red blood cellO-GlcNAc-modified proteins. Through a combination ofapproaches, we have revisited the possibility of O-GlcNAc inPlasmodium and provide evidence of this important PTM in extra-cellular stages of the parasite, specifically the ookinete form,which is involved in parasite establishment in its obligate invert-ebrate vector, the Anopheles mosquito. Plasmodium ookinetes,produced in vitro (in the absence of mosquito cells/tissues) werestained using two anti-O-GlcNAc monoclonal antibodies, RL2 andCTD110.6, and this specific recognition is competed with 1 MGlcNAc. We then characterized the protein banding profile acrossthe asexual and sexual stages of the parasite for both Plasmodiumberghei (murine malaria parasite) and P. falciparum (humanmalaria parasites), and assessed the distribution of this PTM inookinetes by immunofluorescence assay. We report the preliminaryresults stemming from our combined genetic and glycoproteomicapproach to further analyze this PTM in Plasmodium, whichincluded the use of an O-GlcNAse inhibitor in in vivo (murinemalaria) and in vitro (murine and human malaria) studies, immu-noprecipitation of ookinete O-GlcNAc modified parasite proteinsand their identification by tandem mass spectrometry, and the sub-sequent gene knockout and allelic replacement of these modified

proteins as well as the putative enzyme(s) involved in this essentialbiological process.

(31) The Synthesis and Role of Modified GlycoconjugatesComprising the Surface Glycocalyx in the Infectivity

of the Protozoan Parasite Leishmania majorStephen M. Beverley1, Hongjie Guo1, Natalia Novozhilova2,Suzanne Hickerson1, Dia-Eldin Elnaiem3, David Sacks3,

Salvatore J. Turco21Washington University School of Medicine, Saint Louis, MO;

2University of Kentucky Medical Center, Lexington, KY; 3NationalInstitute of Allergy & Infectious Disease, Bethesda, MD

The surface of the Leishmania parasite is dominated by phosphogly-can (PG) glycoconjugates bearing a repeating [Gal(β1,4)Man(α1)-PO4] motif, including lipophosphoglycan (LPG) and proteopho-sphoglycans (PPGs). These play critical roles in the sand fly vector,and following introduction of the infective metacyclic form by biting,during differentiation to the replicative amastigote form, and directingthe immune responses. We have used forward and reverse geneticapproaches to identify genes implicated in LPG/PPG synthesis anddetermine their molecular role and effect on parasite virulence.

In the fly, Leishmania must attach to the midgut to avoidexcretion, but later must release for transmission. In L. major strainFriedlin, this is controlled by stage-specific LPG modifications. TheLPG PG repeats of replicating procyclic parasites are modified byside chain β1,3-galactose residues which bind to the midgut lectinPpGalec. During metacyclogenesis, the Gal residues are cappedwith α1,2-D-arabinopyranose residues, which abrogates binding.

We generated null mutants of a bifunctional D-arabinopyranose/L-fucose kinase/pyrophosphorylase (AFKP80), which were unableto carry out the activation of D-Arap to GDP-D-Arap and lackedLPG Arap modifications. In sand flies, afkp80- survived and differ-entiated to metacyclics like WT. However, fewer free metacyclicswere found in dissected midgets, confirming the critical role ofD-Arap capping for release.

Unexpectedly, afkp80- showed a strong delay in mouse lesionformation. Delayed lesion formation was not seen in afkp80- amas-tigote infections, and surprisingly, metacyclic afkp80- invaded andsurvived in macrophages normally in vitro. All afkp80- phenotypeswere fully restored by re-expression of AFKP80. Several studiesrule out the possibility that the phenotypes reflect exposure of Galside chains, including infectivity tests of galectin KO mice and theuse of L-Fuc as an alternative capping sugar to D-Arap. Instead,LPG Arap -modifications may play an unrecognized role in earlystages of mammalian infections, probably through interaction withhost defenses other than macrophages.

(32) Modulation of Immunity by Helminth Parasites: FromMolecules to Effector Mechanisms

Derek McKayUniversity of Calgary, Calgary, Canada

Helminth (worm) parasites are humans (and their domestic live-stock) evolutionary companion and while some have only a mildeffect on the host, others take a devastating toll on human health:schistosomiasis, neurocysticercosis and elephantiasis are all life-


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threatening diseases caused by helminth parasites. In seeking thisobligate association with their host, helminth parasites must avoidor circumvent host immune responses that are aimed at identifying,inactivating and eradicating the helminthic burden. Consequentlyhelminths have a variety of ways to undermine their host’s immu-nity and promote their own survival: principal amongst these areexcretory/secretory products that directly affect host immune cells.Using the rat tapeworm, Hymenolepis diminuta, we have beenadvancing toward the purification of helminth-derived immunosup-pressive molecules. Data to date illustrate that semi-pure whole-worm extracts of H. diminuta, when applied to human or murine Tcells or macrophages, significantly inhibit immunocyte activationas gauged by proliferation and cytokine production. In the contextof T cell inhibition the bioactive helminth-derived molecule islikely a protein, whereas it appears that a glycan from the helminthis responsible for the suppression of lipopolysaccharide-stimulatedmacrophage activity. The elucidation of these molecules, andothers, from H. diminuta (and parasitic helminths in general) mayserve as the blueprints for the development of new immunomodula-tory drugs. Finally, analysis of the host immune response to infec-tion has the potential to uncover immune signaling events that wecan exploit to intervene and treat idiopathic inflammatory or auto-immune diseases, such as inflammatory bowel disease. We havefound that mice infected with H. diminuta are protected from colitisinduced by intra-rectal administration of dinitrobenzene sulphonicacid, and that interleukin-10 and alternatively activated macro-phages are important components of this anti-colitic effect.Research supported by the Crohn’s and Colitis Foundation of

Canada (CCFC) and the Canadian Institutes for Health Research(CIHR)

(33) Effect of Inhibitors of Sphingolipids Synthesis onLeishmania Growth and (glyco)(sphingo)Lipid Expression

Erica Castro, Helio Takahashi, Anita H. StrausUniversidade Federal de Sao Paulo, Sao Paulo, Brazil

Leishmania expresses specific glycolipids and inositol phosphoryl-ceramide (IPC) in the plasma membrane microdomains, which havebeen related to several biological processes, such as parasite infec-tivity. IPC is the major sphingolipid expressed in promastigotes andalthough present in low concentration in amastigote forms ofLeishmania (Leishmania) amazonensis, treatment of both forms ofparasites with Aureobasidin A, an inhibitor of inositol phosphoryl-ceramide synthase, decreases significantly the parasite growth(Tanaka et al 2007). In the present study it was investigated theeffect of myriocin (an inhibitor of serino palmitoyltransferaseresponsible for first step of sphingolipid synthesis) in the growth,morphology and (glyco)(sphingo)lipid expression of promastigoteforms of Leishmania amazonensis. Addition of myriocin 2.5 and 5µM: i) led to changes in parasite morphology as observed by scan-ning microscopy (ellipsoid to rounded shapes); ii) inhibited by 37%and 45% of promastigotes growth, respectively; iii) reduced signifi-cantly the proportion of IPC from 14.5% (control parasites) to 4%and 2%. Conversely, parasites treated with myriocin increased theexpression of phosphatidylserine - present mainly at the inner leafof plasma membrane - from 6% (control parasites) to 17% and 25%

(2.5 µM and 5 µM myriocin) and lyso-phosphatidylinositol from2.6% (control) to 5.3% and 7.7% (2.5 µM and 5 µM myriocintreated parasites). An increase of glycosylinositol phospholipids(GIPLs) expression was also detected by EIS/MS and by immunos-taining with anti-GIPLs monoclonal antibodies. 5 µM and 12 µMmyriocin decreased by 43% and 77% peritoneal macrophages infec-tivity by promastigotes of L. amazonensis. Myriocin treated culturessupplemented with 3-ketodihidrosphingosine reverted partially pro-mastigote morphological alterations. The absence of GIPLs andIPC in mammalian cells make these molecules potential targets fortherapeutic drugs. Thus, the elucidation of sphingolipid and GIPLroles in parasite biology and in the organization of plasmamembrane, may contribute toward a better understanding thepathobiolgy of leishmaniasis.

Supported by FAPESP, CAPES, CNPq, FADA.

(34) Identification of Glycotransferases Involvedin the Synthesis of Functional O-Man Glycans

Stephanie H. Stalnaker, David Live, Geert-Jan Boons,Kelley Moremen, Lance Wells, Ryan Stuart

University of Georgia, Athens, GA

Dystroglycanopathies, a subset of congenital muscular dystrophies(CMD), are caused by defects in glycosylation of dystroglycan.α-dystroglycan is a key component of the dystrophin-glycoproteincomplex, which links the extracellular matrix and the cytoskeleton.This heavily O-glycosylated protein contains a significant amountof α-linked O-mannose-initiated structures. A recently identifiedphosphorylated trisaccharide, GalNAc β3 GlcNAc β4 (6-P-X)Man, was identified that appears to be extended in a phosphodie-ster linkage and responsible in a LARGE-dependent manner forbinding to extracellular matrix components. We are currentlytrying to determine the entire structure and the enzymes respon-sible for this functional glycan. Currently, recombinant mouseα-dystroglycan is being expressed in C2C12 mouse muscle myo-blasts and purified. Glycans and glycopeptides from the purifiedsample are being examined by means of mass spectrometry. Theglycotransferases responsible for synthesis of the phosphorylatedtrisaccharide-containing moiety are being characterized concur-rently through selective sugar nucleotide radiolabeling with syn-thetic substrates and recombinant enzymes as well as RNAiknockdown of potential candidates in vivo followed by functionalbinding assays. Given that nearly half of all CMD patients do nothave mutations in the currently known involved genes inO-Mannosylation, these efforts should reveal novel candidategenes that may be responsible for CMD.

(35) A Missense Mutation in ST3GAL5 Results in a SevereIntellectual Disability Syndrome Associated with Altered

Glycosphingolipid and O-linked Glycan ExpressionKazuhiro Aoki1, Luigi Boccuto2, Qing Zhang3, Harry Wang3,Frank Bartel2, Xiang Fan1, Robert Saul2, Alka Chaubey2,

Xu Yang3, Richard Steet1, Charles Schwartz2, Michael Tiemeyer11Complex Carbohydrate Research Center, UGA, Athens, GA;

2Greenwood Genetic Center, Greenwood, SC; 3Beijing GenomicsInstitute-Shenzhen, Shenzhen, China


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A missense mutation (p.E332K) was identified in the ST3GAL5(GM3 synthase) gene of two siblings who presented with “Saltand Pepper syndrome,” characterized by altered pigmentation,severe intellectual disability, midface hypoplasia, prognathism,choreoathetosis, abnormal EKG, and scoliosis. This newly ident-ified syndrome is allelic to Old Amish infantile epilepsy syndrome(Simpson et al., Nat Genet 36:1225–1229, 2004). These two con-ditions represent the only identified human diseases resulting froma disruption of ganglioside biosynthesis. ST3GAL5 is a memberof the sialyltransferase family of glycosyltransferases and catalysesthe initial step in the biosynthesis of complex-type gangliosidesusing lactosylceramide as an acceptor substrate. Primary culturesof fibroblasts were grown and harvested in order to assessthe impact of the E332K mutation on glycan expression.Glycosphingolipid (GSL) analysis demonstrated complete loss ofGM3 ganglioside and its biosynthetic derivatives with a concomi-tant increase in globo-series GSLs. Interestingly, the E332Kmutation also impacts protein O-glycosylation, resulting in theproduction of highly sialylated O-linked glycans. To investigateneurodevelopmental consequences of altered glycan expression,we established a zebrafish model of this genetic disorder.Knock-down of zebrafish ST3GAL5 results in altered GSL biosyn-thesis, increased O-linked sialylation, gross changes in brain mor-phology, and increased neural apoptosis. Comparative analysis ofST3GAL5-deficient zebrafish and human fibroblast cultures mayreveal mechanisms of compensatory changes in glycosylation andidentify developmental processes particularly sensitive to alteredglycan expression.

(36) Stem Cell Glycomics Identifies DifferentiationStage-specific Markers and Novel Glycan Functions

Michael PierceUniversity of Georgia, Athens, GA

NA

(37) Multifaceted Role of Heparan Sulfate in MouseEmbryonic Stem Cell Biology

Daniel C. Kraushaar1, Eduard Condac1, Yu Yamaguchi2,Lianchun Wang1

1University of Georgia, Athens, GA; 2The Burnham Inst.,La Jolla, CA

Heparan sulfate (HS) is a highly sulfated polysaccharide and isexpressed abundantly by undifferentiated and differentiatingembryonic stem cells (ESC). In our studies, we investigated theroles of HS in ESC self-renewal and lineage differentiation byexamining EXT1-/- mouse ESCs (mESC) that are deficient in HS.EXT1-/- mESCs retained their self-renewal potential, but failed totransit from self-renewal to differentiation upon removal ofLeukemia Inhibitory Factor. Further study revealed that the aber-rant cell fate commitment was caused by defects in FGF signaling,which retained high expression of the pluripotency gene Nanog inEXT1-/- mESCs. Cell differentiation studies observed that theEXT1-/- mESCs could not form mesoderm as indicated by theaberrant expression of mesoderm marker genes. Examination ofESC differentiation-related signaling revealed that the BMP/Smad/

Id signaling was disrupted and positively correlated with the meso-derm differentiation defect of EXT1-/- mESCs. Restoration ofBMP signaling rescued the mesoderm differentiation defect,showing an essential requirement of HS in BMP-mediated meso-derm differentiation. Taken together, our results demonstrated thatHS essentially regulates self-renewal and lineage differentiation ofmESCs by modulating different signaling pathways, showingmultifaceted roles in ESC biology.

(38) Midas Touch: Heparan Sulfate Proteoglycans Regulatethe Stem Cell Niche via Novel Contact-Dependent Signaling

Hiroshi NakatoUniversity of Minnesota, Minneapolis, MN

The germline stem cell (GSC) niche in the Drosophila ovary pro-vides an excellent model system to study the function of ‘niches’,which support stem cell activity. In the female GSC niche,Decapentaplegic (Dpp; a Drosophila BMP) produced in the nichecells regulates the asymmetric division of a GSC. However, themechanism by which this secreted molecule differentially regulatestwo daughter cells has been a mystery. We found that Dally, aDrosophila heparan sulfate proteoglycan (HSPG) of the glypican-type, is an essential regulator of the GSC niche. Dally is specifi-cally expressed in the niche cells. Dally activates Dpp signaling intrans in a directly contacting GSC. This trans co-receptor activityof Dally could explain the contact-dependency of the GSC niche:When a GSC divides, the daughter cell directly contacting theDally-expressing niche cells remains a GSC (Dpp signaling isON), but the other daughter cell, which has lost contact, willdifferentiate (Dpp signaling is OFF). Thus, the contact-dependentsignaling by HSPGs provides a mechanism to define the physicalspace of the niche.

To further investigate the molecular mechanisms of contact-dependent BMP signaling, we developed a novel in vitro assaysystem using Drosophila S2 tissue culture cells and analyzedHSPG trans co-receptor activity. In this assay, ‘signal-sendingcells’ (expressing Dally) and ‘signal-receiving cells’ (expressingBMP receptors) are co-cultured. Contact between the sending andreceiving cells is visualized by the split-GFP complementationsystem, and BMP signal activation in the receiving cells is moni-tored by phosphorylation of Mad protein. Using this single-cellassay system, we demonstrated that HSPGs in trans enhance BMPsignaling in S2 cells in a contact-dependent manner. Our studyprovides a novel mechanism for contact-dependent signaling, inwhich the complementation of receptors and co-receptorsexpressed on different cells can form an active signaling field atthe interface of two contacting cells.

(39) The Cell Surface Glycan LacdiNAc (GalNAcb1-4GlcNAc)Contributes to Self-Renewal of Mouse Embryonic Stem Cells

by Regulating LIF/STAT3 SignalingShoko Nishihara, Norihiko Sasaki, Kazumi Hirano

Department of Bioinformatics, Soka University, Tokyo, Japan

Self-renewal of mouse embryonic stem cells (mESCs) is main-tained by leukemia inhibitory factor (LIF)/STAT3 signaling.However, this signaling control does not function in mouse


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epiblast stem cells (mEpiSCs) or human ESCs (hESCs), includinghuman induced pluripotent stem cells (hiPSCs). The underlyingmolecular mechanisms that determine this differentialLIF-responsiveness have not been clarified yet. To identify glycansthat were essential for ESC self-renewal, we performed an RNAiscreen using shRNAs for glycan-related genes in mESCs by evalu-ation of alkaline phosphatase activity. Then we found thatLacdiNAc (GalNAcβ1-4GlcNAc) is required for LIF/STAT3 sig-naling. LacdiNAc is frequently present on glycoproteins and gly-colipids in invertebrates but is only present on a limited number ofthose in vertebrates. The roles of LacdiNAc in mammalian cellshave not been fully understood.Undifferentiated state mESCs expressed LacdiNAc at a higher

level than differentiated state cells. Knockdown of β4GalNAc-T3reduced LacdiNAc expression and caused a decrease in LIF/STAT3 signaling that lessened the rate of self-renewal ofmESCs. A biochemical analysis showed that LacdiNAcexpression on LIFR and gp130 was required for the stable local-ization of the receptors with lipid raft/caveolar components, suchas caveolin-1. This localization is required for transduction of asufficiently strong LIF/STAT3 signal. In primed state pluripotentstem cells, hiPSCs and mEpiSC-like cells, LacdiNAc expressionon LIFR and gp130 was extremely weak and the level of local-ization of these receptors on rafts/caveolae was also low.Furthermore, knockdown of β4GalNAc-T3 decreased LacdiNAcexpression and reduced the efficiency of reversion of primedstate mEpiSC-like cells into naive state mESCs. These findingsdemonstrate that the different LIF-responsiveness of naive state(mESCs) and primed state (mEpiSCs, hESCs, hiPSCs) cells isdependent upon the expression of LacdiNAc on LIFR andgp130 and that this expression is required for the induction andmaintenance of the naive state.

(40) Hematopoietic Stem/Progenitor Cell Surface a2,6-sialylGlycans are Constructed by Non-Self ST6Gal-1 via aNon-Canonical ‘Extrinsic’ Glycosylation Pathway

Mehrab Nasirikenari, Christine C. Collins, Joseph T. Y. LauRoswell Park Cancer Institute, Buffalo, NY

The hematopoietic stem/progenitor cells (HSPC), responding tosystemic cues, generate the entire complement of blood celllineages. Previously we demonstrated that mice with deficienciesin the sialyltransferase, ST6Gal-1, produces more granulocytes inresponse to inflammatory stimuli. ST6Gal-1 deficient animals arehematopoietically more robust, having larger pools of LSk (Lin-Sca-1+ cKit+) cells that are highly enriched for HSPCs, and thatalso proliferated and differentiated faster ex vivo. To investigate thelink between ST6Gal-1 and hematopoietic regulation, we exam-ined closer the LSK and LK (Lin- cKit+) fractions. LSK cellsfrom wild-type C57BL/6 animals, but not cells from ST6Gal-1KO animals, were reactive towards SNA (Sambucus nigra) andPSL (Polyporus squamosus) lectins, indicating the presence of cellsurface α2,6-sialyl structurs. However, the endogenous ST6Gal-1gene was silent in wild-type LSK cells, as indicated by theabsence of ST6Gal-1 mRNA by RT-PCR, and the lack ofα2,6-sialyltransferase activity against the synthetic acceptor, Gal

(β1,4)GlcNAc-o-Bn. To interrogate the origin of LSK cell surfaceα2,6-sialylation, bone marrow chimera were constructed.ST6Gal-1 KO donor cells, in thich the endogenous ST6Gal-1 genehas been inactivated, were placed in wild-type ST6Gal-1 normalrecipients. The ST6Gal-1 KO cells acquired SNA-reactivity to thelevel identical to that of wild-type donor cells. On the other hand,wild0-type cells, when placed into ST6Gal-1 KO recipients, haddimmed SNA profiles. Furthermore, addition of recombinantST6Gal-1 strikingly attenuated the proliferation and differentiationof HSPCs in ex vivo cultures. Together, the data strongly indicatedthat HSPCs are modified by ST6Gal-1 of distal origins. This novel‘extrinsic’ ST6Gal-1 glycosylation pathway apparently serves as asystemic regulator of hematopoiesis at the level of stem/uncom-mitted progenitor cells.

(41) Natural Antibodies and Tumor-Associated CarbohydrateAntigens as Target Structures

Satish Kumar Devarapu, Sumathy Jeyaweerasinkam,Reinhard Schwartz Albiez

German Cancer Research Center, Heidelberg, Germany

Natural antibodies (nAb) are immunoglobulin molecules present inthe serum/plasma of individuals in the absence of immunizationby exogenous antigens. These are encoded in germline configur-ation and not been subjected to somatic hypermutation and affinitymaturation. The majority of these nAb of IgM isotype recognizecarbohydrate antigens. These anti-carbohydrate antibodies may inhealthy persons be part of a humoral tumor surveillance immunesystem in that they are able to kill tumor cells. However the intri-cate mechanisms which finally lead to destruction or survival oftumor cells during the attack of nAb is unclear. Identification ofdistinct carbohydrate antigens with anti-tumor cytotoxic capacitymay help to develop a new class cancer immunotherapeutics.

In the present study, serum/plasma samples of healthy individ-uals were screened by flow cytometry to identify natural antibodieswith cytotoxic potentials. As target cells we applied the neuroblas-toma cell line Kelly and the melanoma cell line MeWo. From theFACS analysis we assessed approx. 0.2% of healthy individualsera containing natural antibodies with high anti-tumorcytotoxicity.

Further to study the role of tumor associated carbohydrates aspotential target structures, tumor cells were treated with sialidaseprior to cytotoxicity testing. For most sera tested cytotoxicityincreased after sialidase treatment. Further, overexpression ofdistinct gangliosides in target cells by integration of definedgangliosides into plasma membranes entailed an enhancedcytotoxicity.

Our present study had identified natural antibodies from healthyindividuals and provided first indication for an involvement ofTumor associated carbohydrate as target structure for naturalantibody mediated cell lysis.

(42) Chemical Probes of Glycan Assembly in MycobacteriaLaura Kiessling

University of Wisconsin-Madison, Madison, WI

NA


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(43) Roles of N-glycan in Integrin-Mediated Cell Adhesionand Cellular Signaling

Jianguo GuTohoku Pharmaceutical University, Japan, Sendai, Japan

Integrins play important roles in cytoskeletal organization andintracellular signal transduction to regulate various processes suchas proliferation, differentiation, apoptosis, and cell migration.Although integrin-mediated cell adhesion is based on the bindingof alpha and beta subunits to a defined peptide sequence, thestrength of this binding is modulated by various factors includingthe status of glycosylation of integrin. For example, integrinsundergo glycosylation by N-acetylglucosaminyltransferase III(GnT-III), resulting in the inhibition to cancer metastasis in vitroand in vivo. In contrast, integrins glycosylated byN-acetylglucosaminyltransferase V (GnT-V) to form beta1,6GlcNAc branch, contribute to the promotion of cell migration.Considering integrin contains multiple potential N-glycosylationsites, the determination of these crucial N-glycosylation sites forits biological function is therefore, quite important for an under-standing of the underlying mechanism. The function of theN-glycans was investigated using sequential site-directed mutagen-esis to remove the combined putative N-glycosylation sites onintegrin. We found that the N-glycosylation on beta-propellerdomain of the alpha5 subunit is essential for its hetero-dimer for-mation with beta1 and its biological functions such as cell spread-ing and migration. Interestingly, only one site, the site-4 isessential and effective for GnT-III modification among 14 potentialN-glycosylation sites. Besides alpha5 subunit, we also identifiedimportant N-glycosylation sites of the alpha3 and the beta1 sub-units for their biological functions. These studies may be usefulfor the study of structure and function of super complex formationon cell surface.

(44) The Human Zona Pellucida: An Extracellular MatrixĆthat Mediates Sperm Binding and Immune Recognition?

Gary F. ClarkUniversity of Missouri, Columbia, MO

Previous studies in the human and mouse models indicate thatsperm initiate fertilization by binding to the specialized extracellu-lar matrix of the egg, known as the zona pellucida (ZP). Thisbinding is mediated primarily via a lectin-like interaction, and to alesser extent by protein-protein interactions. Gene knockout exper-iments and inhibition studies with defined carbohydrate sequenceshave confirmed that complex type N-glycans terminated withGalβ1-4GlcNAc (LacNAc) sequences are the major ligands recog-nized during sperm-egg binding in the mouse. These findings indi-cate that a major egg binding protein associated with mouse spermhas a carbohydrate binding specificity that is very similar to thegalectins. By contrast, an undefined carbohydrate ligand that reactswith selectins has previously been implicated in human sperm-eggbinding. Such overlaps indicate that the carbohydrate sequencespresented on the ZP of human and mouse eggs could serve tomediate both sperm binding and immune recognition. This type ofvertical integration could provide a form of immune privilege to

these germ cells both before and after fertilization. The mouseembryo begins to express paternal major histocompatibility anti-gens at the eight cell stage, making it a foreign body very earlyduring its development. Preimplantation embryos encased withinan intact ZP are completely resistant to cytotoxic T lymphocytes(CTLs) directed against paternal MHC antigens, but embryosdenuded of this matrix are rapidly destroyed by these immunecells. How these embryos are protected from such responses is notobvious, since CTLs can transit through basement membranes andother tissues to destroy histoincompatible organ transplants. Thefirst step in establishing proof for this type of immune privilege isto define the precise carbohydrate ligands that mediate gametebinding. Studies focused on this aspect will be presented. (Theefforts of collaborators at Imperial College London, the Universityof Hong Kong, and Academia Sinica are acknowledged.)

(45) Apes, Sugar and Sperm – Glycans as Agents of EvolutionPascal Gagneux

UC San Diego, La Jolla, CA

NA

(46) Increased Polysialylation in Lung Tissue of Patientswith Idiopathic Pulmonary Fibrosis

Christina Ulm1, Poornima Mahavadi2, Sandra Müller1,Susanne Rinné3, Hildegard Geyer1, Rita Gerardy-Schahn4,Martina Mühlenhoff4, Andreas Günther2, Rudolf Geyer1,

Sebastian P. Galuska11Institute of Biochemistry, JLU, Giessen, Germany; 2Departmentof Internal Medicine II, JLU, Giessen, Germany; 3Institute ofPhysiology and Pathophysiology, PU, Marburg, Germany;4Institute of Cellular Chemistry, MHH, Hannover, Germany

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing intersti-tial lung disease of unknown etiology. The disease is characterizedby alveolar destruction, uncontrolled fibroblast proliferation andexcess matrix production, resulting in progressive dyspnea, adecline in lung function and loss of gas exchange properties. Sofar, only pirfenidone has been shown to exert some efficacy in IPFand lung transplantation represents the only option to preventdeath.

The aim of the present study is the analysis of potential differ-ences in glycosylation, in particular polysialylation, of fibroticversus control lung tissue. Polysialic acid (polySia) is adevelopmentally regulated homopolymer of α2,8-linkedN-acetylneuraminic acid residues and is involved in the modulationof several migration as well as proliferation processes. We observedan up-regulation of the polysialyltransferases ST8SiaII andST8SiaIV, the key enzymes of polySia biosynthesis, using quantitat-ive real time PCR in IPF patients. In agreement with an enhancedmRNA expression level of both transferases we detected increasedpolySia levels in tissue samples of IPF patients in comparison todonor tissue by Western blotting. Using a glycoproteomicsapproach we were able to identify NCAM as the polySia carrierwhich could be confirmed by Western blot analysis. Surprisingly,polySia-NCAM was located intracellularly in vesicles of ciliatedbronchiolar epithelial cells as well as clara cells. However, the role


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of polySia-NCAM in the bronchoalveolar system and especiallyduring the development and the pathophysiology of IPF needs to befurther investigated.

(47) Targeted Drug Delivery to Tumor Vasculatureby a Carbohydrate Mimetic Peptide

Toshiaki Shibata1, Kazuhiro Sugihara2, Jun Nakayama3,Minoru Fukuda1, Michiko N. Fukuda1

1Sanford-Burnham Medical Research Institute, La Jolla, CA;2Hamamatsu University School of Medicine, Hamamatsu, Japan;

3Shinshu University School of Medicine, Matsumoto, Japan

Although numerous carbohydrates play significant roles in mam-malian cells, carbohydrate-based drug discovery has not beenexplored due to the technical difficulty of chemically synthesizingcomplex carbohydrate structures. Previously, we identified a seriesof carbohydrate mimetic peptides and found that a 7-mer peptide,designated I-peptide, inhibits hematogenous carbohydrate-dependent cancer cell colonization. During analysis of the endo-thelial surface receptor for I-peptide, we found that I-peptidebound to annexin 1 (Anxa1) (Hatakeyama et al., Proc Natl AcadSci USA 106, 3095-3100, 2009). Since Anxa1 is a highly specifictumor vasculature surface marker, we hypothesized that anI-peptide-like peptide could target anti-cancer drugs to the tumorvasculature. This study identifies IFLLWQR peptide, designatedIF7, as homing to tumors. When synthetic IF7 peptide was conju-gated to fluorescent Alexa 488 (A488) and injected intravenouslyinto tumor-bearing mice, IF7-A488 targeted tumors withinminutes. To monitor tumor growth accurately in vivo, weemployed luciferase-based cancer imaging. The colon cancer cellline HCT116 was infected with lentivirus driving luciferaseexpression, enabling visualization of cells by the Xenogen imager.IF7 was conjugated to the potent anti-cancer drug SN-38. WhenIF7-SN38 was injected into nude mice bearing large HCT116-Luctumors, tumor size was dramatically reduced, whereas tumorgrowth in control mice injected with reverse IF7 conjugated toSN38 was unchanged. Histological examination of tumors treatedwith IF7-SN38 revealed significant necrosis and apoptosis,whereas blood tests showed no sign of side effects. These resultssuggest that IF7 serves as an efficient drug delivery vehicle by tar-geting Anxa1 expressed on the surface of tumor vasculature.Given its extremely specific tumor-targeting activity, IF7 may rep-resent a clinically relevant vehicle for anti-cancer drugs. Supportedby NIH grant P01 CA71932.

(48) Enhancement of Epitelial-Mesenchymal Transition-LikePhenotype in N-acetylglucosaminyltransferase V Transgenic

Mouse Skin Promotes Wound HealingAkiko Ishikawa1, Mika Terao1, Akihiro Kimura1, Arisa Kato1,

Ichiro Katayama1, Naoyuki Taniguchi2, Eiji Miyoshi11Osaka University Graduate School of Medicine, Suita, Japan;

2Riken, Wako, Japan

N-Acetylglucosaminyltransferase-V (GnT-V) catalyzes the β1,6branching of N-acetylglucosamine on N-glycans. GnT-Vexpression is elevated during malignant transformation in varioustypes of cancer; however, the mechanism by which GnT-V

promotes cancer progression is unclear. To characterize the bio-logical significance of GnT-V, we established GnT-V transgenic(Tg) mice, in which GnT-V is regulated by a β-actin promoter. Nospontaneous cancer was detected in any organs of the GnT-V Tgmice. However, GnT-V expression was upregulated in GnT-V Tgmouse skin, and cultured keratinocytes derived from these miceshowed enhanced migration, which was associated with changesin E-cadherin localization, and epithelial-mesenchymal transition(EMT). Further, EMT–associated factors snail, twist, and,N-cadherin were upregulated, and cutaneous wound healing wasaccelerated in vivo. We further investigated the detailed mechan-isms of EMT by assessing epidermal growth factor (EGF) signal-ing and found upregulated EGF receptor signaling in GnT-V Tgmouse keratinocytes. These findings indicate that GnT-V overex-pression promotes EMT and keratinocyte migration in part throughenhanced EGF receptor signaling.

(49) A Systems Approach to Dissecting ImmunityAlan Aderem

Seattle Biomedical Research Institute, Seattle, WA

Macrophages and dendritic cells represent one of the cornerstonesof the innate immune system. They detect infectious organismsand then orchestrate an appropriate host response to them. In orderto precisely define the nature of the threat the immune cell needsto read the molecular bar code that is displayed on the specificpathogen. The molecular bar codes have been referred to asPAMPS and they are recognized by pattern recognition receptors(PRR). The Toll-like receptors (TLRs) and the Nod-like receptors(NLRs) are prototypic PRRs that detect extracellular PAMPS andintracellular PAMPS respectively. Since infectious agents carrymany different PAMPS the information must be integrated in orderto define the specific pathogen and enable the host to formulate anappropriate response. We use the tools of systems biology to ident-ify the molecular networks that lead to this decision-making.Biological results and technological developments will bediscussed.

(50) Downregulation of Fer Tyrosine Kinase SignalingIncreases a-dystroglycan-mediated Cell Adhesion Thereby

Suppresses Tumor FormationTohru Yoneyama, Kiyohiko Angata, Xingfeng Bao,

Sumit Chanda, Minoru FukudaSanford-Burnham Medical Research Institute, La Jolla, CA

Glycans of α-dystroglycan (α-DG), which is expressed at the epi-thelial cell-basement membrane (BM) interface, play an essentialrole in epithelium development and tissue organization.Laminin-binding glycans on α-DG expressed on cancer cells sup-press tumor progression by attenuating tumor cell migration signalfrom the BM (1,2). However, mechanisms controlling laminin-binding glycan expression are not known yet. Here, we usedsiRNA library screening and identified Fer kinase, a non-receptortype tyrosine kinase, as a key regulator of laminin-binding glycanexpression. Fer overexpression decreased laminin-binding glycanexpression, while siRNA-mediated knockdown of Fer kinaseincreased glycan expression on breast and prostate cancer cell


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lines. Loss of Fer kinase function via siRNA or mutagenesisincreased transcription levels of glycosyltransferases, includingPOMT1, β3GnT1, and LARGE, which are required to synthesizelaminin-binding glycans. Consistently, inhibition of Fer expressionincreases laminin-binding glycan thereby decreases cell migrationin the presence of laminin fragment. Fer kinase regulates STAT3phosphorylation and consequent activation, and knockdown ofSTAT3 increased laminin-binding glycan expression on cancercells. However, expression or knockdown of Fes, which is highlysimilar to Fer, does not affect the expression of the laminin-binding glycans. These results indicate that Fer tyrosine kinasespecifically downregulates laminin-binding glycans, therebyincreases cell migration. The results also suggest that Fer kinase isan excellent target for downregulation of tumor progression andtreatment of muscular dystrophy. Supported by NIH/NCI grantPO1 CA71932.

References1. Bao X, Kobayashi M, Hatakeyama S, Angata K, Gullberg D,Nakayama J, Fukuda MN, Fukuda M. Tumor suppressor functionof laminin-binding α-dystroglycan requires a distinctβ3-N-acetylglucosaminyltransferase. Proc Natl Acad Sci U S A;106:12109–12114, 2009.2. Shimojo H, Kobayashi M, Kamigaito T, Shimojo Y, Fukuda M,Nakayama J. Reduced glycosylation of α-dystroglycans on carci-noma cells contributes to formation of highly infiltrative histologi-cal patterns in prostate cancer. The Prostate; 71, 1151–1157, 2011.

(51) Chemical Methods for Biomarker DiscoveryGeert-Jan Boons

The University of Georgia, Athens, GA

Strain-Promoted Alkyne-Azide Cycloadditions (SPAAC) betweencyclooctynes and azides to give stable 1,2,3-triazoles have foundwide utility in labeling proteins, lipids and glycans of living cells,glycoprotein enrichment for proteomics, and tissue reengineering.These reactions have also made entry in materials sciences and hasfor example been employed for the assembly and surface modifi-cation of dendrimers, derivatization of polymeric nanostructures,and patterning of surfaces. Unlike copper-catalyzed azide-alkynecycloaddtions (CuAAC), SPAAC does not require a toxic metal, ishighly efficient even in a very complex milieu and proceed effi-ciently at ambient temperature. We have found that derivatives of4-dibenzocyclooctynol reacts fast with azido-containing sacchar-ides and amino acids and can be employed for visualizing metabo-lically labeled glycans of living cells. Attractive features of DIBOinclude easy access to the compounds by a simple syntheticapproach, nontoxicity and straightforward attachment of a varietyof probes. Furthermore, dibenzocyclooctynes can be generatedphotochemically by short irradiation by UV light of correspondingcyclopropenones thereby providing opportunities for the spatialand temporal controlled labeling of the target substrates. We havealso shown that by employing nitrones as 1,3-dipoles, the rate ofcycloaddition can be further enhanced and this technology made itpossible to selectively tag proteins at the Nterminus. The newchemical technology has been applied to the isolation and

identification of glycoconjugates unique to disease state.Furthermore, we have successfully been able to track cell surfaceglycoconjugates of healthy cells and cell from patients affected bystorage diseases.

(52) Glycobiology Successes and Opportunitiesin Biotechnology and Pharma

John LoweGenentech Inc, San Francisco, CA

NA

(53) Analytical Services and Training at the ComplexCarbohydrate Research Center

Roberto Sonon, Mayumi Ishihara, Krajang Talabnin, Zhirui Wang,Ian Black, Radnaa Naran, Christian Heiss, Parastoo AzadiComplex Carbohydrate Research Center, UGA, Athens, GA

The Analytical Services Laboratory of the Complex CarbohydratesResearch Center (CCRC) of University of Georgia, a non-profitorganization is involved in structural characterization of variety ofglycoconjugates derived from animal, plant or microbial origin.We routinely analyze glycoconjugate samples from universities,federal agencies, research institutions and pharmaceutical from theUS and other countries. Here we will show examples of projects inour laboratory that employs a combination of analytical techniquesfor structural elucidation of glycoproteins and polysaccharides.The service laboratory is well complemented with instrumentssuch as LTQ-Orbi-MS, MALDI-TOF, and high field NMRs inaddition to HPAEC and GC-MS.

For glycoprotein analysis, we provide: identify site(s) of attach-ment of N- and O-linked glycans, release and purification of N-and O-linked glycans, identify the type of N-linked glycans, e.g.biantennary, triantennary, tetraantennary, high mannose, hybrid,and complex, determine sequence, ring size, and anomericconfiguration, identify and determine the points of attachment ofnon-carbohydrate constituents such as phosphate and sulfate andcomposition and linkage analysis.

For polysaccharide analysis, we provide: purification to hom-ogeneity and determination of composition and linkage analysis,sequencing of oligosaccharide or polysaccharide repeating unitsincluding identity, linkage, ring size, and anomeric configurationof each glycosyl residue and identifying and analyzing the lipidmoiety, e.g. lipid A, ceramide, or diacylglycerol.

In addition we have developed methods for purification andsequencing of glycosaminoglycans through various chromato-graphy, MS and NMR techniques.

We also offer annual hands-on training courses for structuralcharacterization of both glycoproteins and polysaccharides

(54) Rapid Glycoprotein Sialic Acid Determination by HighPerformance Anion Exchange Chromatography with Pulsed

Amperometric DetectionDeanna Hurum, Jeff Rohrer

Thermo Fisher Scientific, Sunnyvale, CA

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carbohydrates control glycoprotein bioavailability, function, stab-ility, and metabolism. While over 50 natural sialic acids have beenidentified, two forms are commonly determined in glycoproteins:N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid(Neu5Gc). This determination is performed in two steps; acidhydrolysis releases the sialic acids from the glycoprotein and theyare then quantified by a chromatographic method. There are twoclasses of common chromatographic sialic acid determinationmethods; direct detection methods, such as High PerformanceAnion Exchange Chromatography with Pulsed AmperometricDetection (HPAE-PAD), and those that require analyte derivatiza-tion for detection, such as fluorescent labeling followed byUHPLC.This work describes the development of a 5 min HPAE-PAD

assay for glycoprotein sialic acids after prior acetic acid hydrolysis,which is significantly faster than existing HPAE-PAD sialic acidassays.Calibration ranges were chosen spanning the expected Neu5Ac

and Neu5Gc amounts in five representative sialylated glyco-proteins with varying degrees and types of glycosylation.Response was linear in the investigated ranges, with coefficients ofdetermination ≥0.9995. The method LODs of 0.11 pmol and0.058 pmol for Neu5Ac and Neu5Gc, respectively, allowed sialicacids detection in diluted acid hydrolyzates. Eliminating thesample lyophilization step saved time. Reproducibility was goodwith retention time RSDs of <0.8 and peak area RSDs of <1.98for standards. Recoveries from the five glycoprotein hydrolyzatesamples ranged from 81-96% for Neu5Ac and 82-106% forNeu5Gc, and the determined amounts agreed with publishedvalues, both suggesting method accuracy. This 5 min assay allowsthe rapid, direct, and accurate quantification of glycoprotein sialicacids, providing a convenient method for quick screening ofexpression optimization experiments without the costly and timeconsuming derivatization steps required for UHPLC fluorescencedetection methods.

(55) N-linked Glycosylation on Non-Consensus Protein MotifsAlain Balland1, John Valliere-Douglass1, Paul Kodama1,

Mirna Mujacic2, Catherine Eakin1, Lowell Brady1,Wei-Chun Wang1, Alison Wallace1, Michael Treuheit1,

Pranhitha Reddy21Analytical Sciences, Amgen, Seattle, WA; 2Cell Sciences

and Technology, Amgen, Seattle, WA

We report that N-linked glycosylation can be present on asparagineresidues not adhering to the consensus motif NX(S/T) described inthe literature. Mass spectrometry analysis of recombinant IgG2molecules’ acidic variants separated by cation exchange chromato-graphy showed the presence of other N-linked glycan structures,additional to the well documented glycans found on the canonicalsite in the CH2 domain. This modification was located on aspara-gine 162 of the CH1 domain, in the sequence TVSWN162SGAL,by detailed peptide mapping associated with 18O isotope incorpor-ation during N-glycosidic digestion and MS analysis. We deter-mined that this unexpected modification was not related torecombinant expression and was also present on natural IgG

molecules isolated from human serum. The impact of the aminoacid sequence around this modification was probed by site-directedmutagenesis. Expanding on this original observation, we devel-oped a strategy based on lectin enrichment of glycosylated IgGs,followed by endo F2 treatment and peptide mapping withCID-MS2 / ETD- or CID-MS3 analysis to locate several otherexamples of non-consensus N-linked glycosylation sites. Theresults pointed to a common theme around the modified residue,supporting the concept of a novel protein motif forN-glycosylation. These findings indicate that protein glycosylationis governed by more diversified requirements than previouslyappreciated.

(56) Neutron Structure of Retaining Glycosyltransferase GTABrock Schuman1, Suzanne Fisher2, Svetlana Borisova1,

Leighton Coates3, Paul Langan2,3, Stephen Evans11University of Victoria, Victoria, Canada; 2Los Alamos NationalLaboratory, Los Alamos, NM; 3Oak Ridge National Laboratory,

Oak Ridge, TN

With at most one electron, hydrogen atoms can be difficult toobserve in protein structures by X-ray crystallography even atatomic resolution. This can pose a barrier to the critical evaluationof enzymatic mechanisms that involve proton translocation as theionization states of active site residues cannot always be reliablydetermined by the chemical environment in which they reside.

One such class of enzyme is the glycosyltransferase.Glycosyltransferases catalyze the biosynthesis of oligosaccharidesand glycoconjugates by the transfer of a monosaccharide unit froman activated donor molecule to an acceptor molecule with highstereospecificity. The anomeric stereochemistry of the donor sugarwill either be inverted or retained upon formation of the new gly-cosidic linkage, depending on the enzyme. Although a consensushas been reached on an SN2 mechanism used by inverting glyco-syltransferases, the nature of the retaining mechanism is still amatter of debate.

The model retaining enzyme human ABO(H) blood group Aα-1,3-N-acetylgalactosaminyltransferase (GTA) generates theblood group A antigen by the transfer of N-acetyl-galactosaminefrom UDP-GalNAc to the blood group H antigen. To understandbetter how specific active-site-residue protons and hydrogen-bonding patterns affect substrate recognition and catalysis,neutron diffraction studies were conducted at the ProteinCrystallography Station (PCS) at Los Alamos Neutron ScienceCenter (LANSCE).

This is the first study of a retaining glycosyltransferase usingcombined X-ray and neutron diffraction data. These data providethe first unambiguous assignment of protons and the causativehydrogen-bond patterns in a glycosyltransferase active site.

(57) Glycan Analysis by Solid-Phase Capture and Releaseusing Reversible Reaction to Hydrazide

Shuang (Jake) Yang, Hui ZhangJohns Hopkins University, Baltimore, MD

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biological processes, such as signaling, activation, adhesion,growth and differentiation, surface expression etc. To investigatethe roles of glycosylation to these protein functions, glyco-proteins and glycans have been intensively studied. However,the analysis of glycoproteins and their glycans is underminedby the continuous loss of glycopeptides and glycans in multiplesample preparation processes, resulting in low yield of glycanisolation. In this study, we developed a unique direct approachfor glycan analysis using hydrazone on solid-phase substratesthat were able to capture and concentrate glycans on hydrazidesurface without pre-purification and desalting after glycans werereleased from glycoproteins. The captured glycans on hydrazideimmobilized surface are reversibly released via hydrolysis ofhydrazone bond without interrupting glycan structures. Wedemonstrated the capture-release of N-linked glycans in bothAgarose and magnetic beads. This novel technique was vali-dated on glycan capture and release for standard glycoproteins,and further applied to serum glycan analysis forclinical studies.

(58) Databases for N-Glycosylated Proteins Identified by SolidPhase Extraction of Glycosylated Peptides (SPEG) Coupled

with LC/MSDeniz Baycin Hizal, Yuan Tian, Vishaldeep Sarkaria,

Michael Betenbaugh, Hui ZhangJohns Hopkins University, Baltimore, MD

The alterations in the glycosylation pathway or protein glycosy-lation have been associated with many diseases such as cancer,hereditary, diabetes, cardiovascular, inflammatory, neurologicaland neuromuscular diseases. Glycosylated proteins have beenextensively studied for the improvement of diagnosis andtherapy of these diseases. Glycoproteomics is an emerging tech-nique which provides the qualitative and quantitative analysisof large number of glycoproteins. In this study solid phaseextraction of N-glycosylated peptides (SPEG) method iscoupled with LC/MS in order to identify N-glycosylated pro-teins and the glycosylation sites from human, mouse, droso-phila and zebrafish. In addition to these, the glycoproteins ofChinese Hamster Ovary (CHO) cells are analyzed to improveglycoprotein production in CHO cells. In order to make thisdata publicly available, we established four different databases,UniPep, GlycoFly, GlycoFish, GlycoCHO. We believe that eachof these databases is a significant advancement for the glyco-biology and different research communities. It provides exper-imental evidences of these glycoproteins. UniPep (www.unipep.org) gives access to the N-glycosylated proteins and glycositesof human which have significant importance in biomarker dis-covery for cancer and other human diseases. GlycoFly (http://betenbaugh.jhu.edu/GlycoFly/) and GlycoFish (http://betenbaugh.jhu.edu/GlycoFish/) are for N-glycosylated proteins ofDrosophila melanogaster and zebrafish. These two organismsare widely used genetic models to understand the biologicaland disease processes since their genes can be easily mutated.CHO cell lines are extensively used for the therapeutics

production in pharmaceutical companies. The glycoproteome ofthese cell lines is available in GlycoCHO website. These fourwebsites include around 9000 glycoproteins that are significantfor cell adhesion, development, protein transport and manyother cell functions. Identification of N-glycosylated sites andproteins will provide insights to address the roles of these gly-coproteins and to identify glycoproteins that may be involvedin cancer, cardiovascular, inflammatory, neurological, and devel-opmental diseases.

(59) Monosaccharide Notation in Glycoinformatics: Problems,Pitfalls, and Solutions

Thomas LüttekeJustus-Liebig-University Giessen, Giessen, Germany

The complexity of carbohydrates is not only caused by theirability to form branched structures but also by the large amountof different building blocks, the monosaccharides, the number ofwhich by far exceeds the number of different amino acids foundin proteins or of different nucleotides in DNA or RNA.Monosaccharides are often derived by modification of severalbasic residues, e.g. by deoxygenation, methylation, acetylation,etc. This introduces a further level of complexity, and variousrules have to be followed to create correct names according toIUPAC standards. If not all notation rules are applied correctly,or trivial names are used inconsequently, multiple names existfor the same residue. This does not only hamper data exchangebetween individual carbohydrate databases but can also lead tomultiple entries of one specific glycan chain within one data-base, especially in resources that allow free-text notation ofmonosaccharides. Users who query such databases theoreticallyhave to use all feasible residue names to ensure getting all avail-able information of the glycan they are looking for. In practicethis means that users might miss some information that ispresent in the database if they use not all or “the wrong” namesin their query.

These problems can be solved by using a controlled residuedictionary. Such a dictionary is provided by MonosaccharideDB(www.monosaccharidedb.org). The interface includes routinesthat can read monosaccharide names in several notation schemesand translate them into properties, from which unique notationcan be created – even if the requested residue or the name thatis used to encode it in a specific query is not yet present in thedatabase. This way the use of MonosaccharideDB is not limitedto the actual content of the database but is automaticallyextended to other monosaccharides.

(60) GlycoPattern: A Platform for Motif Discovery and GlycanArray Exploration

Sanjay Agravat, Sharath Cholleti, Tim Morris, Joel Saltz,Xuezheng Song, Richard Cummings, David Smith

Emory University, Atlanta, GA

Microarrays of defined glycan structures represent a highthroughput approach to determine the specificities of glycan-binding proteins (GBPs) including plant and animal lectins,


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antibodies, and viruses. Binding data from tens of GBPs inter-acting with hundreds of glycans generate large amounts of datathat are difficult to manually analyze in terms of identifying aglycan structural motif or determinants required for high affinitybinding by a GBP. It is also challenging to identity what struc-tural characteristics of glycans inhibit binding affinity to a GBPwhen a motif is present on the structure. We have developed aweb-application platform called GlycoPattern that includes algor-ithms and tools to automatically rank candidate ligands, discovermotifs, visualize the glycan array data, compare the binding pat-terns of multiple GBPs over the same glycans and motifs, anda search feature that allows cartoon or linear-code search forglycans on the Consortium for Functional Glycomics (CFG)Array.

(61) Improving Standard N-Glycan Sample Preparation withManual Automation using Microchromatography to Improve

Efficiency, Accuracy, and ReproducibilityToni Hofhine1, Craig Nishida2

1Gilson, Inc., Middleton, WI; 2ProZyme, Inc, Hayward, CA

The search for more efficient tools that streamline the sample prep-aration process for characterization of N-Glycans continues to beat the forefront in the field of glycomics. The goal is to moreeffectively streamline the sample preparation and data analysis toallow for a deeper understanding of the multiple structures andfunctions that result from glycosylation. Adding sugars to proteinsin the process of forming glycoproteins can be complex.Abnormal glycosylation, congenital disorders of glycosylation(CDG), can occur and be linked to a number of diseases. Betterunderstanding of this structure and function relationship can assistwith understanding how these diseases occur and thus developingdisease biomarkers. Monitoring bioprocessing of monoclonalantibodies (Mabs) is another area where product glycosylationis important. Eliminating manufacturing variability frombatch-to-batch is critical, but the traditional laboratory samplepreparation approach can often take too long. Traditionally, samplepreparation for N-Glycans takes more than three days and ofteninvolves specific laboratory technique, not including the time forsample analysis. The transition to manual automation tools com-bined with microchromatography reduces sample preparation toless than 2.5 hours, while providing the added confidence thatN-Glycan sample preparation and the sample data are reliable.Human Immunoglobulin G (hIgG) was mixed with bovine fetuinprior to microchromatography purification and subequent samplepreparation, labeleing and analysis using HPLC with fluorescencedetection. Results indicate that manual automation tools showimproved accuracy and reproducibility between samples whileallowing for more effective and more efficient characterization ofN-Glycans to be performed.

(62) “Efficient Synthesis of Aminopropyl-Blood Group ATriaose in Engineered Escherichia coli”

Randriantsoa Mialy1, Drouillard Sophie2, Fort Sebastien2,Chaud Patricia2, Samain Eric2, Havet Stephane1

1Elicityl, Crolles, France; 2CERMAV-CNRS, Grenoble, France

We have previously reported the large scale synthesis of humanABO(H) blood group antigens by growing metabolically engin-eered Escherichia coli strains (Randriantsoa. M, PhD thesis2009). In the present study, we have extended the method tothe biosynthesis of blood group A antigen triaose(GalNAcalpha-3(Fucalpha-2)Gal) bearing an aminopropyl groupat its reducing end (figure 1). The system is based on high celldensity culture of a recombinant strain overexpressing twoheterologous glycosyltransferase genes: the Helicobacter pylorifutC gene which encodes an alpha-2 fucosyltransferase and thehuman gtA gene which encodes an alpha-3N-acetylgalactosaminyltransferase. The culture was carried out inthe presence of aminopropyl-galactoside which was activelyinternalized into the cytoplasm and converted into the targetcompound. This chemically activated oligosaccharide can beused as standard building block to design neoglycoconjugatesand construct biotechnological tools such as affinity columnsand glycan microarrays.

(63) New Features and Improvements in Carbohydrate 3DStructure Validation

Denis Mokros1, Robbie P. Joosten2, Andreas Dominik1,Gert Vriend3, Thomas Lütteke4

1University of Applied Sciences Giessen-Friedberg, Giessen,Germany; 2Department of Biochemistry, NKI, Amsterdam, TheNetherlands; 3CMBI, NCMLS, Radboud-University Nijmegen,Nijmegen, The Netherlands; 4Justus-Liebig-University Giessen,

Giessen, Germany

More than 5000 entries in the Protein Data Bank (PDB, www.pdb.org, the largest collection of biomolecular 3D-structures) containcarbohydrates. This makes the PDB a valuable resource not onlyfor proteomics but also for glycoscience. Unfortunately, thequality of the carbohydrate moieties is significantly lower than thatof the protein parts of glycoproteins or protein-carbohydratecomplexes in the PDB; many entries contain errors. There areseveral reasons for this, one of them being the lack of validationsoftware. Only recently crystallographers became aware of thisproblem and started to use tools such as PDB CarbohydrateResidue check (pdb-care) to examine the carbohydrate parts. Herewe present an updated version of pdb-care, which in addition tothe residue notation checks that were already performed bythe former version, also detects other problems such as invalidresidues within the N-glycan core structure (e.g. a-D-GlcpNAcinstead of b-D-GlcpNAc), missing LINK records, which oftenresult in “1-deoxy” sugars, or superfluous atoms within glycosidiclinkages.

The results are presented via a new, clearly arranged webinterface for human readability, or as a computer-readable xmlfile to aid automatic validation routines. Suggestions how tocorrect errors are also included in many instances. These areused e.g. within the PDB_REDO project (www.cmbi.ru.nl/pdb_redo/) to enable an automatic correction of some of theproblems.

The interface to pdb-care is available at www.glycosciences.de/tools/pdb-care/.


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(64) High-Resolution Crystal Structure ofN-Acetylmannosamine Kinase: Insights about Substrate

Specificity, Activity and Inhibitor ModellingLong Duc Nguyen1, Jacobo Martinez2, Stephan Hinderlich3,

Hans-Ulrich Reissig2, Werner Reutter1, Hua Fan1,Wolfram Saenger2, Sebastien Moniot4

1Charité - Universitaetsmedizin Berlin, Berlin, Germany; 2FreieUniversitaet Berlin, Berlin, Germany; 3Beuth Hochschule fürTechnik, Berlin, Germany; 4Universitaet Bayreuth, Bayreuth,

Germany

Sialic acids are essential components of membrane glycoconju-gates. They are responsible for the interaction, structure and func-tionality of all deuterostome cells and have major functions incellular processes and diseases. The key enzyme of the biosyn-thesis of sialic acid is UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE). This bifunctional enzymecatalyzes the transformation of UDP-N-acetylglucosamine toN-acetylmannosamine-6-phosphate and has a direct impact on thesialylation of the cell surface. We have solved the crystal structuresof the N-acetylmannosamine kinase (MNK) domain in complexeswith N-acetylmannosamine (ManNAc), MNK/ManNAc (1.64 Å),MNK/ManNAc/ADP (1.82 Å) and MNK/ManNAc-6-Phosphate/ADP (2.10 Å). These new insights on the active center of MNKoffer the necessary structural basis to design agonists and antagon-ists, opening novel ways for sialic acid research, glycan bioengi-neering and cancer therapy.

(65) Glycan Map®Analysis for High-throughput GlycomicProfiling of Clinical and Other Specimens

Hidehisa Asada, Taku Nakahara, Yoshiaki Miura,Thomas Stevenson, Toshiaki YamazakiEzose Sciences Inc., Pine Brook, NJ

While glycosylation is known to play a critical role in any biologi-cal and cellular processes and has been clearly linked to disease,large scale glycomics studies have traditionally been limited bythe lack of practical high-throughput analysis methods. To facili-tate practical functional glycomics and open a pathway to explor-ing the human glycome, Ezose Sciences Inc. has developed aproprietary new high-throughput solution, the GlycanMap® plat-form. GlycanMap® analysis is compatible with a variety ofcomplex biological samples and can deliver both qualitative andquantitative data on N- and O-linked glycans. The GlycanMap®

method relies on robust enrichment of carbohydrates on a solidphase (glycoblotting), followed by quantitative MALDI-TOF massspectrometry and custom bioinformatics. The key assay steps areintegrated into an automated, 96-well robotic assay system and aglycan internal standard and proprietary MALDI matrix compo-sition is used to enable quantitation. The GlycanMap® platformhas been applied to biomarker research using a variety of sampletypes. The GlycanMap® methodology provided good reproducibil-ity, linearity, and sensitivity along with the throughput required foranalysis of the large numbers of clinical samples required for dis-covery of robust biomarkers. N-linked and O-linked glycans were

captured and quantitated from crude biological samples, includingserum/plasma, cerebral spinal fluid, and cell extracts. Treatment-dependent changes in individual glycans were detected, andoverall glycan changes were also projected against the known bio-synthetic pathways to add biological and mechanistic relevance tothe observed glycan changes. Finally, a method for identifyingparent proteins containing glycans of interest was developed usingreverse-glycoblotting and multiple reaction monitoring (MRM)techniques.

(66) Role of Quorum Sensing ΔaviR gene inLipooligosaccharide Structure Modulation in Agrobacterium

vitis F2/5.Cristina De Castro1, Thomas Burr2, Rosa Lanzetta1,

Antonio Molinaro1, Michelangelo Parrilli1, Sandor Sule31University of Napoli, Napoli, Italy; 2Department of Plant

Pathology, Cornell University, Geneva, NY; 3Hungarian Academyof Sciences, Budapest, Hungary

A fundamental role in the plant-bacterium interaction forGram-negative phytopathogenic bacteria is played by membraneconstituents, such as proteins, lipopoly- or lipooligosaccharides(LPS, LOS) and Capsule Polysaccharides (CPS).

In the frame of the understanding the molecular basis of plant-bacterium interaction, the Gram-negative bacterium Agrobacteriumvitis was selected in this study. It is a phytopathogenic member ofthe Rhizobiaceae family and it induces the crown gall diseaseselectively on grapevines (Vitis vinifera).

A. vitis wild type strain F2/5, and its mutant in the quorumsensing gene ΔaviR, were studied. The wild type produces biosur-factants; it is considered a model to study surface motility, and itcauses necrosis on grapevine roots and HR (HypersensitiveResponse) on tobacco. Conversely, the mutant does not show anysurface motility and does not produce any surfactant material;additionally, it induces neither necrosis on grape, nor HR ontobacco. Therefore, the two strains were analyzed to shed somelight on the QS regulation of LOS structure and the consequentvariation, if any, on HR response.

LOS from both strains were isolated and characterized: the twoLOS structures maintained several common features and differedfor few others.

With regards to the common patterns, firstly: the Lipid A regionwas not phosphorylated at C4 of the non reducing glucosaminebut glycosylated by an uronic acid (GalA) unit, secondly: a thirdKdo and the rare Dha (3-deoxy-lyxo-2-heptulosaric acid) moietywas present.

Importantly, the third Kdo and the Dha residues were substitutedby rhamnose in a not stoichiometric fashion, giving four differentoligosaccharide species.

The proportions among these four species, is the key differencebetween the LOSs from both the two bacteria.

LOS from both strains and Lipid A from wild type A. vitis arenow examined for their HR potential in tobacco leaves and grape-vine roots.


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(67) Examining the Role of the ppGalNAc-T Lectin Domainin Modulating ppGalNAc-T Glycopeptide SpecificityThomas A. Gerken1, Leslie Revpredo2, Joseph Thome1

1Case Western Res Univ Sch of Medicine, Cleveland, OH; 2Deptof Chemistry, Case Western Reserve Univ., Cleveland, OH

ppGalNAc-Ts transfer GalNAc from UDP-GalNAc to Ser and Thrresidues on polypeptide substrates thereby initiating mucin typeO-glycosylation. Members of this family (�20) show varying pre-ferences for peptide characteristics including neighboring glycosy-lation, charge and composition. These transferases play a role indevelopment, tumorigenesis, and possibly signaling. Structurally,ppGalNAc-Ts are composed of an N-terminal catalytic domainconnected by a short linker to a C-terminal ricin-like lectindomain. The roles of the catalytic and lectin domains in peptideand glycopeptide recognition and specificity remain unclear. Tofully understand the ppGalNAc-T’s function and biological impor-tance their substrate specificities need be systematically character-ized. We have reported a series of oriented random peptide andglycopeptide substrates for quantifying transferase specific cataly-tic domain preferences. With these substrates, unique preferencedata for all amino acid residues except Thr, Trp and Cys have beenobtained for mammalian ppGalNAc-T1, T2, T3, T5, T10 & T12(see J. Biol. Chem. 286, 14493(2011)). We now extend our studiesto the function of the lectin domain, utilizing a series of randomglycopeptides containing N- or C- terminal placed Thr-O-GalNAcresidues. These substrates are designed to determine the effects ofGalNAc placement on transferase specificity. Preliminary resultssuggest that the presence and N- or C- terminal location of Thr/Ser-O-GalNAc site on these glycopeptides are important determi-nants of the overall catalytic activity of these enzymes and differbetween transferase isoforms. This work represents the firstattempt to systematically characterize the role of the lectin domainin ppGalNAc-T substrate specificity. Supported by NIH-NCI:R01-CA78834.

(68) The Initiation of Mucin Type O-glycosylation:Development of a Web-Based Tool for the Prediction of

ppGalNAc T Isoform Specific O-GlycosylationThomas A. Gerken1, Gerardo Cardenas2, Igor Almeida2,

Ming-Ying Leung21Case Western Res Univ Sch of Medicine, Cleveland, OH;

2University of Texas at El Paso, BBRC, El Paso, TX

Mammalian mucin type O-glycosylation is initiated by a largefamily (�20) of UDP-GalNAc: polypeptide α-N-acetylgalactosami-nyltransferases (ppGalNAc-T’s) that transfer GalNAc fromUDP-GalNAc to Ser and Thr residues of polypeptide acceptors.Characterizing each isoform’s peptide substrate specificity is criticalto understanding their individual properties, biological roles andsignificance. This is particularly important, as several ppGalNAc-Tsplay critical roles in proper development and disease. Recent workalso suggests mucin type O-glycosylation may be sufficiently regu-lated to modulate complex biological processes and perhaps signal-ing. We have recently reported a series of oriented random peptideand glycopeptide substrate libraries for quantitatively determining

the amino acid residue preferences (enhancement values) of thehuman ppGalNAc-T1, T2, T3, T5,T10 & T12 (J. Biol. Chem.281:32403(2006), Glycobiology 18:861(2008), J. Biol. Chem.284:20387(2009) & J. Biol. Chem. 286:14493(2011)). We havefurther shown that the products of the enhancement values flank-ing a site of glycosylation can roughly rank and predict the exper-imentally observed isoform specific glycosylation patterns. Withthe goal of providing wide access, a web-based tool has beendeveloped that utilizes these enhancement values for the predic-tion of ppGalNAc T isoform specific O-glycosylation. Thewebsite (to be announced) allows user input of specific sequences,the transferase isoform(s) to be included and the window of flank-ing residues to be considered. Parameters for all amino acid resi-dues (except Cys, Thr and Trp) including O-GalNAc glycosylatedSer or Thr are available for most of the 6 isoforms currentlycharacterized. This represents the first O-glycan isoform specificprediction tool and should be widely applicable to the study ofmucin type O-glycosylation.

Acknowledgments: NIH grants NCI-R01 CA78834 (to T.A.G.),R01 AI070655 (to I.C.A.) and G12 RR008124 (to the BorderBiomedical Research Center (BBRC), Bioinformatics Program,University of Texas at El Paso).

(69) Double Mutants as Tools for Elucidating N-glycosylationPathways in Caenorhabditis elegans

Shi Yan1, Katharina Paschinger1, Silvia Bleuler-Martinez2,Verena Jantsch3, Iain Wilson1

1Department für Chemie Universität für Bodenkultur, Vienna,Austria; 2Institute of Microbiology, ETH-Zürich, Switzerland,

Zürich, Switzerland; 3MFPL, Dept. of Chromosome Biology, Uni.of Vienna, Vienna, Austria

Recent years, C. elegans began to be more popular in the study ofglycomics. Interestingly, its glycome has been shown to have morecomplicated patterns than those of some higher organisms, e.g.Drosophila. The basis for this must be the presence of multipleglycosylation pathways involved in forming highly variable glycanstructures. Here we report two novel double mutants as tools forelucidating N-glycosylation pathways in vivo: the H2H3 doublemutant, lacking β-N-acetylhexosaminidase-2 (hex-2) and 3 (hex-3),and the F1F6 double mutant, lacking α-1, 3 fucosyltransferase-1(fut-1) and 6 (fut-6). N-glycans of the double mutants werereleased by PNGase F, labeled with 2-aminopyridine and sub-sequently fractionated by HPLC, then analyzed by MALDI-TOFMS/MS. Preliminary data showed that the double mutants possessnovel features. Both of the double mutants have a rather conspicu-ous difference in terms of reversed phase HPLC profiles, in com-parison to their own parent mutants and the N2 wild type. A lossof anti-HRP epitope (core α-1,3-fucose) in an extract of the H2H3double mutant, but not in the single hex-2 and hex-3 mutantextracts provides new evidence in biosynthesis of truncatedN-glycans: HEX-2 and HEX-3 are main contributors in trimmingthe MGn to MM, together with Golgi mannosidase II, in the con-version of Man5Gn to MM. The abundance of a Gal-Fuc modifi-cation on the inner GlcNAc of N-glycan core increased the


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sensitivity to a toxic galectin CGL2. In comparison to the N2 wildtype and fut-1 or fut-6 single mutant, the N-glycosylation patternof the F1F6 double mutant has a general reduction in N-glycanfucosylation as judged by MALDI-TOF MS. Clues may berevealed when the glycan structures in each double mutant arecarefully studied. It has to be noted that unexpected N-glycanstructures were also observed in the double mutants, whichincrease the difficulties of any analyses.

(70) Thio-galactoside Glycolipid Analogs; EnzymaticSyntheses, Their Galactosidase Resistance and Application

for Metabolism Studies in Single CellsYayoi Yoshimura1, Dietlind Adlercreutz1, Karin Mannerstedt1,Warren W. Wakarchuk2, Norman J. Dovichi3, Ole Hindsgaul1,

Monica M. Palcic11Carlsberg Laboratory, Copenhagen V, Denmark; 2NationalResearch Council of Canada, Ottawa, Canada; 3University of

Notre Dame, Notre Dame, IN

Glycosphingolipids (GLSs) are ubiquitous components of cellsurface membranes and are especially abundant in cells of thenervous system. Through repetitive cycles of synthesis and degra-dation, the GSL composition is characteristic for a given cell type.Analysis of GSL metabolism is important for understanding theirroles in biological processes.We have reported a method for studying GSL metabolism in

cell extracts and in single cells utilizing fluorescently labeledGSLs that enter metabolic pathways together with endogenousGSLs. The metabolism of these fluorescently labeled GSLs can bemonitored by capillary electrophoresis with laser-induced fluor-escence detection. However, most of the metabolites observed inthese studies were catabolic products [1, 2].To investigate GSL anabolism, we are exploring the use of 5-thio-

galactosides, in which the ring oxygen is replaced by sulfur. Previousstudies with bacterial and plant glycosidases have shown that 5--thio-pyranosides are resistant to hydrolysis or are hydrolyzed at avery low rate. This hydrolytic stability is attractive for GSL metabolicanalyses. Here, we present the enzymatic syntheses of fluorescentGSL analogues containing terminal 5-thio-galactoside. Furthermore,the hydrolytic stability of these GSL compounds was tested in vitroand in vivo. The results suggest that 5-thio-galactosides are resistantto hydrolysis by mammalian galactosidases and are thus promisingtools for the study of anabolic pathways in single cells.

References[1] Whitmore CD, Hindsgaul O, Palcic MM, Schnaar RL, DovichiNJ. 2007. Anal. Chem. 79:5139–5142.[2] Whitmore CD, Olsson U, Larsson EA, Hindsgaul O, PalcicMM, Dovichi NJ. 2007. Electrophoresis. 28:3100–3104.

(71) Microscale Analytical Platforms for ScreeningCarbohydrate-Active Enzymes

Aarthi Chandrasekaran1,2, Rajiv Bharadwaj1,2, Kai Deng1,2,Paul Adams1,3, Anup Singh1,2

1Joint BioEnergy Institute, Emeryville, CA; 2Sandia NationalLaboratories, Livermore, CA; 3Lawrence Berkeley National

Laboratories, Berkeley, CA

Glycosyltransferases (GTs) and glycosylhydrolases (GHs) areclasses of carbohydrate-active enzymes that are responsible forbiosynthesis, breakdown and modification of complex carbo-hydrates or glycans. By regulating the expression of cellularglycans, these enzymes play a critical role in a number of cellularand pathological processes including cell growth and adhesion,cell signaling, inflammation, cancer and microbial pathogenesis. Inaddition, GT activity regulates cell-wall composition in plantswhile GHs are critical for enzymatic deconstruction of the majorplant cell-wall polysaccharides (cellulose and hemicellulose) intofermentable sugars. Characterizing the activities of carbohydrate-active enzymes is therefore critical for studying disease mechan-isms, for novel biomarker and drug discovery, and for cost-effective production of liquid biofuels from lignocellulosicbiomass. Despite their widespread role in diverse applications, fewhigh-throughput approaches exist for activity screening of theseenzymes. In order to address this need, we have developed analyti-cal platforms for rapid enzyme activity screening in microscalevolumes. In this work, we first describe a sensitive, fluorescence-based array platform for expression and screening of thermophilicGHs in volumes down to 2 µl. Further, we discuss a microfluidiccapillary electrophoresis chip-based platform for quantitativeanalysis of carbohydrate products formed as a result of enzymeactivity. Finally, we describe a multiplexed flow cytometry-basedassay using glycan-microspheres for screening GTs and GHs. Ourmicroscale platforms (i) support high-throughput multiplexedenzyme screening assays (ii) greatly reduce reagent usage therebylowering costs and (iii) enable rapid and sensitive enzyme activitycharacterization. Our platforms are ideally suited for screening oflarge libraries of carbohydrate-active enzymes for drug discoveryand bioenergy applications, identification of enzyme inhibitors,discovery of biomarkers and development of diagnostics.

(72) Cyberinfrastructure for Glycome Research: neoGRIDfor Motif Analysis

Arun DattaNational University, La Jolla, CA

Advances in Cyberinfrastructure, particularly in grid computing,are changing the way scientific research is conducted in all aspectsof science and have led to the generation of seemingly limitless pos-sibilities of national and international collaboration and sharing ofdata for research, education and training. The National ScienceFoundation’s recent XSEDE Project will enable scientists andengineers to utilize advanced computing, digital services, andexpertise through collaborations that could not be achieved before.With a partnership of 17 institutions across the nation, this ExtremeScience and Engineering Discovery Environment (XSEDE), will bethe most advanced, powerful, and robust collection of integratedadvanced digital resources and services in the world. As aChampion of this program, this author invites the Glyco-Community to utilize these resources for advancing their collabora-tive research. In collaboration with XSEDE partnering institutions,this author is developing neoGrid to facilitate this process. It isdeveloped in a virtual hosting environment ‘Quarry’ for workingwith Taverna-based workflow utilizing high-performance


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computing. It is designed to offer a XSEDE-supported collaborativeenvironment for the researchers from multidisciplinary scientificfields to gather data, integrate and analyze using XSEDE resources.In addition, workflows available at KEGG and myGRID will alsobe available in this neoGRID that can be utilized for motif analysisin the glycosyltransferase enzyme family, and drug discoveryresearch using members of this family as target protein(s).Demonstration will be provided how this environment is used forhigh-throughput sialylmotifs analysis across the three kingdoms.

(73) Synthesis of α-D-Glc1,2α-D-Glc1,3α-D-Glc-OCH3

and Overexpression of Processing Alpha Glucosidase IVenkatrao Konasani1, Akihiro Imamura2, Todd Lowry3,

Christine Scaman11University of British Columbia, Vancouver, Canada; 2GifuUniversity, Gifu, Japan; 3University of Alberta, Edmonton,

Canada

Processing alpha-glucosidase-I (Glu-I) is an endoplasmic reticuluminner membrane-bound glycoprotein that initiates N-linked glycanprocessing through hydrolysis of the α(1,2) linked glucose mol-ecule of Glc3Man9GlcNAc2. To further investigate the structureand catalytic mechanism of this key regulatory enzyme, we havesynthesized a new trisaccharide α-D-Glc1,2-α-D-Glc1,3α-D-Glc-OCH3, and cloned and overexpressed the soluble form of theenzyme. The trisaccharide substrate was chemically synthesizedfrom three protected monosaccharide building blocks through theuse of sequential glycosylation reactions. Following the assemblyof the protected substrate, the molecule was deprotected in a singlestep affording the target glycan. CWH41 gene region that encodesfor amino acids 35-833 was cloned into expression vectors pET30aand pPICZalphaB, and over-expressed in Escherichia coli andPichia pastoris as N- or C-terminal 6XHis tagged proteins, respect-ively. Enzyme was purified to homogeneity using Ni-NTA column.The optimal expression of soluble Glu-I was induced in E. coli bythe addition of 1mM isopropyl-beta-D-thiogalactopyranoside at 25°C. Preliminary expression of Glu-I from P. pastoris using 0.5%methanol for 96 hours, yielded 1.6 mg enzyme/l medium with aspecific activity of 1.5 µmoles/min.mg protein. Optimization ofexpression, and kinetic, mechanistic, and structural studies of Glu-Iare currently underway.

(74) Complement L-ficolin Binds to Surface Glycans of HCVand Reduces the Viral Infectivity, and Functions

as an Antiviral OpsoninYinnan Zhao1,2, Yi-Dan Zhou3, Kun Yang4, Xiao-Lian Zhang1,21Wuhan University School of Medicine, Immunology, Wuhan,China; 2State Key Laboratory of Virology, Wuhan, China;

3Jianghan University, College of Life Science, Wuhan, China;4Huazhong Science and Technology Univ Tongji M, Wuhan, China

L-ficolin is a recently identified complement lectin, which recog-nizes bacterial carbohydrates, thereby activate lectin complementpathway. However little is known about the role of L-ficolin inviral infections. This report shows that human L-ficolin specifi-cally binds to surface glycans of HCV, and subsequently activatesthe lectin complement pathway and complement mediated

cytolytic activity. Moreover, we found that L-ficolin could signifi-cantly block the entry and infection of HCV to Huh7.5.1 cellsin vitro in a dose dependent manner, and functioned as an antiviralopsonin in vitro by enhancing phagocytosis of macrophages.These new findings will contribute to the development of L-ficolinas a novel immunotherapy agent against the infection of thisimportant human virus.

(75) Studies on Site-Specific N-Glycosylation of Hemagglutininfrom Influenza AViruse Phil82 and Surfactant Protein D

Nancy Leymarie, Kevan Hartshorn, Mitchell White,Tanya Cafarella, Barbara Seaton, Michael Rynkiewicz,

Joseph ZaiaBoston University School of Medicine, Boston, MA

The influenza A virus accounts for 36,000 deaths, 200,000hospitalizations per year resulting in $10 billion in health carecosts in the US. One aspect of influenza infection is the inhi-bition of the activity of viral protein hemagglutinin (HA) bybinding collectin proteins such as lung surfactant protein SP-D.Although a strong consensus in the literature concerning theinteraction between SP-D and N-glycans in the head group ofHA, limited structural information concerning the glycans hasbeen reported. We explored a comprehensive mass spectrometrystrategy to study HA and SP-D in terms of identification ofN-glycosylation sites and the carbohydrate structures in order tomeet the need for a detailed understanding of influenzapathobiology.

In total, 6 of the 13 theoretical N-glycosylation sites wereconfirmed in HA isolated from H3N2 strain Phil 1982 and asingle site was observed for pig SP-D truncated protein. Siteoccupancy of 100% was observed for almost all of the sites.Depending on the glycosylated site, high mannose and complexN-glycans were observed. At this point of our interpretation,sulfation and the presence of sialic acid have not been ident-ified for H3N2 HA. Hybrid and complex carbohydrates wereclearly demonstrated to be present on the site of glycosylationof the pig SP-D protein. An example for illustrating our resultsis the glycosylation of site N165 of HA. The data indicatedthe presence of N-glycans at this site as composed of abundanthigh mannose carbohydrates: Hex(2-6)-Man3GlcNAc2 with aseries of complex compositions present at lower abundances .This finding supports the implication of high mannoses carbo-hydrates of N165 in the inhibition of the HA activity by col-lectins but indicates that the N-glycans at this site may evolvefurther.

(76) Anti-HIV Effect in vitro of Human Milk GlycoconjugatesItzia Acosta-Blanco1, Sandra Ortega-Francisco1,

Misael Dionisio-Vicuña2, Mario Hernandez-Flores3,Luis Fuentes-Romero3, David Newburg4,

Luis Enrique Soto-Ramirez3, Guillermo Ruiz-Palacios3,Monica Viveros-Rogel3

1Universidad Nacional Autonoma de Mexico, Mexico City,Mexico; 2Universidad Autonoma de Puebla, Puebla, Mexico;

3Instituto Nacional de Ciencias Medicas y Nutricion, Mexico City,Mexico; 4Boston College, Chetnut Hill MA, USA


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Background: Sulfated glycolipids(SGs) and glycosaminoglycans(GAGs)present on the surface of colonic, vaginal epithelial andneuroglial cells bind to HIVgp120, suggesting that these glycocon-jugates may have a role in HIV infection. Objectives: To test theability of the human milk glycoconjugates(GAGs,SGs and F3) toinhibit the infection of T-cell lines and monocytes cell lines byHIV-1 laboratory isolates in vitro. Methods: GAGs,SGs and F3 gly-coconjugates were extracted from 2L of pooled human milk, puri-fied by ion exchange chromatography, thin layer chromatographyand characterized by mass spectrometry. Four laboratory isolates,HIV-1IIIB,HIV-1MN(lympho-tropic virus) and HIV-1ADA-M(macrophage-tropic virus) were used for inhibition assays usingMT2 T cell line, THP-1 and U937 monocytes cell lines. Inhibitionassays were performed by a 2 hr preincubation of serial dilutions ofhuman milk glycoconjugates with each virus before infecting cells.After 5 to 11 days,HIV-1p24 antigen was quantified by ELISA inculture supernatants.Significant inhibition of viral infectivity wasdefined as >80% reduction in p24 concentration with respect to thecontrol. Results: GAGs fraction significantly inhibits THP-1 infec-tion by macrophage-tropic virus HIV-1ADA(89-100%).No signifi-cant inhibitory effect was observed with U937 infection byHIV-1ADAneither with MT2 infection by lympho-tropic virusHIV-1IIIB and HIV-1MN. SGs fraction significantly inhibits MT2infection by lympho-tropic virus HIV-1IIIB and HIV-1MN(85-100%) but no significant inhibitory effect was observed withthe HIV-1ADA isolate.F3 fraction did not inhibit the infection ofHIV-1 lympho-tropic and macrophage-tropic isolates. Conclusions:GAGs and SGs from human milk inhibit significantly infection ofT-cell lines and monocytes cell lines by HIV-1 laboratory isolates invitro.The magnitude of inhibition seems to be virus strain-specificand glycoconjugates have a dose-effect inhibition of HIV-1 infec-tion.Glycoconjugates from human milk are a unique group ofnatural compounds that may provide a new basis of blocking HIVinfection and they may qualify as efficient microbicides.

(77) Yeast-Binding Lectin with Antibacterial Activityfrom Scapharca broughtonii

Changqing Tong1, Wei Li1, Liang Kong2, Min Qu2, Qiao Jin2,Pavel Lukyanov3

1Northeast Forestry University, Harbin, China; 2Dalian OceanUniversity, Dalian, China; 3Pacific Institute of Bioorganic

Chemistry, Vladivostok, Russia

A mannose-specific lectin (SBL) with molecular mass of 29 kDawas isolated from the sea clam Scapharca broughtonii by chrom-atography on DEAE-52 and Sephadex G-100. SBL showedagglutination activity against yeast (Saccharomyces serevisiae) andantibacterial activity to bacteria, Escherichia coli, Shewanella sp.and Staphyloccocus aureus. The effects of SBL to bacteria NOSwere investigated by Griess regent. Results showed that SBLexhibited promotion effect on the NO production of E. coli andStaphyloccocus aureus, and inhibition effect on the NO productionof Shewanella sp. Bacterial nitric oxide synthases (NOS) produceNO by catalyzing the oxidation of arginine. NO mediated cellresistance to antibiotics. SBL did not show NO-mediated antibioticresistance for Shewanella sp.. Supported by Natural Science

Foundation of China (31071612) and Natural Science Foundationof China (21075012).

(78) A GlcNAc/GalNAc-specific Lectin from the AscidianDidemnum ternatanum with Effects on the Scacharomyces

cerevisiae in Ethanol Fermentation ProcessWei Li1, Changqing Tong2, Wei Zhang1, Min Qu1, Qiao Jin1,Irina Chicalovets3, Valentina Molchanova3, Pavel Lukyanov3

1Dalian Ocean University, Dalian, China; 2Northeast ForestryUniversity, Harbin, China; 3Pacific Institute of Bioorganic

Chemistry, Vladivostok, Russia

Previously we isolated GlcNAc/GalNAc-specific lectin (DTL-A)from the ascidian Didemnum ternatanum. The purified DTL-Awasdigested with trypsin and analyzed with nanoESI-MS/MS. Fivepeptide sequences (LIDTNTGKP, YLDTNTGKI, KLNDLEDALQQAKE, GEMCEEPILSGI and VGMGSILMDHCDVG)were obtained and subjected to the UniProt Knowledgebase(European Bioinformatics Institute) using the WU-BLAST2network service in a search for proteins that atched the amino acidsequences of DTL-A, but no sequences were retrieved. In studies ofits influence on fermentation, DTL-A reveals to be a stimulator forSaccharomyces cerevisiae ethanol production. Ethanol productionincreased from 1.24 to 1.71% (v/v), when DTL-A concentration infermentation broth was increased from 0 to 0.0213 mg/L at 12 h.DTL-A shows potent effect on ethanol production from glucose bythe S. cerevisiae. Supported by the Natural Science Foundation ofChina (31071612) and Natural Science Foundation of China(21075012).

(79) Three Recognition Mechanisms for Agaricus bisporusAgglutinin– Glycan Interactions

Albert M. Wu1, Jia-Hau Liu21Chang-Gung Univ., College of Med., Tao-Yuan, Taiwan;

2National Taiwan University Hospital, Taipei, Taiwan

For the GalNAcα1→ specific Agaricus bisporus agglutinin (ABA)from an edible mushroom, the mechanism of polyvalent Galb1→3/4GlcNAcb1→ complex in ABA–carbohydrate recognition hasnot been well defined since Gal and GlcNAc were weak ligands.By enzyme-linked lectinosorbent and inhibition assays, it is shownthe polyvalent Galb1→ 3/4GlcNAcb1→ in natural glycans alsoplay vital roles in binding and proposed that four different intensi-ties of glycotopes (Galb1-3GalNAcα1-, GalNAcα1-Ser/Thr andGalb1-3/4GlcNAcb1-) construct three recognition systems at thesame domain. This peculiar concept provides the most comprehen-sive mechanism for the attachment of ABA to target glycans andmalignant cells at the molecular level.

(80) A Role for the ST3Gal-VI Sialyltransferases in SelectinLigand Formation in vivo

Won Ho Yang1, Claudia Nussbaum2, Prabhjit K. Grewal1,Markus Sperandio2, Jamey D. Marth1

1Sanford-Burnham Med. Res. Inst., Santa Barbara, CA;2Ludwig-Maximilians-University, Munich, Germany

The binding between E-, P-, and L-selectins and their oligosac-charide ligands termed sialyl-Lewis-X that control lymphocyte


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trafficking to lymph nodes and neutrophil recruitment to vascularendothelium during inflammation. Selectin ligands are attached tovarious glycoproteins including PSGL-1 and others. Upon theirsynthesis and transport, these nascent glycoprotein templates aremodified by one or more of six different α2,3-sialyltransferasesdetected in the mammalian genome and denoted ST3Gal-I-VI.Previous studies established that ST3Gal-IV contributed to E- andP-selectin ligand formation, and to some extent L-selectin ligandsynthesis as well. However absence of ST3Gal-IV failed to com-pletely abolish selectin ligands as judged by continued ligandpresence in the absence of sialidase treatment. Cells lackingST3Gal-I, -II, and –III continued to make selectin ligands nor-mally while ST3Gal-V, which has specificity for glycolipids, wasnot tested. In this study, we report that ST3Gal-VI plays a signifi-cant but partial role in E-, P- and L-selectin ligand biosynthesis invivo among neutrophils and lymph node high-endothelial venules.Moreover, we show that the deficiency of both ST3Gal-IV andST3Gal-VI induces a much greater loss in selectin ligand synthesisthan the absence of either alone, resulting in more severe defect inneutrophil recruitment during inflammation and lymphocytehoming to secondary lymphoid organs. Particularly, E-selectinligand synthesis on neutrophils was almost completely eliminatedby the absence of ST3Gal-IV and ST3Gal-VI. However, weobserved an unexpected retention of all selectin ligands, especiallyfor P- and L-selectin in the doubly-deficient cells, while thisremaining expression was eliminated by sialidase treatment. Ourfindings demonstrate that ST3Gal-VI contributes to selectin ligandsynthesis in vivo, and together with ST3Gal-IV they play animportant role in homing and inflammation responses. The unex-pected retention of selectin ligands in the absence of both sialyl-transferases suggests that a third sialyltransferase can compensatefor the absence of ST3Gal-IV and ST3Gal-VI.

(81) Development of Peptide Mimics of Ligand for anti-GM1Antibody and Cholera Toxin B Subunit

Robert Yu1, Seigo Usuki1, Han-Chung Wu2, Dawn O’Brien11Georgia Health Sciences Univ, Augusta, GA; 2Academia Sinica,

Taipei, Taiwan, ROC

Autoantibodies against ganglioside GM1 have often been shownto be present in sera of patients with Guillain-Barré syndrome(GBS). The antibody activity is presumed to be elicited by preced-ing infection of Campylobacter jejuni (C. jejuni) through a mol-ecular mimicry mechanism between the lipo-oligosaccharide(LOS) antigens and endogenous GM1. The present study wasundertaken to generate structural mimics of the carbohydrateepitope of GM1 for using in a disease treatment strategy. For thispurpose, we used cholera toxin B subunit (CTB) for selectingimmunoreactive GM1-like peptide mimics from a dodecamerphage display library. Six GM1-like peptides could be selected bybiopanning and recognition with CTB. Furthermore, their degreeof mimicry was confirmed with GM1. Five of the 6 peptidesshowed inhibitory activity for binding of GM1 with CTB. Sincebinding of CTB is not limited to GM1 but also to several otherligands, we compared the relative inhibitory activities of thesepeptides for binding of fucosyl-GM1, GD1b, and LOSGM1 with

CTB. At the concentration of IC50, these peptides showed that theinhibitory activity for fucosyl-GM1 and LOSGM1 was similar tothat for GM1, but that for GD1b was significantly lower. Further,we tested the inhibitory activity of 6 peptides on binding of GM1with anti-GM1 rabbit antibody (anti-GM1Ab). UnlikeCTB-binding, inhibition of anti-GM1Ab required higher concen-trations of peptides to reach their IC50 values. Interestingly, one ofthe 6 peptides inhibited binding of GM1 with anti-GM1 Ab, butnot with CTB; while another one showed the opposite effect.Despite of considerable variations in binding mode and affinity,our studies revealed that some of the peptide mimics selectedusing CTB could serve as effective inhibitors for binding of GM1with anti-GM1 Ab. Further studies are in progress in employingthe peptide mimics in neutralizing pathogenic anti-GM1 Ab inpatients with GBS.

(82) New Fucooligosaccharides from Human Milk: Occurrenceof LeYAntigenVladimir Piskarev

Institute of Organoelement Compounds (INEOS), Moscow,Russian Federation

Human milk is a rich source of oligosaccharides containing impor-tant fucoantigens - H type 1, LeA, LeX, and LeB. To find newnatural ligands for H-specific fucolectins, we have studied humanmilk from the donors of blood group H (O) specificity, both secre-tors and non-secretors. Using combination of gel-chromatographyand normal phase (amino) HPLC, followed by lectin affinity chrom-atography on Laburnum anagyroides bark agglutinin (LABA) andPerca fluviatilis lectin (PFL), and reversed phase (C18) HPLC, anumber of individual oligosaccharides were isolated from hexa-,hepta-, octa-, nano-, and decasaccharide fractions. By combinationof monosaccharide analysis, chemical hydrolysis, glycosidasedigestion, 1H-NMR (600-MHz), and MS, their structures weredetermined. LABA-binding oligosaccharides were found to containLeY antigen, and lacto-N-neodifucohexaose I (IV2Fuc,III3Fuc-nLcOse4) was found to be the main LeY oligosaccharide in humanmilk. It was also found in milk of blood group A donors. MinorLeY –containing oligosaccharides included lacto-N-hexaose andlacto-N-neohexaose derivatives. Among PFL-binding oligosacchar-ides a few novel H type 1 oligosaccharides were found, namelymonofucosyl(1-2)isolacto-N-octaoses and difucosyl(1-2,1-2)iso-lacto-N-octaose. Lectin-negative fractions contained lacto-N-trifucoheptaose (IV2Fuc,III4Fuc,I3Fuc-LcOse4), monofucosyl(1-4)lacto-N-hexaose (MFLNH II), and difucosyl(1-2, 1-4)lacto-N-hexaose (DFLNH(c)).

(83) Binding Free Energy Calculations of Dimannoseto Cyanovirin-N High and Low Affinity Sites usingthe Jarzynski’s Equality and Umbrella SamplingSai Kumar Ramadugu1, Hemant Kumar Kashyap1,

Giovanna Ghirlanda2, Claudio Margulis11University of Iowa, Iowa City, IA; 2Arizona State University,

Tempe, AZ

Cyanovirin-N (CVN) is a small (101 amino acids) cyanobacteriallectin that binds to dimannose termini of N-linked oligomannose


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on HIV envelope glycoprotein, gp-120. The protein encompassestwo homologous binding domains, A and B, which differ slightlyin their specificity for trimannose and dimannose respectively. Ithas been shown that dimannose binds to domain B with a Kd of0.1 µM, 10-fold lower than to domain A. Here we present freeenergy of binding calculations of dimannose to the high and lowaffinity binding sites of CVN using the umbrella sampling tech-nique (US) and the Jarzynski equality (JE) ΔF = -1/β lnexp<-βW>). Our goal is to establish which protocol will be themost efficient and accurate for this particular system. The long-term objective is to predict how mutations will affect binding tothe target dimannose, and to other oligomannoses found ongp120. While these studies are still at a preliminary stage, someinsightful information can be derived from our available compu-tational data. Force versus distance/extension plots enable us toprobe which key residues are involved in tight interaction with theligand. In the high affinity site Asp41, Ser52, Asn53, Thr57 andLys74 that hydrogen bond with 3, 4, 3’ and 4’ hydroxyls ofdimannose appear to be important. Key hydrogen-bond inter-actions in the low affinity site are between 3, 4, 3’ and 4’hydroxyls of dimannose and Gln6, Glu23, Agr24, Asn93 andAsp95.

(84) Lectins as Pattern Recognition Molecules: The Effectsof Epitope Density in Innate Immunity

Curtis BrewerAlbert Einstein College of Medicine, Bronx, NY

The innate immune response of multicellular organisms involvesbinding of soluble and membrane bound host molecules to thesurface of pathogenic organisms including viruses, bacteria, para-sites and fungi. Host molecules involved in innate immunityinclude glycan binding receptors (lectins), Toll-like receptors,nucleotide-binding oligomerization domain (NODs) and NK cellreceptors. Until recently, it was believed that the epitopes recog-nized by these molecules were uniquely associated with the patho-genic organisms, and, hence, the term pattern recognitionreceptors (PRRs) was used to describe their binding specificity.However, an expanding number of lectin classes are becomingrecognized as members of PPRs, and it is apparent that many ofthe glycan epitopes recognized on foreign pathogens are present inthe host and involved in normal lectin functions including devel-opmental and immune regulation. Hence, the molecular basis forpattern recognition of carbohydrate epitopes on pathogens bylectins is in question. Recent molecular studies have suggested thatthe density of glycan epitopes in individual multivalent carbo-hydrates and in glycoprotein receptors is a key mechanism for reg-ulating the affinity of lectins and their effector functions.Examples of density dependent recognition of glycan receptors bylectins in biological systems are presented, as well as the mechan-isms and magnitude of these effects on the affinities of lectins forclustered glycan epitopes. The molecular basis for lectin patternrecognition of carbohydrate epitopes on the surface of pathogensappears to be part of a general mechanism of lectins binding todensity dependent glycan expression.

(85) Monoclonal Antibody WN1 222-5 Binds a ConservedCore Region of Enterobacterial LPS

Kathryn Gomery1, Sven Müller-Loennies2, Cory L. Brooks3,Lore Brade2, Paul Kosma4, Franco Di Padova5, Helmut Brade2,

Stephen V. Evans11University of Victoria, Victoria, BC; 2Research Center Borstel,Borstel, Germany; 3University of Alberta, Edmonton, AB; 4Univ.of Natural Resources & Applied Life Sciences, Vienna, Austria;5Novartis Institutes for BioMedical Research, Basel, Switzerland

WN1 222-5 is a broadly neutralizing monoclonal antibody (mAb)shown to recognize a protective epitope within the core regioncommon to enterobacteria including E. coli, Salmonella andShigella, whether displayed on bacterial surfaces or as free LPS.There is a long-standing hypothesis that a monoclonal antibodywith specificity to the conserved inner core region would be protec-tive against a wide range of bacteria (Chedid, Parant et al. 1968),and WN1 222-5 is the only antibody reported thus far to possessthis quality. The structure of the WN1 222-5 antigen-binding frag-ment (Fab) in complex with a large section of the core region oflipopolysaccharide (LPS) has been solved to 1.72 Å resolution,revealing a compact core LPS structure and showing that the heavychain contributes most to binding. Many intra-molecular inter-actions in the form of hydrogen bonds in the ligand assist in the for-mation of a conformational epitope. The conserved tri-heptose coreregion, associated phosphate residues and Kdo region are critical tointra-molecular binding, where they are involved in 9 of 10 totalinteractions, and also in intermolecular binding where they contrib-ute 11 of 13 interactions. Outer core sugars help in the formation ofthe epitope but are not critical; LPS distal outer core sugars aremore significant for intra-molecular binding, whereas sugars closerto the conserved inner core region are more implicated in intermole-cular binding. Complementarity determining region (CDR) H2 isthe most involved region in the combining, with CDR H3 alsobeing predominant. The light chain plays little contribution tobinding, with only one hydrogen bond from the CDR L3 region.

Chedid, L., M. Parant, et al. (1968). ‘A Proposed Mechanism forNatural Immunity to Enterobacterial Pathogens.’ J Immunol(100):292–301.

(86) The Role of lman2la, an L-type Lectin Gene,in the Escape Behavior in Zebrafish

Kazuhide Asakawa1,2, Koichi Kawakami1,21National Institute of Genetics, Mishima, Japan; 2Graduate

University for Advanced Studies(SOKENDAI), Mishima, Japan

An important feature of reactive behaviors is the ability to spatiallylocalize a sensory stimulus and act accordingly. Such sensory-motor transformations must be precisely coordinated in escapebehaviors, which determine whether an animal can survive a pred-atory attack.

Zebrafish embryos, when a tactile stimulus is applied, exhibit anescape response with an initial turn away from the stimulus. Inorder to understand the molecular mechanism that establishes thelaterality of the escape response, we performed a genetic screen toidentify transposon insertion mutant that displayed an altered


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escape direction. From the pilot screen, we identified a recessivemutant that showed the initial turn towards the tactile stimulus at ahigh frequency. We found that, in the mutant, the transposon wasinserted in the lman2la gene, which encodes an L-type lectin thatis evolutionarily conserved among vertebrates and implicated inglycoprotein trafficking. To understand in which tissue lman2lafunctions, we performed phenotypic rescue experiments using theGal4-UAS gene expression system to express the wild-typelman2la gene in the lman2la mutant animal in a tissue specificmanner. First, by performing Gal4 enhancer/gene trap screening,we collected transgenic lines that expressed the yeast Gal4 tran-scription factor in various tissues. Then, to induce the lman2laexpression in the Gal4-expressing cells, we established theUAS-lman2la line, which carried the lman2la gene downstream ofUAS (the target DNA sequence for Gal4). We found that, whenthe wild-type lman2la gene was ectopically expressed in the hind-brain by the Gal4-UAS system, the initial turn towards the tactilestimulus was rarely observed in the lman2la mutant, indicatingthat the lman2la expression in the hindbrain rescued the mutantphenotype. These observations indicate that lman2la has a func-tion in the hindbrain and imply an unexpected link betweenLman2la-mediated glycoprotein trafficking and the laterality ofescape response.

(87) Invariants Vα14 Natural Killer T Cell Activationby Edible Mushroom Acidic Glycosphingolipids

Yasunori Kushi1, Yuusuke Suzuki1, Hirofumi Nozaki2,Saki Itonori3

1Col. of Sci. and Tech., Nihon University, Tokyo, Japan; 2NationalInstitute of AIST, Tsukuba, Ibaraki, Japan; 3Faculty. of Liberal

Arts & Education, Shiga Univ., Otsu, Shiga, Japan

Natural killer T (NKT) cells recognize CD1d loaded with glyco-sphingolipid (GSL) antigens. After being stimulated by GSL anti-gens, NKT cells immediately secrete cytokines. The variety ofcytokines release from NKT cells can promote immunoresponsesagainst tumors and micorbial infections, and regulate autoimmuneresponse, though a puzzling aspect of these responses is that bothTh1 and Th2 cytokines can be secreted in response to the stimu-lation. The fungal GSLs are exogenous antigens for NKT cells;therefore, we focused on acidic glycosphingolipids (AGLs) ofedible mushrooms (Hypsizigus marmoreus and Pleurotus eryngii)and reported that AGLs induced interferon-γ (IFN-γ) andinterleukin-4 (IL-4) release from murine T cells in aCD11c-positive cell-dependent manner and more over that AGLsof mushroom presented by murine CD1d-transfected rat basop-holic leukocytes induced interleukin-2(IL-2) release from iNKThybridoma cells. AGL-1, one of the AGLs, containing mannose atthe non-reducing ends, induced CD1d-dependent IL-2 release.Although α-galactosylceramide (α-GalCer) presented byCD11c-positive cells induced both IFN-g and IL-4 release, all ofthe presented AGLs presented by CD11c-positive cells and AGL-1presented by B cells induced IL-4 release from iNKT hybridomacells. Above results suggested that edible mushroom AGLs mightcontribute to the retention of immunohomeostasis through theminimun induction of iNKT cell activation in vitro and in vivo.

References1. Nozaki and Kushi. Biochem. Biophys. Res. Comm. 373, 435(2008).2. Nozaki and Kushi. Biol. Pharm. Bull. 33, 580–584 (2010).

(88) Mannose-Binding Lectin Activity of Lactobacilliand Its Potential in Pathogen Exclusion

Shweta Malik1, Sarah Lebeer1, Mariya Petrova1, Jan Balzarini2,Jos Vanderleyden1

1KULeuven-CMPG, Heverlee, Belgium; 2Rega Institute forMedical Research, K.U.Leuven, Leuven, Belgium

Various pathogens infect host cells via binding to specific glycansat the host surface by virtue of carbohydrate-binding proteins,named lectins, on their cell surface. Probiotic bacteria have beenshown to antagonize pathogens. An important mechanism of pro-biotic action is the exclusion/ inhibition of these pathogens by theproduction of antimicrobial compounds and/or competition foradhesion sites. Probiotic bacteria also have lectins at their cellsurface. These lectins could play a role in pathogen exclusion by(1) competitively binding to the same glycans on the host surface,thereby blocking pathogen adhesion or (2) by binding glycans onthe pathogenic surfaces, thereby blocking virulence mechanismssuch as invasion. In this project, the mannose-binding lectinactivity of lactobacilli is explored for its potential to excludemannose-binding/mannose-containing pathogens such asEscherichia coli, Candida albicans and HIV. Hereto, a total of 70selected Lactobacillus strains isolated from various sources werescreened for the presence of MBLs based on the yeast aggluti-nation assay. Seven strains were found to coaggregate withSaccharomyces cerevisiae in a calcium-independent manner thatcould be inhibited by methyl-alpha-D-mannopyranoside. Thestrain with the highest activity for agglutination exhibited also astrong auto-aggregating phenotype. Its aggregation is inhibitedupon treatment with mannose-specific inhibitors and is abolishedby proteinase K treatment. The strain is capable of forming bio-films on abiotic surfaces and adheres well to human intestinal andvaginal epithelial cell lines. In-depth molecular studies are beingcarried out to identify the genes encoding the lectin, overexpressthe lectin in E. coli and construct the corresponding Lactobacillusknock-out mutants. Coaggregation ability of this strain with patho-gens will be investigated to evaluate its potential as a competitorfor colonization of the host surface. The ultimate aim is to bio-chemically characterize the putative lectins involved in pathogenexclusion.

(89) Regulatory Functions of Activation-Dependent Reductionof N-glycolylneuraminic Acid in Mouse T Cells

Yuko Naito-Matsui, Hiromu Takematsu, Keisuke Murata,Yasunori Kozutsumi

Kyoto University, Kyoto, Japan

Sialoglycans play important roles in the immune cell recognitionevents. Sialic acids, occupying the outermost part of glycans,occur mainly in two forms, N-acetylneuraminic acid (Neu5Ac)and N-glycolylneuraminic acid (Neu5Gc). Neu5Gc is a majorsialic acid species in mouse lymphocytes, and biosynthesized from


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Neu5Ac by CMP-Neu5Ac hydroxylase (Cmah). We reported thatactivated B cells undergo repression of Neu5Gc by means ofCmah suppression, which is probed by GL7 monoclonal antibody,a marker for germinal centers. The GL7 positivity in activated Bcells could be interpreted as downregulation of CD22/siglec-2ligand. Meanwhile, GL7 also bound to Concanavalin A(ConA)-stimulated T cells. Here we hypothesized that activated Tcells also repress Neu5Gc to regulate T cell function. This questionis important because T cells lack CD22/siglec-2.Indeed, Neu5Gc expression was activation-dependently sup-

pressed in T cells. To address the physiological significance of thisNeu5Gc repression, we analyzed the T cell phenotypes ofNeu5Gc-deficient Cmah knockout mice. When stimulated withConA or anti-CD3 plus anti-CD28 antibodies, Neu5Gc-lacking Tcells showed increased expression of activation markers, CD25and CD69 and augmented proliferation.T cells regulate immune responses by interacting with other cells,

such as B cells and dendritic cells, both of which express CD22/siglec-2. Due to the Neu5Gc suppression, CD22/siglec-2 ligandwas decreased and sialoadhesin/siglec-1 ligand was concomitantlyincreased upon T cell activation. Therefore, Neu5Gc repressionmight regulate immune responses by modulating trans siglec-ligandinteraction, in addition to the above regulation of T cell activation.

(90) NMR Analysis of an Anti-Carbohydrate AntibodyMLS128 Single-Chain Fv Fragment toward Elucidation

of the Multivalent Recognition MechanismGanesh Prasad Subedi1, Tadashi Satoh1, Shinya Hanashima1,

Akemi Ikeda1, Hiroshi Nakada2, Reiko Sato3, Mamoru Mizuno3,Noriyuki Yuasa4, Yoko Fujita-Yamaguchi4, Yoshiki Yamaguchi1

1Structural Glycobiology Team, RIKEN ASI, Wako, Japan; 2KyotoSangyo University, Kyoto, Japan; 3The Noguchi Institute, Tokyo,Japan; 4Tokai University School of Engineering, Kanagawa,

Japan

Tn antigens are aberrantly overexpressed in adenocarcinomas andhas been a major target for developing therapy against cancer.MLS128 is an anti-carbohydrate antibody which recognizes the Tnantigen consisting of both the two and three consecutivelyarranged GalNAc-O-Ser/Thr at surface of cancerous cells. To elu-cidate the details of multivalent recognition mechanism behind theevent, we prepared a corresponding single-chain Fv fragment(scFv) of MLS128 antibody and analyzed by NMR.Overproduction of recombinant proteins in E. coli is often ham-

pered by their failure to fold correctly, leading to their accumu-lation in an insoluble form and requiring refolding processes. Toovercome the issue, we developed a novel expression system usingpCold-PDI vector in Origami E. coli strains. The system success-fully provided the recombinant MLS128-scFv protein having twodisulfide bonds. ITC analyses revealed that scFv retains fullbinding activity towards synthetic glycopeptides having Tn3 unit.For the atomic level analyses of the binding event, NMR exper-

iments using stable-isotopes labeled scFv were performed with theligand glycopeptides (Tn2 and Tn3). Several NMR signals derivedfrom scFv were perturbed upon an addition of ligands, whichclearly indicates that the peptide ligands bind to the specific

binding sites. We will discuss their molecular basis on the multi-valent GalNAc-Thr recognition mechanism.

(91) Imidazolium Compounds: A New Generationof Galactosyltransferase Inhibitors

Yin Gao, Jason Vlahakis, Walter A. Szarek, Inka BrockhausenQueen’s University, Kingston, Canada

Galactosyltransferases (GalT) extend the glycan chains of mamma-lian glycoproteins by adding Gal to terminal GlcNAc residues,and thus build the scaffolds for biologically important glycanstructures. Inhibitors of GalT have the potential to reduce celladhesion in inflammation, cancer and metastasis. Gram negativebacteria also have GalT that can extend the oligosaccharide struc-tures of O-antigens which are virulence factors. We have pre-viously reported hydrophobic GlcNAc derivatives as substrateanalog inhibitors which inhibited mammalian beta4-GalT. Here,we showed that positively charged imidazolium compounds,having aliphatic lipid chains of 10 to 20 carbon lengths, formpotent inhibitors of mammalian beta3-GalT 5. Selected imidazo-lium compounds also inhibited other GalT, as well as bacterialenzymes. Imidazolium compounds were shown to bind toUDP-Gal but this binding was not directly related to their abilityto inhibit GalT. The structures of these inhibitors do not appear tobe related to glycosyltransferase substrate structures and representa new class of glycosyltransferase inhibitors with potential asanti-cancer, anti-inflammatory and anti-bacterial drugs. This workwas supported by a Discovery grant from NSERC (to I.B.).

(92) Mutational Analyses of Human acetyl-CoA:Glucosamine-6-phosphate N-acetyltransferase, hGNA1.

Dileep G. Nair, Yifan Wang, John Allingham,Tassos Anastassiades, Inka BrockhausenQueen’s University, Kingston, Canada

Human acetyl-CoA: Glucosamine-6-phosphate N-acetyltransferase1 (hGNA1) catalyses the acetylation of glucosamine-6-phos-phate (GlcN6P) to form N-acetylglucosamine-6-phosphate(GlcNAc6P), a major intermediate in the biosynthesis of UDP-GlcNAc. Despite a low sequence homology, the numerousmembers of the GNAT superfamily share a conserved structuralfold, consisting of five beta sheets surrounded by four alpha helices.Previous mutational studies of hGNA1 showed that the E156Amutant was inactive and exhibited impaired GlcN-6PO4 binding.The E156D mutant was partially active. In order to study theenzyme and its mutants further, we developed a novel, sensitive andefficient fluorescence-based assay and showed that the apparent Kmvalue for acetyl-transfer of hGNA1 was 84 ± 17 uM. We are inter-ested in the specificity of the hGNA1 and in identifying aminoacids involved in donor and acceptor substrate binding. We there-fore focused on residues located near the binding pocket of thedonor substrate, acetyl coenzyme A (AcCoA) and targeted con-served and non-conserved residues that possibly affect the dimen-sions and properties of the AcCoA binding pocket. Six mutantswere produced and purified, and showed variable activities,suggesting their roles in substrate binding or catalysis. Pro159 wasfound to be essential for catalysis while several Leu residues


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significantly influenced the activity. Thus, these residues appear toaffect the catalytic activity without changing the overall structure ofthe enzyme protein. This work was supported by an NSERC grantSTPGP 381564-09.

(93) Slit3 C-terminal Binds Heparin and Neutralizes Heparin’sAnticoagulant Activity

Eduard Condac1, Heather Strachan1, Christian Heiss1,Darryl Johnson1, Parastoo Azadi1, Ron Orlando1, Job Harenberg2,

Kelley Moremen1, Lianchun Wang11University of Georgia, Athens, GA; 2Ruprecht-Karls University

Heidelberg, Mannheim, Germany

Slit3 belongs to the Slit family of proteins that are guidance mol-ecules directing the migration of various cell types like neurons,leucocytes, endothelial cells. They are also involved in organogen-esis, morphogenesis and angiogenesis. All Slit proteins share acommon structure: a tandem of four leucine-reach repeatsdomains, followed by six to nine EGF repeats, a laminine G-likedomain and a cystine knot-like domain at the C terminus. Theleucine-rich repeat domain 2 is involved in the interaction withRobo proteins and heparan sulfate enhances this interaction. It hasbeen demonstrated that the C terminal domain of Drosophila Slitand of human Slit 2 can bind heparin/heparan sulfate. Here weshow that the C-terminal fragment of human Slit3 binds heparinwith high affinity. Characterization studies observed that thebinding depends on both the degree and type of sulfation ofheparin. N-sulfation and 6-O-sulfation proved to be more impor-tant than 2,3-O-sulfation and carboxyl groups. An oligosachacar-ide of 6 to 8 monomers with a specific sulfation pattern appears tobe the minimum heparin sequence to bind Slit3 C-terminal.Interestingly, we determined that Slit3 C-terminal is an antagonistof heparin binding to antithrombin III and it also reduce the antith-rombin dependent inactivation of factor Xa, suggesting that Slit3C-terminal may neutralize at least partially the antithrombin-dependent anticoagulant action of unfractionated heparin.

(94) Structural Investigation of Chlamydiaceae-SpecificAntibodies

Omid Haji-Ghassemi1, Sven Müller-Loennies2, Lore Brade2,Paul Kosma3, Roger Mackenzie4, Helmut Brade2, Stephen Evans1

1University of Victoria, Victoria, Canada; 2Research CenterBorstel, Borstel, Germany; 3University of Agricultural Sciences,Vienna, Austria; 4National Research Council, Ottawa, Canada

The lipopolysaccharide of Chlamydiaceae-family of pathogenscontain a unique 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) tri-saccharide of the sequence Kdo-(2→ 8)-Kdo-(2→ 4)-Kdo.Several murine monoclonal antibodies raised through immuniz-ation with glycoconjugates of Kdo trisaccharide have been crystal-lized in the presence of antigen and their structures determined tohigh resolution using single-crystal x-ray diffraction methods. Thestructures reveal the mechanisms employed by these antibodies todisplay differential specificity and adaptability, and correlate withbinding data obtained through enzyme-linked immunosorbentassays and surface plasmon resonance experiments. While a fewof the antibodies bind the Kdo Chlamydiaceae-family specific

antigen with high affinity and specificity, a number of other anti-bodies were able to accommodate a range of LPS-derived antigensand antigen fragments. Antibodies that recognize a broad range ofKdo derived antigens utilized an induced fit binding pocket withinteractions distributed between the light and heavy chains. In con-trast, antibodies that are specific to the Kdo trisaccharide immuno-gen utilized a groove dependent on the heavy chain. Comparisonof the primary structure of the antibodies gives insight into howthe humoral immune system evolved to recognize carbohydratesurface features of common pathogens.

(95) Functional Characterization of Histoplasma capsulatumLipid Rafts

Tanil Lacerda, Marcos Toledo, Anita Straus, Helio K. TakahashiUniversidade Federal de Sao Paulo, Sao Paulo, Brazil

The structural organizational of detergent resistant plasma mem-brane lipid rafts of yeast forms of Histoplasma capsulatum and therelationship of these lipid raft components with fungal ability toinfect mouse alveolar macrophage was analyzed by a series ofexperiments with methyl-beta-cyclodextrin (mβCD) and solubleergosterol or cholesterol. It was observed the presence of twotypes of lipid rafts: i) ergosterol-dependent lipid rafts andii) glycosphingolipid enriched/dependent lipid rafts. About 40% ofergosterol and 25% of glycosphingolipids (GSLs) of H. capsula-tum yeasts are present in membrane microdomain fractions resist-ant to treatment with 1% Brij 98 at 4°C. Specific proteins werealso enriched in these lipid rafts, particularly: Pma1p, a fungalplasma membrane proton ATPase and microdomain proteinmarker; a (glyco)protein of 30kDa, able to bind to laminin and a50kDa protein recognized by mAb anti-α5-integrin. Removal ofergosterol of H. capsulatum by mβCD reduced the fungal abilityto infect alveolar macrophages by 45% and displaced Pma1p andthe 30kDa (glyco)protein from lipid raft fractions to detergentsoluble fractions. On the other hand, mβCD treatment did not dis-place GSLs or α5-integrin-like 50kDa protein from lipid raft frac-tions to detergent soluble fractions. Reinsertion of ergosterol butnot cholesterol into membranes of mβCD-treated yeasts restoredtheir ability to infect alveolar macrophages indicating that sterolspecific structural features are required for functional organizationof microdomains. Among the several components detected in thedetergent insoluble membranes of H. capsulatum it is noteworthythe presence of GM1-binding proteins (approximately 18 and 28kDa) suggesting that H. capsulatum may express a gangliosidebinding protein possibly involved in the fungal-host interaction.Studies to characterize the roles of ergosterol, glycosphingolipidsand specific proteins in fungal infectivity, regulation of membraneproperties and signaling processes may represent new therapeuticapproaches to histoplasmosis.

Supported by FAPESP, CNPq and CAPES.

(96) Exploiting Multivalency: A Designed Dimer of CV-NShows Improved anti-HIVActivity

Giovanna Ghirlanda1, Brian Woodrum1, Melissa Ruben1,Barry O’Keefe2

1Arizona State University, Tempe, AZ; 2NCI, Frederick, MD


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Natural lectins often achieve the high affinity and specificityrequired for biological function through multivalent interactions.Antiviral lectins, for example, typically encompass multiple glycanbinding sites, or form dimers. Although the exact mechanism ofaction is not known, it is generally accepted that they bind glycanson the viral envelope glycoprotein and inhibit the cell-virionfusion step necessary for infection.Cyanovirin-N (CV-N) is an algal lectin with potent antiviral

activity against HIV-1, Ebola, and other viruses. CV-N containstwo glycan binding domains, A and B, which display slightlydifferent specificities for Man3 and Man2 termini respectivelywithin the N-linked oligomannose core. Work in our lab andothers has established that the presence of two binding sites, eitherthrough the presence of intact domains A and B in wild type, orthrough the formation of domain swapped dimers in mutants inwhich one of the domains has been impaired, is necessary foractivity. We hypothesize that the activity of CV-N could be furtherimproved by further increasing its multivalency.To test whether the antiviral activity of Cyanovirin could be

augmented, we engineered a covalent dimer in which one fullsequence of CVN is spliced in between the two repeats of asecond CVN sequence. The new construct, NestedCVN, encom-passes four glycan binding sites: two copies of domain A and twocopies of domain B (AA’BB’). Biophysical characterization ofNestedCVN shows that the protein is well folded, and has athermal denaturation profile similar to wt CVN. NestedCVN bindsgp120 with tighter affinity than wt CVN, as evaluated by ELISA.Most importantly, the neutralization activity of NestedCVNtowards HIV-1 is increased 3-fold compared to wild-type CVN.These results suggest that inhibition of viral entry by antivirallectins may be significantly augmented via designed multivalentconstructs.

(97) Lectenz®: Carbohydrate-Binding BiomoleculesEngineered via Computational Modeling and Directed

EvolutionKausar N. Samli1, Loretta Yang2, Robert J. Woods1,3

1The University of Georgia, Athens, GA; 2Glycosensors &Diagnostics LLC, Athens, GA; 3The National University of

Ireland, Galway, Ireland

Glycosylation is an enzymatic process by which proteins are post-translationally modified by the covalent attachment of glycans(complex carbohydrate structures). Not only do glycans serve asimportant disease biomarkers, but they also impact the pharmaco-logical properties of therapeutic biologics. Approximately 28% ofthe therapeutics approved by the U.S. Food and DrugAdministration in 2010 were biologics, compared to 10% in 2005.The research and development process for therapeutic biologicsfaces unique challenges compared to small molecule development.For example, glycan heterogeneity can impact batch-to-batchconsistency, immunogenicity, pharmacokinetics, activity and clear-ance. Given that more than two thirds of therapeutic biologics areglycosylated recombinant proteins, new tools for glycosylationanalysis during bioprocess monitoring are required. Reported hereis the development of a novel class of lectin-like carbohydrate-

binding biomolecules which are derived from carbohydrate-processing enzymes and are called Lectenz®. As proof-of-concept,by employing an interdisciplinary strategy that merges compu-tational modeling and directed evolution, N-glycan bindingLectenz® biomolecules have been designed, engineered andcharacterized. The design of Lectenz® is initiated in silico todetermine optimal carbohydrate-enzyme interactions using molecu-lar dynamics simulations. Simulated structure/function relation-ships are validated by generating focused biocombinatoriallibraries for selection and downstream characterization ofLectenz® candidates. Surface Plasmon Resonance is utilized todetermine binding kinetics. Lectenz® biomolecules may beutilized as recognition elements in biosensors and multiplexedbead-based assays for monitoring of glycosylation states in glyco-proteins of interest, a key requirement for bioprocess monitoringand disease related biomarker detection.

(98) Pro-Inflammatory Phenotype Associated with ST6Gal-1Deficiency is Reversed by Hepatic Expression of a Secretory

ST6Gal-1 TransgeneMark B. Jones, Mehrab Nasirikenari, Joseph T.Y. Lau

Roswell Park Cancer Institute, Buffalo, NY

The sialyltransferase ST6Gal-1 constructs the α2,6-sialyl linkagecommon on many mammalian cell surface and secreted proteins.Our laboratory has demonstrated that deficiency in ST6Gal-1,specifically in pool of enzyme expressed in the liver and releasedinto systemic circulation, resulted in a generally pro-inflammatorystate, and more acute Th1 and Th2 responses to inflammatorystimuli. ST6Gal-1 deficiency also resulted in a generally morerobust hematopoietic capacity, with an expanded and more activehematopoietic stem and progenitor cell pool in the marrow. Inanimals with inactivated liver-specific promoter of ST6Gal-1 gene(Siat1ΔP1), the pro-inflammatory phenotype is accompanied byonly minor alterations to serum glycoprotein glycosylation,suggesting a direct contribution of the secreted ST6Gal-1 enzyme.To confirm the biologic relevance of the circulatory form ofST6Gal-1, we constructed a secretory form of the recombinantST6Gal-1 transgene, controlled by a heterologous but still liver-specific promoter, the α1-antitrypsin (ATT) promoter. The trans-gene, Tg-ST6G1-sc, when placed in the Siat1ΔP1 background,abrogated the more robust hematopoietic phenotype associatedwith ST6Gal-1 deficiency as assessed by clonogenic assays and exvivo liquid cultures. Acute LPS-induced pulmonary inflammationwas also reduced to wild-type levels. Together, the data presentedhere further serves to reinforce the hypothesis that extracellularST6Gal-1, particularly in circulation, is an important systemic reg-ulator of inflammation and in hematopoietic activity.

(99) Reengineering the Glycan Binding Pocket of Cyanovirinby Directed Evolution

Melissa Ruben, Jason Maxwell, Brian Woodrum,Giovanna Ghirlanda

Arizona State University, Tempe, AZ

Lectins bind a variety of carbohydrate targets and mediate manybiological activities. The lectin Cyanovirin (CV-N) binds


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specifically to di- and tri-mannose termini of oligomannoseglycans on the envelope glycoprotein of various viruses, includinggp120 of HIV, resulting in potent antiviral activity. CV-N is an11kDa protein containing two homologous domains, each forminga glycan binding site that binds oligomannose with micromolar tonanomolar affinity. Our research aims to improve CV-N’s affinityfor target oligomannoses and design a method for producingCV-N mutants specific to other glycan targets.To facilitate binding studies, our lab mutated CVN to have one

active binding site (P51G-m4-CVN). The structure ofP51G-m4-CVN bound to dimannose revealed which residues areinvolved in binding. Seven residues were subjected to saturationmutagenesis in a T7 phage display library to explore the sequencespace of the binding domain. The library was screened againstgp120 and RNAse B, used as a negative control. From this library,we isolated five CV-N mutants. Each mutant is well folded andstable at physiological conditions, showing that P51G-m4-CVNcan tolerate a large number of mutations. We assessed themutants’ ability to bind gp120 by ELISA, obtaining EC50s in thehigh nanomolar to low micromolar range, which compares wellwith the EC50of 0.9 µM obtained for wt CV-N. A quantitativeassessment of the mutants’ affinity and specificity for dimannoseby ITC is in progress.Our results show that the binding pocket of CV-N can be suc-

cessfully remodeled, while conserving high affinity and specificityfor the target dimannose. This approach can be extended to theselection of CV-N based lectins with altered specificities.

(100) Carbohydrate-Dependent Uptake of Glycopolymers byMacrophages and the Implications for Targeted Drug Delivery

Eun-Ho Song, Matt Manganiello, Yu-Hua Chow,Anthony J. Convertine, Lynn M. Schnapp, Patrick S. Stayton,

Daniel M. RatnerUniversity of Washington, Seattle, WA

Targeted delivery of biologic drugs is believed to be crucialtowards improved therapeutic efficacy and reduced dosage require-ments. However, the design of targeted drug vehicles still awaitsthe development of efficient drug carriers that can deliver thera-peutic agents to designated tissues or cells. Carbohydrates are notonly the most abundant biomolecules in nature, but also promisingmaterials for targeted delivery of therapeutics in cell-specificmanner. As a first step in investigating specificity of carbohydratesbinding toward targeted immune cells such as macrophages, wehave synthesized several structurally well-defined reversibleaddition-fragmentation chain transfer (RAFT) glycopolymers (e.g.,mannose, galactose and N-acetyl-glucosamine) displaying multi-valent ligands for increasing affinity toward the macrophagemannose binding receptor (MMRs). Additional functionality hasbeen added to the glycopolymers, including pyridyl disulfidependant moieties for subsequent bioconjugation and fluorescentlabeling. Cellular uptake analysis of glycopolymers in-vitro(bone-marrow-derived macrophages) and in-vivo (alveolar macro-phages) clearly demonstrated uptake of glycopolymers driven bycarbohydrate-binding specificity and multivalent interactions.Furthermore, uptake of glycopolymers by discrete populations of

activated macrophages (M1 vs M2) supports our hypothesis thatMMR-specific carbohydrates can be preferentially internalized byM2 macrophages. These results not only provide valuable tools forinterrogating multivalent carbohydrate-MMR interactions, but alsooffer insights into the rational design of targeted delivery platformsfor cell-targeted therapeutics.

(101) Insights into the Mechanism of OncofetalThomsen-Friedenreich (TF) Antigen Recognition by Galectin-3

Svetlana Yegorova1, Maria C. Rodriguez1, Dmitriy Minond1,Jesús Jiménez-Barbero2, Luis Calle2, Ana Ardá2,

Hans-Joachim Gabius3, Sabine André3,Karina Martinez-Mayorga1, Austin B. Yongye1, Mare Cudic11Torrey Pines Inst. for Molecular Studies, Port St. Lucie, FL;

2Center for Biological Research, Madrid, Spain;3Ludwig-Maximilians-University, Münich, Germany

The functional significance of glycosylation changes during theonset of cancer and its progression to metastatic stage of diseasehas significant implications for understanding and treating thismalignant disease. Recent findings indicate that galectins,β-galactoside-specific lectins, play an important role in cancerinitiation and progression. Oncofetal TF carbohydrate antigen(galactose-β-1,3-N-acetylgalactosamine) of the cancer-associatedMUC1 glycoprotein has been identified as natural ligand forgalectin-3. In this study we have examined the structural basis ofTF antigen recognition by galectin-3. We hypothesize that specificrecognition may involve either the amino acid or the local peptidesequence to which TF antigen is attached.

NMR spectroscopy and molecular modeling were used to eluci-date the molecular recognition of Thr O-linked TF antigen bygalectin-3. Saturation transfer difference (STD) NMR experimentsconfirmed the strongest interactions of galectin-3 with the non-reducing terminal galactose residue as expected for the galectinfamily. The interactions with the protons of GalNAc were smallerbut still significant. Surprisingly, the methyl protons of the Thrresidue were strongly saturated, suggesting tight contact withgalectin-3. Two-dimensional transferred nuclear Overhauser effectspectroscopy (TR-NOESY) NMR experiments and molecularmodeling indicated possible differences in conformation popu-lations between free-states and bound-states. The thermodynamicparameters of galectin-3 binding to Thr-TF antigen were deter-mined by isothermal titration calorimetry (ITC). In addition, thebinding affinities of galectin-3 towards its natural ligands,MUC1-derived glycosylated peptides that carry one or multiple TFantigens, were assessed by an AlphaScreen-based assay.

The results obtained from this study provide a better understandingof galectin-3 interaction with the TF antigen. This information willfacilitate the development of specific reagents for the detection of theTF code and intervention in tumor progression and metastasis.

(102) Golgi Phosphopotein 3 Regulates Cell Adhesionto Selectins and ICAM-1 by Controlling Golgi Retention

of C2GnT1Mohamed F. Ali, Vishwanath B. Chachadi, Pi-Wan Cheng

Nebraska Medical Center, Omaha, NE


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C2GnT1 is known to be involved in the production of core 2associated sialyl Lewis x (sLex), which serves to guide leukocytetrafficking and cancer metastasis in a regulated fashion.Localization of C2GnT1 at its Golgi compartment is required forthis enzyme to carry out its glycosylation function. However, themechanism of the Golgi localization of this enzyme is not known.Previously, by yeast-two-hybrid screen, we identified GolgiPhosphopotein 3 (GOLPH3), an oncoprotein overproduced inmany cancers, as a binding partner of C2GnT1. We also showedthat GOLPH3 colocalized with C2GnT1 in the Golgi. In thisstudy, we show that GOLPH3 is required for Golgi localization ofC2GnT1 through which GOLPH3 controls leukocyte rolling andadhesion. The biotinylated cytoplasmic tail of C2GnT1 was shownto pull down GOLPH3 from the KG1a cell lysate and recombinantGOLPH3 as well. This property was lost when the three basicamino acids in the tail were replaced with alanine. Also, knock-down of GOLPH3 mRNA or mutation of the three basic aminoacids in the cytoplasmic tail of C2GnT1 abolished Golgi localiz-ation of C2GnT1 and rendered the cells unable to generate core 2associated sLex and thus compromised cell adhesion to immobi-lized E or P-selectin under dynamic flow condition. We alsoshowed that binding of soluble E-selectin to PSGL-1-associatedcore 2 sLex on the leukemic cells activated a signaling cascade,starting with phosphorylation of Syk, followed by activation ofintegrin and culminated in increased adhesion of the cells toICAM-1 under flow. We conclude that GOLPH3 regulates core 2sLex-mediated cell adhesion to selectins and ICAM-1 by control-ling the Golgi localization of C2GnT1.

(103) Use of Siglec-F-Fc and a Novel IgY AntibodyRecognizing 6’-sulfated-sialyl Lewis X to Identify Endogenous

Lung Ligands for Siglec-FTakumi Kiwamoto1, Ho Jeong Na1, Mary Brummet1, M. G. Finn2,David Smith3, Vu Hong2, Zinaida Polonskaya2, Nicolai V. Bovin4,

Sherry Hudson1, Bruce Bochner11The Johns Hopkins University School of Medicine, Baltimore,MD; 2The Scripps Research Institute, La Jolla, CA; 3EmoryUniversity School of Medicine, Atlanta, GA; 4Institute ofBioorganic Chemistry, Russian Academy, Moscow, Russian

Federation

Background: Siglec-F, an inhibitory receptor on mouse eosino-phils, is thought to bind –a2,3-linked sialylated glycoproteinsexpressed by airway epithelium, but this natural ligand is incom-pletely characterized. Objectives: We hypothesize that mouseairway cells make Siglec-F ligands that are 6’-su-sLeX - contain-ing structures. We also hypothesize that primary airway epithelialcell cultures will express sialidase-sensitive cytokine-inducibleSiglec-F ligands. Methods: The CFG glycan microarray (version4.2) was used to identify glycans that bind to Siglec-F.Distribution of Siglec-F ligands was studied in mouse lung viaimmunohistochemistry. To detect 6’-su-sLeX, a novel polyclonalIgY antibody was generated. To characterize the Siglec-F ligand,primary mouse tracheal epithelial cells (mTEC) were isolated andcultured from C57BL6 mice. In some experiments, cells werestimulated with either IL-4 or IL-13 (50ng/ml for 4 or 24 hours).

Expression of ligands by mTEC was also studied via immunocyto-chemistry and Western blotting. Results: Among ≈ 450 glycanstested, Siglec-F-Ig fusion protein (Siglec-F/Fc) recognized only6’-su-sLeX and its non-fucosylated form. The IgY antibody had asimilar binding pattern in the array and blocked binding ofSiglec-F/Fc to these two glycans. In immunohistochemistry,Siglec-F/Fc bound airway epithelial cells and binding was blockedby the 6’-su-sLeX IgY antibody. A Siglec-F/Fc binding, sialidase-sensitive band (≈225 kDa) was detected in mTEC lysates byWestern blot. In immunocytochemical staining, there was a sizableintracellular pool of this ligand. IL-4 and IL-13 stimulationenhanced Siglec-F ligand levels. Another Siglec-F/Fc binding,sialidase-sensitive band (≈460 kDa) was also detected in mTECsupernatants by Western blot and was increased by IL-4.Conclusions: Siglec-F ligands containing 6’-su-sLeX and/or itsnon-fucosylated form are expressed by mouse lung epithelium.mTEC, especially after IL-4 treatment, make and secrete a highmolecular weight sialylated Siglec-F ligand and appear well suitedfor subsequent studies whose goal is to perform full structuralcharacterization of this Siglec-F ligand.

(104) Distinct Flow Cytometric Lectin-Binding Profiles ofUrinary Exosomes and Purified Tamm-Horsfall Protein

Susan Gallogly1, Anja Krüger1, Shirley Hanley1, Jared Gerlach1,Marie Hogan2, Christopher Ward2, Lokesh Joshi1,

Matthew Griffin11National University of Ireland Galway, Galway, Ireland;2Department of Medicine, Mayo Clinic, Rochester, MN

Background/Aims: Exosome-like vesicles (ELV) from urine arerich in proteins originating from kidney cells. Isolation and analy-sis of urinary ELVs is of high interest for the study of kidneydisease pathophysiology and the development of diagnostic andprognostic assays. The glycosylation characteristics of ELV pro-teins are of particular interest but have been little studied and maybe difficult to distinguish from the glycosylation pattern ofco-purified Tamm Horsfall protein (THP). In this study, we aimedto develop a lectin-based flow cytometry (FC) assay to analyzeand compare the glycosylation profiles of urinary ELVs and puri-fied THP. Methods: ELVs were enriched from healthy urinesamples by concentration (CON) and ultracentrifugation (UC) andwere fluorescently labelled with PKH26 or DiI. THP was purifiedby repeated salt precipitation and centrifugation followed by dialy-sis. For FC, labelled ELVs or THP were adsorbed to 4 µm latexbeads, incubated with a panel of 22 biotinylated lectins followedby streptavidin-PECy7 then analyzed using a FACSCanto® cyt-ometer and FlowJo® software. Results for each lectin wereexpressed as fold-change of fluorescence compared to lectin-stained, non-ELV-coated beads. Results: Concentration-dependentadherence of CON- and UC-enriched ELVs and of AF647-labelledTHP to the beads was confirmed by FC detection of fluorescencein the relevant channels. ELV- and THP-coated beads each demon-strated quantifiable binding of multiple lectins by FC analysis withfold-changes in fluorescence varying from <1 to >2000. The fol-lowing lectin binding patterns were observed: No binding to ELVor THP (n = 2), ELV = THP (n = 5), ELV > THP (n = 10), ELV <


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THP (n = 5). Of interest, 5 lectins were identified which demon-strated high-level binding to ELVs with no or minimal binding toTHP. Conclusions: A bead-based FC assay allows for rapid, sensi-tive analysis of lectin-binding by proteins displayed on the surfaceof urine ELVs. Compared with purified THP, healthy urine ELVshave multiple distinct lectin affinities.

(105) Modular Labeling of Monoclonal Antibodies using ClickChemistry

Carolyn Demarco, Ruth Deveny, Robert Aggeler, Courtenay Hart,Tamara Nyberg, Brian Agnew

Life Technologies, Inc., Eugene, OR

Standard antibody labeling techniques can sometimes lead to adecrease or loss of binding affinity due to active site blockagewhen using amine or cysteine-reactive labeling compounds.Monoclonal antibodies are particularly susceptible to active-siteblockage as they contain only a single amino acid sequence.Additionally, standard labeling techniques are often labor-intensive, poorly reproducible, and require a significant amount ofhands-on expertise. Here we describe two metabolic techniques forthe labeling of monoclonal antibodies with clickable azide handlesusing a simple “mix and incubate” copper-free dibenzocyclooctyne(DIBO)-mediated click chemistry reaction. In the first method wefed mouse hybridomas with azide sugars that specifically incorpor-ate into the heavy chain region of the antibody. In the secondmethod, cultured cells were fed L-azidohomoalanine (AHA), amethionine analog that can incorporate into both heavy and lightchains. The azide-tagged monoclonal antibodies were purifiedfrom cell supernatants and conjugated to a variety ofAlexa-Fluor®-DIBO detection reagents. The functionality of thelabeled antibodies was assessed by Western Blot, fluorescenceimaging, or flow cytometry. We found metabolic labeling to be asimple and robust alternative to standard labeling techniques formonoclonal antibodies. Metabolic incorporation of azide sugarsyielded a degree of labeling (DOL) of 1-2, and metabolic incor-poration of AHA yielded DOLs of 3-5. Using several differentantibody expressing cell lines, both methods resulted in functionalazide-tagged antibodies that were clicked to a variety ofAlexa-Fluor®-DIBO dyes. The method provides a highly reprodu-cible, robust, and versatile workflow to metabolically label func-tional monoclonal antibodies with a clickable azide tag. Metaboliclabeling with azide sugars allows for the labeling of antibodies faraway from the antigen binding domain ensuring full antigenbinding activity.

(106) Study of Fluorinated SLeX – Selectin Binding by ITCand SPR

Gizem Akçay2, John Ramphal1, Phillip Calabretta1,Anh-Dai Nguyen1, Krishna Kumar2, Daryl Eggers1, Roger Terrill1,

Marc d’Alarcao11San Jose State University, San Jose, CA; 2Tufts University,

Medford, MA

Cell surfaces are decorated with complex glycans that carry out abroad range of biological functions including cell recognition, cellsignaling, and cell adhesion. Particular interest has been focused

on the glycan, Sialyl Lewis X (SLeX), which is involved in leuko-cyte rolling adhesion prior to extravasation, as a potential thera-peutic target for the prevention of inflammation and metastasis. Ithas been shown previously that unnaturally fluorinated sialic acidscan be incorporated into cell surface glycans via glycoengineeringand that this modification reduces cellular adhesion to E- andP-selectin-coated surfaces. Since the metabolic engineeringapproach likely leads to modification of all cell surface sialic acid-bearing glycans and may alter the cell in other ways, the mechan-ism of reduced cellular adhesion is difficult to ascertain. To decon-volute this effect, we have undertaken a study whereby structurallyhomogeneous fluorinated SLeX molecules are being prepared byorganic synthesis and their binding to purified E and P-selectin isbeing measured by SPR and ITC. Our progress on these studies aswell as on model studies with a fucosylated disaccharide and afucose-binding lectin will be presented.

(107) Immunological Functions of Cholesteryl a-glucosidesin Helicobacter pylori-associated InflammationYuki Ito1,5, Jose Luis Vela2, Fumiko Matsumura1,

Hitomi Hoshino3,6, Heeseob Lee1,7, Motohiro Kobayashi3,Xingfeng Bao1, Thomas Borén4, Rongsheng Jin1, PeterH. Seeberger1, Jun Nakayama3, Mitchell Kronenberg2,

Minoru Fukuda11Sanford-Burnham Med Res Inst, La Jolla, CA; 2La Jolla Inst forAllergy & Immunol, La Jolla, CA; 3Shinshu Univ Grad Sch ofMed, Matsumoto, Japan; 4Umeå Univ, Umeå, Sweden; 5Sci ResCtr, Yamaguchi Univ, Ube, Japan; 6Nat’l Ctr of Geriatrics and

Gerontol, Obu, Japan; 7Pusan Nat’l Univ, Busan, Korea

Helicobacter pylori (H. pylori) uniquely contains cholesterylα-glucosides (αCGs) as the major components of its cell wall.Cholesteryl-α-glucoside (αCGL) is synthesized by cholesterolα-glycosyltransferase (αCgT), and αCGL is a precursor forcholesteryl-acyl-α-glucoside (αCAG) and cholesteryl-phosphatidyl-a-glucoside (αCPG). We reported that mucin-type core 2-branchedO-glycans expressing α1,4-N-acetylglucosamine residues suppressbacterial growth by inhibition of αCGs synthesis (Kawakubo et alScience 2004, Lee et al BBRC 2006).

To determine roles of αCGs, we analyzed amino acid substi-tutions in αCgT of H. pylori from 24 Japanese clinical isolates.Recombinant αCgT showed diverse activity from 15 to 174% com-pared to the αCgT in the wild-type H. pylori 26695. Analysis of themutated αCgT activity and histological features revealed that theenzyme activity is associated with the progression of gastricatrophy. We generated αCgT deficient H. pylori (αCgTΔ), andαCgThigh and αCgTlow, which expressed higher and lower αCgTactivity than wild-type, respectively. αCgTΔ exhibited significantlydelayed growth and morphologic abnormality caused by loss of allof αCGs. These results suggested that αCGs are essential for H.pylori growth.

We investigated further functions of αCGs in immune response.Phagocytosis and activation of dendritic cell were observed at thesame degrees in the wild-type H. pylori and all mutants, regardlessof αCgT activity. However, response by invariant natural killer T(iNKT) cells was positively correlated with the activity of αCgT. In


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synthetic compounds of αCGs, αCPG most activated iNKT cells.Moreover, monoacyl-αCPG was the most potent antigen in vivoassay. More H. pylori strain was retrieved from the stomach ofiNKT deficient (Jα18−/-) mice than wild-type mice. Furthermore,expression level of cytokines in αCgThigh infected Jα18−/- mice wassignificantly decreased than αCgThigh infected wild-type mice. Ourfindings suggested that recognition of αCGs by iNKT cells arenecessary for bacterial clearance and stimulation immune cellresponse in various cell lineages in Th1/Th2/Th17 cells. Furtheranalysis is currently in progress. Supported by NIH grantsPO1CA71432, CA33000 and Toyobo Biotechnology FoundationFellowship.

(108) Small Molecule Recognition of Tumor-AssociatedCarbohydrate Antigens sLex/a

Jean-Philippe Pitteloud, Maria C. Rodriguez, Predrag CudicTorrey Pines Institute, Port St. Lucie, FL

The structure of carbohydrates, which decorate the cell surface ofhigher organisms change with the onset of cancer and inflam-mation, representing, therefore, highly promising targets for newand more-selective anticancer drugs and for early diagnosis.Arguably, the most clinically important glycosylation changes

involved in tumor progression and metastasis is overexpression ofsialylated Lewis oligosaccharide antigens, sLex and sLea. Thesecancer-associated cell surface changes are recognized by the selec-tin family of carbohydrate binding proteins. However, due to itscomplexity and weak binding affinities, sLex/a recognition by theirnatural receptors is still poorly understood. On the other hand,studies on synthetic carbohydrate receptors could make significantcontributions to a better understanding of this process and lead tothe development of new chemotherapeutics and chemosensors. Wehave designed and synthesized novel 1,8-naphthyridine macrocyc-lic compounds that bind sialic acid and sLex/a with affinities com-parable to those reported for lectin/monosaccharide complexes.These small molecules can inhibit anti-sLex mAb binding to sLex

antigen in a competitive ELISA assay and inhibit adhesion of thesLex expressing Hep G2 cells to E-selectin. In addition, some ofthese compounds were able to recognize structural nuances of gly-cosylation in vivo as shown by binding to leukocytes isolated fromthe mouse model of breast cancer but not from the healthy mouse.Obtained experimental data have demonstrated that1,8-naphthyridine based macrocyclic compounds can be valuablemolecules for probing the structure and function of carbohydrateantigens.

(109) How the Autophagy Receptor NDP52 Defendsthe Cellular Cytosol against Bacterial Invasion

Natalia Von Muhlinen, Teresa Thurston, Natalia von Muhlinen,Michal Wandel, Masato Akutsu, Agnes Ágnes Foeglein,

David Komander, Felix RandowMRC Laboratory of Molecular Biology, Cambridge,

United Kingdom

Autophagy is a degradation mechanism that engulfs cytosolicmaterial into a double membrane called the autophagosome.ATG8, an ubiquitin-like protein in yeast, and its mammalian

orthologues become covalently attached to the double membraneand are widely used as autophagy markers. Under starvation con-ditions, autophagy engulfs cytosolic material non-specifically, butautophagy is also able to selectively target misfolded proteins,organelles, and intracellular pathogens. However, how autophagyspecifically targets invading pathogens remains unclear.

Recently, we reported that NDP52 is an autophagy receptor thatdefends the cytosol against invading bacteria. In my poster, I willaddress how carbohydrate recognition contributes to antibacterialdefence and how NDP52 achieves its role as an autophagy recep-tor. The interaction between NDP52 and the mammalianATG8-like proteins will be discussed, and the question of whetherthe human ATG8 orthologues perform distinct biological functionswill be addressed.

(110) The Fine-Specificity of Mannose-Binding andGalactose-Binding Lectins Revealed using Outlier-Motif

Analysis of Glycan Array DataKevin Maupin, Daniel Liden, Brian Haab

Van Andel Research Institute, Grand Rapids, MI

Glycan-binding proteins are commonly used as analytical reagentsto detect the levels of specific glycan structures in biologicalsamples. A detailed knowledge of the specificities of glycan-binding proteins is required for properly interpreting their bindingdata. A powerful technology for characterizing glycan-bindingspecificity is the glycan array. However, the interpretation ofglycan array data can be difficult due to the complex fine-specificities of certain glycan-binding proteins. We developed asystematic approach, called Outlier Motif Analysis, for extractingfine-specificity information from glycan array data, and we appliedthe method to the study of four commonly-used lectins: twomannose binders (concanavalin A and lens culinaris) and twogalactose binders (bauhinea purpurea and peanut agglutinin). Thestudy confirmed the known, primary specificity of each lectin andalso revealed new insights into their binding preferences. LCA’smain specificity may be non-terminal, alpha-linked mannose witha single linkage at its 2’ carbon, which is more restricted thanprevious definitions. We found broader specificity for BPL thanpreviously reported, showing that BPL can bind terminalN-acetylgalactosamine and penultimate beta-linked galactoseunder certain limitations. PNA may bind terminal Galα1,3Gal, aglycolipid motif, in addition to terminal Galα1,3GalNAc, acommon O-linked glycoprotein motif. These results could be usedto more accurately interpret data obtained using these well-studiedlectins. Furthermore, this study demonstrates a systematic andgeneral approach for extracting fine-specificity information fromglycan array data.

(111) When Two Biomarkers of Thyroid Cancer Meet:Interaction between galectin-3 and Thyroglobulin

Tarun Kanti Dam1, Robert K. Brown1, Molly Wiltzius1,Melanie Jokinen1, Sabine Andre2, Herbert Kaltner2,

Hans-Joachim Gabius21Michigan Technological University, Houghton, MI;2Ludwig-Maximilians University, Munich, Germany


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Galectin-3 and thyroglobulin (Tg), expressed in thyroid tissues,serve as reliable biomarkers of thyroid cancer. TheN-acetyllactosamine (lacNAc) binding endogenous lectingalectin-3 is considered as a biomarker because of its tumor-specific expression in thyroid tissues. Tg is a biomarker of follicu-lar and papillary thyroid cancer. Human Tg contains 10 highmannose, 18 complex and 4 hybrid glycan chains. The glycan pro-files of Tg change upon malignant transformation. For example,Tg of metastatic papillary carcinoma contains repeating lacNAcunits in linear unbranched chains or highly branched chains.Presence of high number of lacNAc moieties on Tg, especially intransformed thyroid tissues, and concurrent increased expressionof galectin-3 in the same vicinity could facilitate binding inter-action between these two biomarkers in vivo. Such interaction maypotentially have profound functional implication in the pathologyof thyroid gland. Although these two biomarkers have beenstudied intensively, very little is known about their mutual inter-action. In the present study, we found that human galectin-3 bindsto Tg with nanomolar affinity (Ka = 1.5 x 10−7 M−1). Each Tgmolecule binds to 25-30 galectin-3 molecules. The truncatedversion of galectin-3 also interacts with Tg (Ka = 2.7 x 10−6 M−1).We have used bovine and porcine Tg as they possess the samenumber of complex-type N-glycans as those of human Tg.Binding thermodynamics of galectin-3 for Tg from both sourceswere comparable. Porcine Tg contains fucosylated and sulphatedcomplex-type N-glycans similar to those found in papillarythyroid carcinomas. Data obtained with porcine Tg indicate thatgalectin-3 can strongly interact with Tg in papillary carcinomas.Elongation of the lacNAc chains of the transformed Tg, asreported in certain thyroid cancers, is expected to further increasetheir affinity for galectin-3 in vivo. The significance of such high-affinity interactions between galectin-3 and Tg is discussed.

(112) Mechanistic Basis of Polysaccharide Recognitionby Lectins

Tarun Kanti Dam, Molly Wiltzius, Robert K. BrownMichigan Technological University, Houghton, MI

Mannan and galactomannan are highly immunogenic componentsof fungal cell wall. Because of their immunogenic properties,these polysaccharides are considered as adjuvants. Binding ofthese polysaccharide antigens to host receptors such as toll-likereceptor and C-type lectins, elicits host response. Mannan is recog-nized by a variety of host receptors including mannose receptor,DC-SIGN, Dectin-2, TLR4, mannose binding lectin, SP-A, SP-D,Pentraxin and anti-mannan antibodies. However, the mechanisticbasis of this recognition process is not properly understood. Ingeneral, a physical model for polysaccharide recognition by lectinsis not readily available. Such a model is essential to understandthe polysaccharide-initiated host response and the adjuvant proper-ties of polysaccharides. In the present study, we have investigatedthe mechanism of lectin binding by mannan and galactomannan.Interactions of these two polysaccharides with the mannosespecific lectin concanavalin A (ConA) have been studied byhemagglutination inhibition assay and isothermal titration calori-metry. The results show that mannan binds to ConA with

nanomolar affinity. The binding stoichiometry is high. Over 40ConA molecules are complexed with one molecule of mannan.ConA-mannan interaction shows concentration and time dependentprecipitation. The association constant of galactomannan for ConAis less than that of mannan. The thermodynamic binding datasuggest that ConA binds to these polysaccharides through the“bind and jump” mechanism as previously recorded in lectin-mucin interactions. The “bind and jump” mechanism results inhigh affinity lectin binding by linear biopolymers such as mucinsand polysaccharides.

(113) Evidence for Direct Cross-Talk between Two EssentialNutrient-Sensing Enzymes: O-GlcNAc Transferase (OGT)

and AMP-activated Kinase (AMPK)John Bullen1, Jeremy Balsbaugh2, Dietbert Neumann3,Grahame Hardie4, Jeffrey Shabanowitz2, Donald Hunt2,

Gerald Hart11Dept. of Biol. Chem., Johns Hopkins Hospital, Baltimore, MD;2Dept. of Chem., Univ. of Virginia, Charlottesville, VA; 3Dept. of

Molecular Genetics, Maastricht Univ., Maastricht, TheNetherlands; 4College of Life Sciences, Univ. of Dundee, Dundee,

Scotland

O-linked b-N-acetylglucosamine (O-GlcNAc) is a highly dynamicSer/Thr specific post-translational modification of cytoplasmic andnuclear proteins, and is regulated by the concerted actions of twoenzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA).O-GlcNAcylation is a nutrient-sensitive process involved in theregulation of various signaling pathways mediating transcription,cellular metabolism, growth and proliferation. Activation ofAMP-activated kinase (AMPK), a vital monitor of cellular energyhomeostasis, is also a nutrient-sensitive process that regulates cel-lular metabolism, growth and proliferation. AMPK and OGT sharemany downstream targets with the net effect to prolong cell viabi-lity under metabolic stress, suggesting significant overlap in bothupstream and downstream regulation of AMPK and OGT signalingpathways. Despite this, no studies have investigated whetherAMPK and OGT are capable of directly regulating each other. Wedemonstrate that acute inhibition of O-GlcNAc cycling disruptspharmacological and physiological activation of AMPK and thatboth alpha (catalytic) and all 3 gamma (AMP/ADP-sensing regu-latory) subunits of AMPK are O-GlcNAcylated. Furthermore,O-GlcNAcylation of the gamma1 subunit directly correlates withboth the time and extent of AMPK activation. To further assessthe possibility of cross-talk between OGT and AMPK, we demon-strate that acute pharmacological activation of AMPK increasesnuclear localization of OGT and alters OGT’s substrate selectivity.AMPK also phosphorylates OGT on Thr 444 in vitro; a residuethat lies in close proximity to regions that may facilitate OGT’snuclear localization and substrate selectivity. Collectively, theseresults demonstrate significant cross-talk between two essentialnutrient sensing processes, possibly via direct signaling betweenAMPK and OGT. (Supported by NIH R01 DK61671, R01CA42486 and R24 DK084949. Dr. Hart receives a share ofroyalty received by the university on sales of the CTD 110.6 anti-body. Terms of this arrangement are managed by JHU).


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(114) Human Cosmc and T-synthase are TranscriptionallyRegulated by SP1 Transcription Factor

Rongjuan Mi1, Yingchun Wang1, Xiaokun Ding1, Irma Van Die2,Arlene B. Chapman1, Richard D. Cummings1, Tongzhong Ju1

1Emory University School of Medicine, Atlanta, GA; 2VUUniversity Medical Center, Amsterdam, Netherlands

Mucin type O-glycans, such as those based on the core 1 structure(Galβ1-3GalNAcα1-R), play important roles in many biologicalprocesses, such as cell adhesion, angiogenesis, immunity. TheT-synthase (core 1 β3galactosyltransferase) is responsible forsynthesizing core 1 O-glycan disaccharide (also known as Tantigen), which is the precursor for most complex O-glycans.Interestingly, a specific molecular chaperone Cosmc is required forbiosynthesis of the active T-synthase. Recent studies have shownthat both Cosmc and T-synthase are transcriptionally suppressed inthe B cells from the patients with IgA nephropathy (IgAN). Yet,how these two genes are transcriptionally regulated is not known.Here we characterized the critical cis-elements required for Cosmcand T-synthase transcription. The upstream regions of humanCosmc and T-synthase lack a conventional TATA box but containGC rich sequences or CpG islands: two for Cosmc (cCpG-I andcCpG-II), and one for T-synthase (tCpG). By luciferase reporterassay, we have identified 150 bp DNA fragment in the cCpG-II forCosmc which contains two SP1 transcription factor bindingsequences close to the transcription starting site is the promoter.Similarly, a 170 bp DNA fragment in the tCpG which also con-tains two SP1 binding sequences is the promoter for T-synthase.Site direct mutagenesis demonstrates that both SP1 sequences forCosmc and T-synthase are essential to their promoter activity.Furthermore, CHIP analyses confirm that the putative SP1sequence in the promoter associates with the SP1 transcriptionfactor for both Cosmc and T-synthase. These results demonstratethat human Cosmc and T-synthase are transcriptionally active at abasal level by SP1, which explains the ubiquitous expression ofthese two functionally related genes. Understanding the mechan-ism for how these two genes are transcriptionally regulated willaid us both in understanding these genes’ function and in elucidat-ing their roles in human diseases, such as IgAN and cancer.

(115) Molecular Mechanism of Cosmc Function in theBiosynthesis of Active T-synthase

Rajindra Aryal, Tongzhong Ju, Richard CummingsEmory University School of Medicine, Atlanta, GA

Cosmc (Core 1 β3-galactosyltransferase Specific MolecularChaperone) is a chaperone in the endoplasmic reticulum (ER) thatfacilitates folding of T-synthase (Core 1 β3-galactosyltransferase)both in vivo and in vitro. The T-synthase is an important branchpoint enzyme in the Golgi apparatus that adds galactose (Gal)from UDP-Gal to the Tn antigen (Galβ1-3GalNAcα1-Ser/Thr) toform Core 1 O-glycans, which can be further modified to give riseto extended O-glycans. In the absence of Cosmc in the ER,T-synthase aggregates there in inactive oligomeric complexes,whereas purified, active, soluble T-synthase is a noncovalenthomodimer. We explored mechanistically how Cosmc helps to

facilitate the folding of the T-synthase. We found that Cosmcdirectly interacts with misfolded T-synthase, but not with nativeT-synthase or other denatured proteins, to form a relatively stable,noncovalent complex facilitating the folding of T-synthase andacquisition of activity. The Cosmc chaperone cycle of binding andrelease is regulated by misfolded T-synthase itself, which bindscompetitively to Cosmc to displace refolded T-synthase. Thiscycle is not regulated by potential ER chaperone regulatingfactors, which include ATP, calcium, redox, and pH. These find-ings have significant impact on our understanding of mucin-typeO-glycan biosynthesis and diseases associated with aberrantexpression of these glycans

(116) CD68’s Glycosylation is Modified duringRANKL-Induced Osteoclastogenesis

Jason Ashley, Xu FengUniversity of Alabama at Birmingham, Birmingham, AL

CD68 is a heavily glycosylated member of the lysosome-associatedmembrane protein (LAMP) family. Unlike most LAMPs, which arecharacterized primarily by N-linked glycans, CD68 is mostlyO-glycosylated in a manner similar to that of mucins. We have foundthat CD68 is expressed by both bone marrow macrophages (BMMs)and the large, multinuclear, bone-resorbing osteoclasts into whichthey differentiate and that expression of CD68 is essential to thenormal morphology and efficient function of the osteoclast. In ourinitial studies, we found that the electrophoretic migration rate ofCD68 in protein extracts from osteoclasts was accelerated comparedto CD68 from bone marrow macrophages. Our understanding ofboth osteoclastogenesis and previous studies of CD68 led us tohypothesize that CD68’s pattern of glycosylation is altered duringosteoclast formation, and this alteration is dependent upon the osteo-clast differentiation factor Receptor Activator of Nuclear Factor κBLigand (RANKL). To test the first part of our hypothesis, we immu-noprecipitated CD68 from both BMMs and osteoclasts and subjectedthe precipitated protein to blotting with a panel of lectins. We found areduction in the reactivity of Galanthus nivalis agglutinin and com-plete elimination of peanut agglutinin reactivity to the osteoclast-derived CD68. There was a slight increase in Maackia amurensisagglutinin binding, though the change was subtle enough to demandmore sensitive confirmation. These findings confirm that osteoclastsand BMMs express different glycoforms of CD68. Further, usingBMMs that express mutant forms of the RANKL receptor (RANK)we found that the altered migration of CD68 and, likely, its alteredglycosylation are dependent upon multiple intracellular RANK sig-naling motifs. Future work will determine the nature and configur-ation of CD68’s osteoclast glycoform and the specific signalingpathways and enzymes responsible for its formation. With this infor-mation, we will be equipped to explore the functional significance ofCD68’s RANKL-induced alternative glycosylation.

(117) Targeted Knockouts of Enzymes of O-GlcNAc Cyclingand Epigenetic Regulation

John A. Hanover, Peng Wang, Chithra Keembiyehetty,Salil Ghosh, Michelle Bond, Michael Krause, Dona Love

NIDDK/NIH, Bethesda, MD


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The nutrient-sensing hexosamine signaling pathway terminating inO-GlcNAc cycling is emerging as a key epigenetic regulator ofgene expression in mammals. Mounting evidence suggests thatO-GlcNAc cycling sits atop a robust regulatory network maintain-ing higher-order chromatin structure, epigenetic memory andimprinting. We have focused on transcriptional impact of interfer-ing with O-GlcNAc cycling in the worm, fly and mouse. InC. elegans, O-GlcNAc marks the promoters of over 800 develop-mental, metabolic, and stress-related genes. These O-GlcNAcmarked genes show a 5’ bias in the distribution of RNAPolymerase II (Pol II) suggestive of enhanced promoter-proximalpausing. In response to starvation or feeding, levels of O-GlcNAcat promoters remain nearly constant due to dynamic cyclingmediated by OGT and the O-GlcNAcase. However, in viablemutants lacking either of these enzymes of O-GlcNAc metab-olism, the nutrient-responsive O-GlcNAcylation of promoters isdramatically altered. Blocked O-GlcNAc cycling leads to a strikingnutrient-dependent accumulation of O-GlcNAc on RNA Pol II.O-GlcNAc cycling mutants also show an exaggerated,nutrient-responsive redistribution of promoter-proximal RNA PolII isoforms and extensive transcriptional deregulation. Thesestudies in the nematode have been confirmed in the fly and themouse. Our findings suggest a complex interplay between theO-GlcNAc modification at promoters and the kinase-dependent‘CTD-code’ regulating RNA Pol II dynamics. In the mouse, weobserve deregulation of Hox gene expression and associated skel-etal and hematopoietic stem cell abnormalities. Therefore,O-GlcNAc cycling represents a concrete mechanism by whichnutritional information may be transmitted across generations inthe intrauterine environment. The hexosamine signaling pathwaymay, therefore, be a key contributor to the metabolic deregulationresulting from prenatal exposure to famine, or the ‘vicious cycle’observed in children of mothers with type-2 diabetes and meta-bolic disease.

(118) Ectopic Expression of Core 3 Synthase in HumanPancreatic Cancer Cells Suppresses Tumor Formation

and MetastasisPrakash Radhakrishnan, Paul M. Grandgenet, Ashley M. Mohr,

Stephanie K. Bunt, Fang Yu, Michael A. HollingsworthUniversity of Nebraska Medical Center, Omaha, NE

Core 3 derived glycans, a major type of O-glycans expressed innormal epithelial cells of the gastrointestinal tract, are downregu-lated during malignancy. To investigate functions of core 3glycans, we ectopically expressed β3-N-acetylglucosaminyltrans-ferase-6 (core 3 synthase) in human pancreatic cancer cells(Capan-2 and FG). Pancreatic cancer cells expressing core 3synthase showed reduced in vitro cell proliferation, migration andinvasion compared with vector control cells. We also found thatcore 3 O-glycans altered expression of α2β1 integrin, decreasedactivation of focal adhesion kinase, and altered lamellipodia for-mation. Orthotopic injections of FG cells expressing core 3synthase into pancreases of nude mice produced significantlysmaller tumors and decreased metastasis to the surrounding tissuescompared to vector control FG cells. Additionally, we observed

that the addition of GlcNAc residue by core 3 synthase led toextension of Tn epitope on MUC1 and the down regulation ofREG1α. These findings indicate that expression of core 3 derivedO-glycans in pancreatic cancer cells suppresses tumor formationand metastasis through modulation of mucins and extracellularmatrix proteins glycosylation patterns, as well as down regulationof growth factors and growth factor receptors.Keywords: Core 3 synthase, β3GnT-6, pancreatic cancer, Tnepitope and MUC1

(119) GAG-specific Endoglycosidase Assay using 35S-LabeledProteoglycans

Cheryl Ethen, Miranda Machacek, Brittany Prather,Zhengliang Wu

R&D Systems Inc, Minneapolis, MN

Glycosaminoglycans (GAGs) are linear amino-polysaccharidesfound in the extracellular matrix and on the cell membrane. Theyinclude heparan sulfate, heparin, chondroitin sulfate, dermatansulfate, keratan sulfate, and hyaluronan. The majority of GAGsexist as components of functional proteoglycans. GAGs play rolesin numerous cellular events, including cell growth, migration andsignaling through interaction with various growth factors, cyto-kines and other extracellular matrix proteins. Regulation of GAGsynthesis and degradation is essential to these related cellularevents. In mammals, GAG degradation is accomplished byGAG-specific endoglycosidases. For example, heparan sulfate isdegraded by heparanase while hyaluronan is degraded by SPAM1and hyaluronidase 1. These enzymes are key to furthering ourunderstanding of GAG degradation and subsequent cellular events.However, unlike bacterial GAG-specific lyases, the products ofthese enzymes do not have UV absorption, posing a great chal-lenge to the study of these enzymes.

Here, we describe a novel enzymatic assay for mammalianGAG endoglycosidases using 35S-labeled recombinant proteogly-cans. Proteoglycans that contain GAG substrates are first radio-labeled with 35S using recombinant sulfotransferases, and thelabeled substrates are subsequently treated with GAG endoglycosi-dases. The reactions are separated using SDS-gel electrophoresis.The gels are dried and radioimages are obtained. This methodallows us to visually monitor the progress of an enzymatic reac-tion. Given the high sensitivity of radioisotope labeling, nanogramlevels of GAG degradation can be detected. As examples, humanheparanase is assayed using recombinant syndecan-4 as the sub-strate, bovine SPAM1 and human hyaluronidase 4 are assayedusing recombinant serglycin as the substrate.

(120) Phosphoproteomic Analysis of Drosophila EmbryosDeficient in Neural-Specific Glycosylation

Varshika Kotu, Peng Zhao, Lance Wells, Michael TiemeyerComplex Carbohydrate Research Center, University o, Athens, GA

Appropriate glycan expression is essential for development andnormal tissue function. A family of structurally related N-linkedglycans known as HRP epitopes are preferentially expressed inDrosophila neural tissue, providing a platform for exploring mech-anisms that control tissue specific glycosylation. We previously


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generated and characterized a Drosophila mutant called sugar-freefrosting (sff ) which affects HRP-epitope expression in the embryo-nic nervous system. The sff mutation mapped to the Drosophilahomologue of a serine/threonine kinase known as SAD-1 in otherinvertebrate and vertebrate species. In mid-stage Drosophilaembryos, confocal analysis demonstrated that the sff mutationalters Golgi compartmental distributions such that glycoproteinglycosylation is shifted in favor of greater glycan complexity anddecreased HRP-epitope expression. In order to further characterizethe molecular mechanism underlying altered neural glycanexpression, we have undertaken differential phosphoprotemicanalysis of wild-type and sff mutant embryos. Our results haveidentified a pool of phosphoproteins that fall into one of four cat-egories: 1) candidate Sff substrates relevant for neural glycosyla-tion, 2) candidate Sff substrates unrelated to glycosylation, 3)substrates of other kinases that are relevant for neural glycosylationand whose activities are modulated by Sff, or 4) substrates ofother kinases unrelated to glycosylation but whose activities aremodulated by Sff. These categories can be distinguished by inves-tigating genetic interactions between Sff and mutants in each ofthe identified phosphoproteins and by comparing mapped phos-phorylation sites to specific kinase consensus sequences. Ourongoing efforts are directed towards understanding the functionalsignificance of the identified phosphoproteins in the context ofneural specific glycosylation.

(121) The Skp1 Prolyl 4-hydroxylase of DictyosteliumContributes Glycosylation-Independent and -Dependent

Effects on O2-dependent Development without Affecting Skp1Turnover

Dongmei Zhang, Hanke van der Wel, Jennifer M. Johnson,Christopher M. West

University of Oklahoma Health Sciences Center,Oklahoma City, OK

A primary mechanism of O2-sensing in animals involves theaction of HIFα-type prolyl 4-hydroxylases (PHDs) on the tran-scriptional factor subunit HIFα, leading to its poly-ubiquitinationby the E3VBCUb-ligase and degradation in the 26S-proteasome.Previously thought to be restricted to animals, we have found thata homolog of HIFα-type PHDs also functions in O2-sensing,reduces the O2-requirement for development in the social amoebaDictyostelium. Dictyostelium lacks HIFα and P4H1 modifies adifferent target, Skp1, an adaptor of the SCF-class of E3 ubiquitin(Ub) ligases related to the E3VBCUb-ligase that targets animalHIFα. Normally, the HO-Skp1 product of the P4H1 reaction iscapped by a GlcNAc sugar that is then extended by a cytoplasmicglycosylation pathway that assembles a pentasaccharide at Pro143.To analyze the effect of Skp1 hydroxylation in the absence of gly-cosylation, we disrupted gnt1 predicted to encode the Skp1α-GlcNAcT. Biochemical studies confirmed absence of glycosyla-tion and metabolic labeling studies showed that Skp1 accumulatedat a normal level with a normal half-life, indicating a distinct func-tion for hydroxylation of Skp1 compared to HIFα. TheO2-requirement was partially but not completely restored tonormal, revealing both glycosylation-independent and -dependent

roles for hydroxylation. Skp1 was the only substrate detectedwhen Gnt1 was restored to mutant extracts, consistent with otherevidence for the importance of Skp1 in O2-signaling. ThoughSkp1 stability is not affected by hydroxylation, the stability ofother proteins may be affected via influence on E3SCFUb-ligaseactivity.

(122) Dependence of G-Protein Coupled Receptor AgonistsTransactivation of Trk Tyrosine Kinase and TOLL-Like

Receptors on Neuraminidase-1 and MatrixMetalloproteinase-9 Cross-Talk

Samar Abdulkhalek1, Schammim Ray Amith1, Preethi Jayanth2,Merry Guo1, Myron Szewczuk1

1Queen’s University, Kingston, Canada; 2McMaster University,Hamilton, Canada

G-protein coupled receptor (GPCR) Ga proteins have been shown toplay a role in neurotrophin Trk tyrosine kinase receptor andTOLL-like receptor-mediated cellular functions, but the mechanism(s) behind their interactions with these receptors has not been fullyelucidated. Recently, we reported an unprecedented membrane siali-dase mechanism initiated by nerve growth factor (NGF) binding toTrkA to potentiate GPCR-signaling via membrane Gαi subunit pro-teins and matrix metalloproteinase-9 (MMP-9) activation to induceNeu1 activation in live primary neurons and TrkA- andTrkB-expressing cell lines. The role of GPCR agonists in the acti-vation of Neu1 and MMP-9 cross-talk to transactivate Trk andTOLL-like receptors was investigated. The data indicate that GPCRagonists bombesin, bradykinin, lysophosphatidic acid (LPA), choles-terol, angiotensin-1 and -2, but not thrombin, induce sialidase activityin live BMC-2 macrophage cells and primary bone marrow (BM)macrophage cells from WT mice but not with Neu1-deficientprimary macrophage cells from Neu1-CathepsinA knockdown mice.Oseltamivir phosphate, pertussis toxin, galardin, piperazine, MMP-9inhibitor, anti-MMP-9 and anti-Neu1 antibodies applied to liveTrkA-PC12 cells inhibited sialidase activity and TrkA phosphoryl-ation associated with bombesin treated cells. Using immunocyto-chemistry and NFκB-dependent secretory alkaline phosphatase(SEAP) analyses, bombesin induced NFκB activation in BMC-2 andRAW-blue macrophage cells. Oseltamivir phosphate, galardin, piper-azine and anti-MMP-9 antibody inhibited NFκB activation inducedby bombesin-stimulated macrophage cells. Silencing MMP-9 usingsiRNA transduction into RAW-blue macrophage cells significantlyinhibited bombesin-induced Neu1 activity and NFκB activation.These findings uncover a molecular organizational GPCR signalingplatform to potentiate Neu1 and MMP-9 cross-talk on the cellsurface that is essential for the transactivation of Trk tyrosine kinaseand TOLL-like receptors and subsequent cellular signaling.

(123) Pathway to Diet– and Obesity–Associated Diabetesthrough Attenuation of Pancreatic Beta Cell Glycosylation and

Glucose TransportKazuaki Ohtsubo1, Mark Chen2, Jerrold Olefsky2, Jamey Marth31RIKEN ASI Disease Glycomics, Ibaraki, Japan; 2University ofCalifornia, San Diego, San Diego, CA, USA; 3Sanford-BurnhamMedical Research Institute/UCSB, Santa Barbara, CA, USA


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A connection between diet, obesity, and diabetes exists amongmultiple species and is the basis of an escalating human healthproblem. The factors responsible provoke insulin resistance andpancreatic β cell dysfunction but remain to be fully identified. Wereport that a high fat diet leading to obesity and diabetes recapitu-lates the effect of free fatty acid stimulation of normal islet cellcultures, resulting in nuclear exclusion and reduced expression ofbeta cell FOXA2 and HNF1α transcription factors. The resultingdeficit of GnT–IVa glycosyltransferase expression in β cellsinduced metabolic disease signs including hyperglycemia,impaired glucose tolerance, hyperinsulinemia, hepatic steatosis,and diminished insulin action in muscle and adipose tissues.Protection from disease was achieved by enforced β cell-specificGnT–IVa protein glycosylation and involved the regulation of βcell glucose transporters with the preservation of glucose transport.This pathogenic mechanism was observed in human islet cellsfrom normal and Type 2 diabetic donors, and illuminates apathway to disease implicated in the diet- and obesity-associatedcomponent of Type 2 diabetes mellitus. (Ohtsubo et al., 2011,Nature Medicine, in press)

(124) Identification of Specific Sequences in thePolysialyltransferase, ST8Sia IV (PST), that are Required

for Substrate RecognitionJoseph Zapater, Deirdre Foley, Karen Colley

University of Illinois at Chicago College of Medic, Chicago, IL

The polysialylation of the neural cell adhesion molecule (NCAM)by the alpha-2, 8-polysialyltransferases (polySTs), ST8Sia IV(PST) and ST8Sia II (STX), modulates cell-cell adhesion, isrequired for proper brain development, and is involved in synapticplasticity, neuronal regeneration, memory formation, and tumorinvasiveness. Our evidence suggests that an initial recognition stepmediated by polyST-NCAM protein-protein interactions is requiredfor efficient NCAM polysialylation. In support of this hypothesis,we found that specific sequences within the NCAM FN1 domainare required for NCAM binding and subsequent polysialylation ofN-glycans in the neighboring Ig5 domain. In this work, we ident-ified sequences in PST required for NCAM recognition. A full-length catalytically inactive PST (PST H331K) and a series oftruncated PST proteins consisting of the first 62 to 140 aminoacids were created for competition studies. All PST proteins weretransiently expressed in SW2 small cell lung carcinoma cells, con-taining endogenous PST and polysialylated NCAM. Our resultsshow that only SW2 cells expressing PST H331K, PST140 (mem-brane bound or soluble) or PST127 demonstrated an overalldecrease in polysialic acid expression when compared to untrans-fected cells, suggesting that these proteins compete with endogen-ous PST for NCAM recognition. We previously demonstrated thatreplacing R82 and R93 in PST with alanine decreased its ability topolysialylate NCAM without similarly affecting enzyme autopoly-sialylation. Here we show that a similar decrease in neuropilin-2polysialylation is observed when this protein is co-expressed withPST R82A/R93A. Interestingly, we found that creating the R82A/R93A mutation in PST H331K compromised this protein’s abilityto compete with PST for NCAM recognition. Taken together,

these results suggest that R82 and R93 in the PST polybasicregion play a role in polyST substrate recognition.

(125) Excessive Aggregation of Ganglioside GM2 CanTransmit Proliferation Signals via c-Src Kinase Directly

Nagako Kawashima1, Naoki Fujitani2, Daisuke Tsuji3, Kohji Itoh3,Yasuro Shinohara2, Ken-ichi Nakayama1

1National Institute of Advanced Industrial Science, Sapporo,Japan; 2Grad Sch of Adv Life Science, Hokkaido Univ., Sapporo,Japan; 3Grad Sch Pharm Sci., Tokushima Univ., Tokushima,

Japan

Sandhoff disease is a progressive neurodegenerative disordercaused by mutations in the HEXB gene which encodes theβ-subunit of N-acetyl-β-hexosaminidase A and B, resulting in theaccumulation of the ganglioside GM2.

Previously, we reported that remarkable faster growth rate ofastrocyte cells from Sandhoff disease model mice (ASD) whencompared with cells established and isolated from wild-type mice(AWT). This phenomenon was caused by c-Src and ERK acti-vation due to accumulated ganglioside GM2 in the lysosomes.These results may indicate that c-Src would be directly activatedby accumulated GM2, but specifics have not proved clearly.

This time, we analyzed : i) interaction between GM2 and intra-cellular c-Src by western blotting of GEM/raft fraction and immu-noprecipitaion with anti-GM2 antibody, ii) localizaion ofaccumulated GM2 and c-Src by immunofluorescein staining, iii)fatty acid length of accumulated GM2 in lysosome byMALDI-TOF MS. We confirmed that c-Src of ASD cells werelocalized in GEM/raft fractions. And we observed that accumu-lated GM2 was directly interacted to c-Src and co-localized inlysosome membrane. The result of ceramide moiety analysis ofGM2 were revealed that length of fatty acids were not differentfrom AWT and ASD, indicating densely-packed GM2 was enoughto interact to cytosolic kinases c-Src, and Glycosphingolipids(GSLs) aggregation could transmit proliferation signals. Theseresults indicate that excessive accumulated GSLs have function ofsignal transduction directly at not only cell surface membranes butalso intracellular organella membranes as like lysosome.

(126) Mucin-Type O-glycosylation is Required for DigestiveSystem Formation and Function in Drosophila

Liping Zhang, Kelly Ten HagenNIH/NIDCR, Bethesda, MD

Mucins are high molecular weight glycoproteins that serve as themain constituent of the mucus-protective layer lining the gastroin-testinal tract, conferring protection from chemical, microbiologicaland mechanical damage. Recent studies have shown that loss ofmucins or mucin-type O-glycosylation results in abnormal intesti-nal barrier formation and predisposition to colitis and colon cancerin mice. The glycosylation of mucins is initiated by an evolutiona-rily conserved family of glycosyltransferases encoded by thepGalNAcT genes in mammals and pgant genes in Drosophila.Here we demonstrate that one pgant member, pgant4, is requiredfor digestive system formation and function in Drosophila. pgant4is expressed specifically in a subset of Drosophila foregut cells


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(PR cells) that are responsible for secreting the peritrophic mem-brane of the digestive system. Mutations in pgant4 or RNAi topgant4 result in lethality. pgant4 mutant larvae displayed abnormalproventriculus formation, with larger and irregularly-shaped PRcells. Immunostaining revealed that one component of peritrophicmembrane accumulated in punctate structures in the mutant PRcells. Food digestion assays demonstrated an abnormal pH andaccumulation of food in the mutant larval gut system. Western blotusing the lectin specific to O-glycans showed the loss ofO-glycoproteins in the pgant4 mutant foregut. We propose thatPGANT4 is responsible for glycosylating certain mucin proteinswithin gastrointestinal tract which play important roles in establish-ing proper digestive system structure and function. This study pro-vides new evidence that mucin-type O-glycosylation is requiredfor digestive system function and a new platform to investigate therelationship between abnormal O-glycosylation and the predisposi-tion to colitis and colon cancer seen in mammals.

(127) The Effect of O-GlcNAcylation on hnRNP A1from Colorectal Cancer

Shira Koren, Galit Yehezkel, Liz Cohen, Adi Kliger,Isam Khalaila

Ben-Gurion University of the Negev,ISRAEL, Beer-Sheva, Israel

The heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1)isoform, which is a member of hnRNP A/B subfamily, was foundto be over expressed in almost all examined human colorectalcancer as well as lung cancer tissues. It is one of the majorpre-mRNA binding proteins and is involved in telomere lengthen-ing, cell signaling and tumor development. Furthermore, it isinvolved in regulating gene expression at both transcriptional andtranslational levels. Although hnRNP A1 is predominantly nuclear,it shuttles rapidly between the nucleus and the cytoplasm, assistedby the translocation protein transportin1, and plays its differentroles in accordance to its location. HnRNP A1 translocation isregulated partially through phosphorylation on serine residues atits c-terminal domain. Recently, we have shown that in addition tophosphorylation, hnRNP A1 is also modified by O-linkedN-acetylglucosaminylation (O-GlcNAcylation). We also confirmedthe association of hnRNP A1 with transportin1 and found thatO-GlcNAcylation affects this interaction. We have shown thatinduced phosphorylation¬ of hnRNP A1 leads to accumulation ofboth hnRNP A1 and transportin1 in the cytosol of colorectalcancer cells. However, elevated O-GlcNAcylation levels of hnRNPA1 counteract the phosphorylation effect. Phosphorylation andO-GlcNAcylation may compete on the same serine or threonineresidues. In addition, one modification could interrupt the otherwhen residing on adjacent sites. Such counter interplay betweenthese modifications could influence the translocation of the proteinby transportin1 as well as its function or stability.Mapping hnRNP A1 O-GlcNAcylation sites and elucidation of

their effect on hnRNP A1 function and its interaction with trans-portin1 are under investigation. This research will significantlycontribute to the understanding of hnRNP A1 in cell physiologyand cancer development.

(128) O-GlcNAc Modification of Proteins and CancerMetastasis

Isam Khalaila, Galit Yehezkel, Liz Cohen, Adi Kliger,Ela Finkelstein

Ben-Gurion University, Biotechnology Engineering, Beer-Sheva,Israel

Aberrant post-translation modifications, such as phosphorylation,of cellular proteins play a key role in cancer development andmetastasis. A variety of nucleocytoplasmic proteins were found tobe modified through a β-O-linkage of N-acetylglucosamine(GlcNAc) to serine or threonine residues, similarly to proteinphosphorylation. However, (unlike phosphoryltaion,) only twoenzymes catalyze the addition and the removal of O-GlcNAc:O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respect-ively. Aberrant patterns of O-GlcNAcylation on key proteins havebeen documented in pathological conditions such as diabetes neu-rodegenerative diseases and cancer. Therefore, we hypothesize thatO-GlcNAcylation of nucleocytoplasmic proteins is altered duringmetastasis of human colorectal cancer and thus contributing to thetransformation of tumor cells towards metastasis. The levels ofO-GlcNAcylation and its enzymatic machinery in primary andmetastatic colorectal cancer clones were followed. The expressionof OGA was silenced by RNA interference, and the effect of thismanipulation on gene expression, and cellular phenotype, wasexamined. Our results show that OGA level was found to be sig-nificantly lower in metastatic cells and in OGA-silenced cells, con-sistently with elevated O-GlcNAcylation level. OGA-silencingaltered cell morphology and gene expression in a manner support-ing our hypothesis.

(129) A Sialidase Specificity Assay Reveals Restricted GlycanTolerance by NEU2

Randy Parker, Jennifer KohlerUniversity of Texas Southwestern Medical Center, Dallas, TX

Sialidases comprise a class of enzymes widely distributed innature. The hydrolytic enzymes remove sialic acids from the term-inal positions of glycans on glycoproteins and glycolipids.Sialidases are implicated in numerous normal and pathologicalprocesses from immune activation to metastasis, yet how theseenzymes exert their effects remains elusive due to the fact that theendogenous substrates are largely unknown. In order to ascertainthe contribution of glycan structure to sialidase activity, wedesigned a sialidase substrate specificity assay based on thePeriodate oxidation and Aniline catalyzed Ligation (PAL) tech-nique for specifically labeling sialosides. Our new assay was usedto analyze the activity of the human cytosolic sialidase NEU2against a panel of diverse glycans containing variations in sialicacid identity, linkage, and identity of the underlying glycan. Weshow that NEU2 strongly prefers sialic acids in an α2,3 linkageover an α2,6 linkage. Additionally, NEU2 acts on hydroxylatedbut not deaminated sialic acids. We also demonstrate that NEU2 issensitive to the identity of the sub-terminal glycan. These resultshave important implications for identifying NEU2’s cellular sub-strates and uncovering its mechanism of action.


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(130) Evaluation of the Metabolic Responses Relatedto Fructose as a Primary Carbon Source

Juliana Sacoman, Lauren Badish, Rawle HollingsworthMichigan State University, East Lansing, MI

Fructose is now a major dietary sugar and its effect on cellularphysiology is a matter of great concern. Fructose and glucoseinteract with different enzymes and the gene functions they induceand pathways they participate in (except for glycolysis) are differ-ent. High levels of fructose through normally quiescent pathwayswill lead to different physiological and biochemical endpoints. Inorder to assess how divergent the metabolic paths of cells arewhen exposed separately to these two sugars we used microarrayanalysis to determine the extent to which genes are up or down-regulated soon after exposure to either sugar. We used an affinitymatrix to obtain further insight on the molecular interactions thatdrive fructose metabolism. To accomplish this, an analog offructose-6-phosphate, the first metabolic product of fructose, wascovalently linked to a matrix to allow the capture of proteinspecies relevant to the metabolism of this sugar.The microarray studies showed a marked divergence in gene

expression between cells exposed either to fructose or glucose. In thefirst case, genes related to glycine betaine and C1 metabolism were themost up-regulated. Pentose metabolism genes were also significantlyup-regulated. Genes involved in the formation and transport ofglycerol-3-phosphate were suppressed. In contrast, the mostup-regulated genes in cells exposed to glucose coded for enzymeswhose functions are still unknown. The majority of the down-regulatedgenes in cells exposed to glucose encode for Krebs cycle enzymes.The affinity matrix captured proteins that bind to

fructose-6-phosphate or to substrates structurally similar to it or thathave common substructures. These include enzymes involved innucleotide synthesis, DNA binding proteins, aldolases, and carbo-hydrate processing enzymes. These results indicate that the meta-bolic fate of cells cultured in fructose is radically different fromthose cultured in glucose.

(131) Disruption of Protein O-glycosylation Alters FGFSignaling by Modulating Basement Membrane Composition

E Tian, Matthew Hoffman, Kelly Ten HagenNational Institutes of Health, Bethesda, MD

Cellular microenvironments are a complex array of componentsthat influence cell signaling, proliferation and differentiation, butthe diverse factors that regulate the composition of the extracellularmatrix (ECM) are not completely understood. Recent studies havedemonstrated that protein O-glycosylation can influence the com-position of the ECM in Drosophila melanogaster. Here we demon-strate that this conserved protein modification affectsFGF-mediated cell proliferation during mammalian organogenesisby influencing the secretion of basement membrane (BM) proteins.Mice deficient for an enzyme (Galnt1) that adds sugars to proteinsduring early development of submandibular glands resulted inintracellular accumulation of major BM components, increasedendoplasmic reticulum stress, decreased FGF signaling, reducedAKT and MAPK phosphorylation, and reduced epithelial cell

proliferation. Exogenous addition of BM component laminin toGalnt1 deficient glands rescued FGF signaling and the growthdefects in a β1-integrin-dependent manner. Our work demonstratesthat O-glycosylation influences the composition of the ECM inmammalian organ development, with resultant effects on cell sig-naling, proliferation and organ growth. These results highlight aconserved role for O-glycosylation in the establishment of cellularmicroenvironments and have implications for the role of thisprotein modification in both development and disease.

(132) Fringe Effects on Delta1-induced Notch Signaling areDifferentially Regulated by Galactose

Xinghua Hou, Yuko Tashima, Pamela StanleyAlbert Einstein College of Medicine, Bronx, NY

Notch signaling is modulated by Fringe β3N-acetylglucosaminyl-transferases. We previously showed that the action of Lunatic Fringe(Lfng) or Manic Fringe (Mfng) inhibits Jagged1-induced Notch sig-naling in CHO cells. However, this inhibitory effect depends on theaddition of Gal to the GlcNAc transferred by Fringe. We also showedthat mice lacking B4galt1 exhibit subtle Notch signaling defectsduring somitogenesis. In co-culture Notch signaling assays, Lfngenhances Delta1-induced Notch signaling in CHO and Lec1 cells.We now show that enhancement of Delta1-induced Notch signalingby Lfng did not occur in either Lec8 or Lec20 CHO mutants, defec-tive in the addition of Gal to O-fucose glycans. Correction of Lec20with a B4galt1 cDNA reversed this affect for Lfng. By contrast,Mfng promoted Delta1-induced Notch signaling better in Lec8 andLec20 cells that transfer Gal poorly, than in CHO cells with Gal onO-fucose glycans. This effect was corrected by expression of aUDP-Gal transporter cDNA in Lec8 cells. To investigate mechanism,flow cytometry binding assays of soluble Notch ligands were per-formed. The binding of Jagged1-Fc was reduced by Lfng and Mfngin Lec1 cells that have Gal on O-fucose glycans, but not in Lec8cells, consistent with signaling assays. Lfng caused increased bindingof Delta1-Fc to Lec1 but binding to Lec8 was highly variable. Mfngroutinely caused enhanced binding of Delta1-Fc to Lec8, but Mfngalso enhanced Delta1-Fc binding to Lec1. This Mfng-enhancedbinding to Lec1 was apparently not sufficient to cause increasedDelta1-induced Notch signaling in Lec1. In summary therefore, Galadded to O-fucose glycans differentially affected the ability of Lfngversus Mfng to enhance Delta1-induced Notch signaling. This mayreflect differential modification by Lfng and Mfng of Notch EGFrepeats on a particular Notch receptor, or on the different Notchreceptors expressed by CHO cells.Supported by NIH grantRO195022 to PS.

(133) Brain Specific Expression of GnT-IX (GnT-Vb)is Controlled by Epigenetic Chromatin Regulationand Transcriptional Factors CTCF and NeuroD1

Yasuhiko Kizuka1, Shinobu Kitazume1, Minoru Yoshida2,Naoyuki Taniguchi1,3

1Systems Glycobiology Research Group, RIKEN ASI, Wako,Japan; 2Chemical Genomics Research Grop, RIKEN ASI, Wako,Japan; 3RIKEN-Osaka Univ. Alliance Lab., ISIR, Osaka Univ.,

Osaka, Japan


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Glycan expression is well-controlled by many factors includingtissue- or cell-specific expression of glycosyltransferase. However,the molecular mechanism is still unclear by which glycosyltrans-ferase genes are regulated in a tissue-specific manner. Here wefocused on a recently cloned brain-specific GlcNAc transferase,GnT-IX (also called GnT-Vb), which is involved in branching ofO-mannose glycan. We examined the expression mechanism ofthis brain specific glycosyltransferase.We employed epigenetic approach because epigenetic mechan-

isms, such as DNA-methylation and/or histone modifications, playkey roles for gene regulation. Actually, our ChIP (chromatinimmunoprecipitation) analysis revealed that histone modificationsbut not DNA methylation are involved in brain-specific expressionof GnT-IX. Moreover, we found that forced histone acetylationevoked GnT-IX expression even in 3T3 fibroblast in which thisgene is not expressed originally, indicating that GnT-IX gene isepigenetically regulated.Meanwhile, we searched for pivotal transcription factors which

are required for GnT-IX transcription in activated chromatin inneural cells. Using reporter assay, EMSA (electro-mobility shiftassay) and ChIP analyses, we identified the most important cisDNA element and two transcriptional factors, CTCF and NeuroD1which bound this element to activate GnT-IX transcription. Wealso found that GnT-IX transcription in neuroblatoma cells wasdown-regulated by CTCF- and NeuroD1-depeletion, indicating theimportance of these factors. In addition, the binding of CTCFhighly depended on chromatin activation status. Taken together, itsuggests that CTCF- and NeuroD1-dependent epigenetic mechan-ism governs the brain-specific expression of GnT-IX. We foundthat other neural glycosyltransferase genes are also regulated byepigenetic histone modifications, suggesting that epigenetic regu-lation is a general mechanism of tissue-specific glycan expression.

(134) Total Internal Reflection Fluorescence (TIRF)Microscopy for the Study of Carbohydrate-Carbohydrate

InteractionsAngelika Kunze1, Waqas Nasir1, Marta Bally2, Fredrik Hook2,

Goran Larson11Sahlgrenska Academy University of Gothenburg, Gothenburg,

SE; 2Chalmers University of Technology, Gothenburg, SE

Cell adhesion mediated by protein-protein interaction (PPI) andcarbohydrate-protein interaction (CPI) is well established. A thirdmechanism mediating cell adhesion, carbohydrate-carbohydrateinteraction (CCI) has been introduced and explored mainly in thegroup of Dr. S I Hakomori. CCIs can be of homotypic (e.g. inter-action between two identical glycosphingolipids (GSL)) or hetero-typic (interaction between two different GSL) nature. A typicalexample of a homotypic CCI is the specific Lex-Lex interaction,which is observed in autoaggregation of embryonal carcinomacells or embryonic stem cells in the presence of calcium.Most techniques come to their limit when low affinity CCIs are

studied. We present TIRF (total internal reflection fluorescence)microscopy as a sensitive nano-technique to study time resolvedCCIs between GSL-modified SLB (solid supported lipid bilayers)

and liposomes in microtiter plates. SLBs containing Lex andPOPC are prepared on glass. In the next step liposomes of definedcomposition and labeled with a fluorophore are added. Due theevanescent field of the TIRF microscope these liposomes aredetected only when they come in close proximity (<150 nm) orattach directly to the GSL doped SLB. With this approach we areable to monitor Lex-Lex interaction in real time with a single lipo-some resolution. We demonstrate that Lex-Lex interaction isspecific and highly sensitive to calcium as well as magnesium.Furthermore, we show the effect of cholesterol on this interaction.

(135) N-glycosylation Enzymes in B Cells are Regulatedby Innate and Adaptive SignalsAlison Mahan1,2, Galit Alter2

1Harvard University GSAS, Cambridge, MA; 2Ragon Institute ofMGH, MIT, and Harvard, Charlestown, MA

The N-linked glycan at asparagine-297 of the IgG heavy chainprofoundly affects the capacity of the antibody to recruit an arrayof innate immune effector functions. While therapeutic antibodiesare produced with specific glycan structures in vitro, little isknown about how B cells naturally modulate antibody glycanstructures. In this study we aimed to define the specific mechan-ism(s) by which B cells alter antibody glycans, with the ultimategoal of designing vaccines that elicit distinct sets of antibodyeffector functions.

B cells purified from healthy leukophereses were stimulatedwith innate pattern recognition receptor agonists (TLRs 2-9) in thepresence or absence of direct B cell receptor (BCR) activation(anti-immunoglobulin). After 14 hours of stimulation, transcrip-tional analysis of glycosylation enzymes was performed usingquantitative-PCR.

Stimulation of B cells with innate or adaptive signals inducedsignificant changes in glycosyltransferase and glycosidaseexpression, which may correlate to changes in N-glycan structure.Interestingly, while stimulation of TLRs 2-6 altered only a few gly-cosylation enzymes, stimulation with nucleic-acid sensing TLRs7-9 reduced expression of GlcNac-transferases, possibly generatingantibodies with shorter glycan branches. Additionally, sialyltrans-ferase genes were downregulated after stimulation of TLRs 7-9,possibly decreasing terminal sialic acids. Interestingly, BCR stimu-lation resulted in a significant reduction in fucosyltranferase andgalctosyltransferase transcripts, thus potentially decreasing corefucose and galactose additions.

Taken together, these data suggest that both BCR and/or innateimmune receptor triggering significantly changes specific glycanmodifying enzyme expression. Interestingly, stimulation ofnucleic-acid sensing receptors, which detect intracellular pathogensmost vulnerable to innate immune recruiting antibodies, resulted inreduced enzyme production possibly producing shorter glycanswith enhanced innate immune recruiting activity. These data implythat specific inflammatory signals lead to the production of anti-bodies with different effector functions, which, in the context of avaccine, could promote the production of antibodies with particu-lar effector functions.


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(136) An O-GlcNAc Cycling Complex Associates with theRibosome in Response to Proteasome Inhibition

Quira Zeidan, Gerald W. HartJohns Hopkins University School of Medicine, Baltimore, MD

The O-GlcNAc modification of proteins is emerging as a mainregulator of every aspect of cell function: transcription, cell cycleprogression, trafficking, signaling, and tolerance to stress, amongothers. Little is known about the role of O-GlcNAc in the processof protein synthesis. We have previously shown that ribosomalproteins are O-GlcNAcylated and that O-GlcNAc transferase(OGT) and O-GlcNAcase (OGA) associate with the translationmachinery under conditions of active protein synthesis. The 26Sproteasome is the macromolecular complex in charge of the degra-dation and turnover of most nucleocytoplasmic proteins.Proteasomal activity is tightly intertwined with translation since itregulates the recycling and availability of amino acids for use inprotein synthesis. Pharmacological inhibition of the proteasomeleads to a rapid shut-down of translation and consequent celldeath, a strategy being currently used for the treatment of severaltumors. Inhibition of the proteasome in HepG2 cells with MG132leads to a rapid reduction in protein synthesis as measured bymetabolic labeling with [35S]-methionine. This is accompanied bythe rapid association of OGT and OGA with ribosomes and theincreased O-GlcNAcylation of many ribosome-associated factors.This association is rapidly reversed upon removal of MG132 andit occurs with three other different proteasome inhibitors. Thebinding of OGT to the ribosome under these conditions correlateswith its increased auto-O-GlcNAcylation. Recent findings suggestthat cells are able to recover from prolonged proteasome inhibitionby restoring protein translation to an extent, which has impli-cations in the recovery of tumor cells from chemical therapy withthese agents. We propose that O-GlcNAc cycling is a key regulatorof the switch between pause and recovery of protein synthesisduring conditions of proteasome inhibition. NIH R01CA42486,R01DK61671 and R24DK084949. Dr. Hart receives a share ofroyalty received by the university on sales of the CTD 110.6antibody.

(137) O-GlcNAcylation of HIPK1 Regulates Its Functiontowards p53 during Fas-mediated Apoptosis

Ronald Copeland1, Jeremy Balsbaugh2, Donald Hunt2,Gerald W. Hart1

1John Hopkins University School of Medicine, Baltimore, MD;2The University of Virginia, Charlottesville, VA

O-linked β-N-acetylglucosamine (O-GlcNAc) is an inducible,abundant, and dynamic post-translational modification consistingof the addition of a single N-acetylglucosamine moiety to serineand/or threonine residue of target proteins. O-GlcNAcylation isknown to play important roles in the regulation of numerous cellu-lar processes including cell signaling, cell cycle progression, andtranscription/translation by modulating proteins’ functions.Homeodomain-interacting protein kinase-1 (HIPK) has been foundto be elevated in many breast cancer cell lines. HIPK1 has alsobeen implicated in the phosphorylation of p53 and in the

regulation of apoptotic signaling events by potentially modulatingp53 and/or Mdm2 functions. However, the HIPK1 dependentphosphorylation site(s) on p53 and their possible function remainsunclear. Herein, begin to investigate how O-GlcNAcylation ofHIPK1 might play roles in apoptosis via its regulation of p53function using Fas-mediated apoptosis as a model. In the presentstudy, we demonstrate that HIPK1 interacts with OGT and isO-GlcNAcylated. Upon Fas stimulation, HIPK1 O-GlcNAcylationdecreases in a time dependent manner. Using mass spectrometry,we identified three sites for O-GlcNAcylation of HIPK1 in vivoand confirm these sites using site-directed mutagenesis. Alongwith a decrease in O-GlcNAcylation, we also observe a decreasein HIPK1 interaction with p53 upon Fas stimulation. Interestingly,mutating the O-GlcNAc sites on HIPK1 also result in a decreasein the HIPK1/p53 interaction. Preliminary data from in vitrokinase assays suggest that O-GlcNAcylation of HIPK1 maypossess the potential to regulate its ability to autophosphorylate.Taken together, results from this study suggest thatO-GlcNAcylation of HIPK1 may regulate its function duringFas-mediated apoptosis by affecting its ability to bind p53 and itskinase activity. Current studies are aimed at determining whetherO-GlcNAcylation of HIPK1 affects its ability to directly phosphor-ylate p53 and the identification of these currently unknown site(s).

(138) COG Complex Specifically Regulates the Maintenanceof Golgi Glycosylation Machinery

Irina Pokrovskaya1, Rose Willett1, Richard Smith1, Willy Morelle2,Tetyana Kudlyk1, Vladimir Lupashin1

1Department of Physiology and Biophysics UAMS, Little Rock,AR; 2University of Lille I, Villeneuve d’Ascq, France

Cell surface lectin staining, examination of Golgi glycosyltrans-ferases stability and localization, and MALDI-TOF analysis wereemployed to investigate Conserved Oligomeric Golgi(COG)-dependent glycosylation defects in HeLa cells. BothGriffonia simplicifolia-II (GS-II) and Galanthus nivalus (GNL)lectins were found to be specifically bound to plasma membraneglycoconjugates of COG-depleted cells, indicating defects inactivity of medial and trans-Golgi-localized enzymes. In responseto siRNA-induced depletion of COG complex subunits, severalkey components of Golgi glycosylation machinery, includingMAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mis-localized. MALDI-TOF analysis of total N-linked glycoconjugatesindicated a decrease in the relative amount of sialylated glycans inboth COG3 KD and COG4 KD cells. In agreement to a proposedrole of the COG complex in retrograde membrane trafficking, allCOG subunit-depleted HeLa cells were deficient in the BrefeldinA- and Sar1 DN-induced redistribution of Golgi resident glycosyl-transferases to the ER. The retrograde trafficking of medial- andtrans-Golgi-localized glycosylation enzymes was affected to alarger extent, strongly indicating that the COG complex regulatesthe intra-Golgi protein movement. COG complex deficient cellswere not defective in Golgi re-assembly after the Brefeldin Awash-out, confirming specificity in the retrograde trafficking block.Finally, the lobe B COG subunits COG6 and COG8 were localizedon trafficking intermediates that carry Golgi glycosyltransferases,


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indicating that the COG complex is directly involved in traffickingand maintenance of Golgi glycosylation machinery.

(139) A Role for O-fucosylation in Quality Controlof Thrombospondin Type-I Repeat Folding

Deepika Vasudevan1, Hideyuki Takeuchi1, Elaine Majerus2,Robert S. Haltiwanger1

1Stony Brook University, Stony Brook, NY; 2Washington UniversitySchool of Medicine, Louisiana, MO

O-Fucosylation of Thrombospondin type-I repeats (TSRs) byprotein O-fucosyltransferase 2 (Pofut2) is essential for embryonicdevelopment in mice. Although the molecular mechanism explain-ing this phenotype is not known, several proteins containing TSRs(e.g. members of the ADAMTS family of extracellular proteases)fail to secrete in the absence of O-fucosylation, suggesting that thelethality may be due to lack of secretion of these proteases. Wehave previously demonstrated that Pofut2 localizes to the ER,despite lacking a KDEL sequence, and modifies only properlyfolded TSRs in vitro. TSRs contain six-conserved cysteine resi-dues that form three specific disulfide bonds when properlyfolded. These observations allow us to draw a model whereinPofut2 plays a role in the quality control of TSR folding andO-fucosylation serves as a signal for ER exit and subsequentsecretion of the proteins. We are testing aspects of this model now.Preliminary proteomic studies on Pofut2 suggest that the enzymeis present in a complex with several other proteins that may assistin protein folding in addition to ER-retention of the enzyme. Ourcurrent experiments focus on identifying factors that are directlyinvolved in ER retention of the unfucosylated proteins. We arealso examining when and where O-fucosylation occurs usingpulse-chase studies in the presence of 6-alkynyl-fucose, abioorthogonal compound that can be labeled with biotin. Finally,we are examining whether the number of TSRs or the sequence ofindividual TSRs has a greater effect on the secretion of proteins inthe absence of O-fucosylation. These experiments will help struc-ture and refine the current model for Pofut2 in the quality controlof TSR folding. This work was made possible by NIH grantCA12307101.

(140) Glycosaminoglycans, New Regulators of α-synucleinAggregation and Apoptosis in Parkinson Disease

Sonia BoufalaUniversité Paris Est Créteil, Créteil, France

The cause of Parkinson disease (PD) that affects dopaminergicneurons remains mysterious, although evidences support mito-chondrial dysfunctions and α-synuclein accumulation as majorevents. Recently, cathepsin D (cathD), a lysosomal proteaseinvolved in apoptosis, was described to be the major enzymeimplicated in α-synuclein degradation. As glycosaminoglycans(GAGs) are implicated in both, regulation of cathD activity andregulation of protein aggregation in case of pathology, we investi-gated if GAGs can be located in the intracellular compartmentwhere they may interacted with cathD to concomitantly regulateboth apoptosis and α-synuclein aggregation. In our cellular PDmodel, the absence of sulfated GAGs increased cathD activity and

limited α-synuclein accumulation. For the first time, the regulationof α-synuclein accumulation and apoptosis activation by cathDwere demonstrated to be under the dependence of intracellularGAGs. Results with heparin, heparan and chondroitin sulfatefurther suggested that GAGs specific sequences or sulfationpatterns could be responsible of this regulation. This open newinsight on intracellular GAGs functions and new fields of investi-gation for glycobiological approaches in neurobiology.

(141) The Decrease of O-GlcNAcylated Protein Levelis Crucial for Human Hepatoma Cell Death Induced

by Protozoan Parasite Entamoeba histolyticaYoung Ah Lee, Deulle Min, Seong Hoon Kim,

Myeong Heon ShinYonsei University College of Medicine, Seoul, South Korea

Entamoeba histolytica is an enteric tissue-invasion protozoan para-site that causes amoebic colitis and occasionally liver abscess inhumans. E. histolytica leads to host cell apoptosis by the inductionof various intracellular signal mechanisms such as caspase-3,calpain and tyrosine dephosphorylation in host cells. These modu-lations triggered by E. histolytica are closely associated with para-sitic immune evasion mechanism as well as host defense. Likephosphorylation, O-GlcNAcylation also has been thought to con-tribute the various cellular signal processes including apoptosisand proliferation. However, there are no information about therelationship between O-GlcNAcylation and Entamoeba-inducedhost cell death. In this study, at first of all, we investigated theintracellular events in HepG2 cell death triggered by E. histolytica.Co-incubation of HepG2 cells with Entamoeba remarkablyincreased DNA fragmentation and LDH release compared to cellsincubated with medium alone. In addition, E. histolytica inducedthe decrease of tyrosine phosphorylation and the cleavage ofcaspases-3 and calpain in HepG2 cells. Next, we examined therelationship between O-GlcNAcylated protein level in HepG2 cellsand Entamoeba-triggered cell death. Adherence of live E. histoly-tica trophozoites caused the decrease of O-GlcNAcylated proteinlevel in HepG2 cells within 2 min. However, Entamoeba-induceddeGlcNAcylation in HepG2 cells was dramatically reduced in cellspretreated with O-GlcNAcase inhibitors, PUGNAc or streptozoto-cin. In addition, DNA fragmentation and LDH release triggered byE. histolytica were strongly inhibited by pretreatment of host cellswith PUGNAc, suggesting that the deGlcNAcylation in host cellplays an important role in E. histolytica-induced HepG2 cell death.This study shows a potentially novel immune evasion mechanismto induce host cell death utilized by E. histolytica.

(142) Combining Essential Dynamics and Binding Statisticsto Rationalize Glycosaminoglycan Sulfation

by N-Sulfotransferase and MutantsTarsis Gesteira1, Laércio Pol-Fachin2,

Vivien Jane Coulson-Thomas3, Hugo Verli2, Helena Nader11Federal University of São Paulo, Sao Paulo, Brazil; 2Federal

University of Rio Grande do Sul, Porto Alegre, Brazil; 3Universityof Cincinnati, Cincinnati, OH


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Heparan sulfates (HS) and heparin are highly sulfated glycosamino-glycans (GAGs). The sulfation patterns along the GAG chainsdictate their functional role. N-Deacetylase-N-sulfotransferase 1(Ndst1) catalyzes the initial modification of heparan sulfate andheparin removing acetyl groups from subsets ofN-acetylglucosamine units and the subsequent sulfation of theresulting free amino groups. Glycosaminoglycan sulfotransferasestransfer the sulfuryl group from donor 3’-phosphoadenosine5’-phosphosulfate (PAPS), yielding 3’-phosphoadenosine5′-phosphate(PAP) and sulfated sugar chains. Recent reports have demonstratedthe influence of many residues in this mechanism, noteworthy, twolysine residues that stabilize the transition state by interacting withthe bridging oxygen between the sulfate and phosphate groups ofPAPS. Crystallographic analyses showed that both cytosolic andmembrane-bound sulfotransferases are single a/b globular proteinswith a characteristic five-stranded parallel b-sheets. The b-sheetconstitutes the PAPS-binding site and the core of the catalytic site,both of which are composed of conserved residues in the cytosol,as well as membrane-bound STs. However, the precise catalytic rel-evance of the boundary residues through the hydrophobic cleft isstill unclear, as well as, their significance to the glycan recognitionand sulfation. A molecular docking and molecular dynamicsapproach was used to study in detail the catalytic site of humanNDST and decipher the precise catalytic relevance of the boundaryresidues through the hydrophobic cleft, as well as, the role of criti-cal amino acid residues for ligand binding.

(143) O-GlcNAc and the Progression of Prostate CancerXin Liu1,2, pengyuan yang2, Gerald W. Hart1

1John Hopkins University School of Med, Baltimore, MD; 2FudanUniversity, Shanghai, China

Prostate cancer is one of the most common forms of cancer in menand the second leading cause of cancer deaths in men in the UnitedStates. O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitousand dynamic post-translational modification that exists on serine andthreonine residues of nuclear and cytoplasmic proteins. This modifi-cation is regulated by O-GlcNAc transferase (OGT), which attachesO-GlcNAc to proteins, and O-GlcNAcase (OGA), which removesO-GlcNAc. O-GlcNAc is regulating virtually all cellular processes aswell as playing roles in the various diseases, including Alzheimer’sdisease, diabetes, and cancer. The role of O-GlcNAc in the regulationof prostate cancer remains unclear. Herein we begin to investigatehow the regulation of O-GlcNAc may affect prostate cancer usingnormal prostate epithelial cell (PrEc) and prostate cancer cell lines(LNCaP and PC-3) as models. Using western blotting techniques, weshow that O-GlcNAc, OGT, and OGA are highly elevated in LNCaPand PC-3 when compared to PrEc. Interestingly, LNCaP exhibitedthe highest levels of O-GlcNAc, OGT, and OGA. OGT and OGA aredifferentially localized in lines with different metastatic properties.Additionally, 2D O-GlcNAc profiles were demonstrated to have sig-nificant differences among the three cell lines. Current studies are onthe way to answer how O-GlcNAc affects the biological propertiesincluding shapes, proliferation, invasion and metastasis of the cells.Supported by NIH CA42486 and the Patrick C. Walsh ProstateCancer Research Fund.

(144) Structural and Functional Studies of the DehydrogenasesRequired for the Biosynthesis of

2,3-Diacetamido-2,3-dideoxy-D-mannuronic AcidJim Thoden, Hazel Holden

University of Wisconsin, Madison, WI

The lipopolysaccharide or LPS is the major structural componentof the outer membrane of Gram-negative bacteria. It is a complexglycoconjugate consisting of lipid A, the core polysaccharide, andthe O-antigen. Lipid A serves to anchor the sugar components ofthe LPS to the bacterial cell membrane whereas the O-antigen,which extends farthest away from the bacterium, contributes to thewide varieties of bacterial strains observed in nature. There isgrowing evidence that O-antigens play important biological rolesincluding, but not limited to, virulence, effective colonization ofhost tissues, protection from phagocytosis and serum-mediatedkilling, and resistance to antimicrobial peptides.

O-antigens often contain some highly unusual dideoxy sugarsincluding 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, here-after referred to as ManNAc3NAcA. This particular sugar hasbeen observed, for example, in the B-band O-antigen ofPseudomonas aeruginosa O:3a,d, and in the A-band trisaccharideof the bacterium Bordetella pertussis, the causative agent ofwhooping cough.

The dehydrogenases that are the topic of this investigation cata-lyze the second step in the biosynthesis of ManNAc3NAcA,namely the oxidation of the C-3′ hydroxyl group on theUDP-linked sugar to a keto moiety and the reduction of NAD+ toNADH. Interestingly, there are two distinct classes of theseenzymes: those that require α-ketoglutarate for the regeneration ofNAD+, and those that simply release NADH. We will refer tothese classes as A and B, respectively. The Class A members arefound, for example, in P. aeruginosa and Thermus thermophilus,whereas the Class B members have been identified in B. pertussisand Chromobacterium violaceum.

We have determined the structures of the enzymes in complexwith NAD(H) and their UDP-linked sugars. Details concerning thestructures and reactions mechanisms of these dehydrogenases willbe presented.

(145) Glycobiology of the Probiotic Lactobacillus rhamnosusGG (LGG): A Network-Based Approach

Hanne Tytgat, Aminael Sánchez-Rodríguez, Sarah Lebeer,Geert Schoofs, Tine Verhoeven, Sigrid De Keersmaecker,

Kathleen Marchal, Jos VanderleydenKULeuven - CMPG, Heverlee, Belgium

Interactions between bacteria and host cells are often mediated byglycoconjugates such as exopolysacharides, glycosylated teichoicacids or glycoproteins on the bacterial cell wall. This is also thecase for probiotic bacteria, where much remains to be discoveredabout the role of the cell wall glycome in the molecular mechan-isms underlying their beneficial functions.

In this study, the objective was to generate knowledge on theglycoproteome of the model probiotic Lactobacillus rhamnosusGG (LGG). A systematic analysis of the protein-coding genes of


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LGG resulted in the identification of putative protein glycosyl-transferases, the key enzymes of glycosylation processes. In a laterphase these genes will be targeted by knock-out analysis and func-tionally assessed in glycoproteome assays. The ultimate goal is toidentify the targets of the protein glycosyltransferases and unraveltheir role in the beneficial functions of LGG.As the genome of LGG is of quite recent date (2009) and as

much remains to be discovered about the functional role of itsgenes, it is tricky to base the identification of protein glycosyltrans-ferases solely on gene annotations. Therefore we developed a toolto systematically assess the genome of a prokaryotic organism byapplying a series of filters that result in the selection of genes thatencode putative protein glycosyltransferases. The basic idea is toscore genes based on the biological processes in which their func-tional partners are involved in the cell. The information was derivedfrom reconstructed functional networks (STRING database). This‘guilt by association’-approach renders a global overview of puta-tive protein glycosyltransferases and information on their possiblefunctional partners. This new tool was also applied to the modelorganism of prokaryotic glycosylation, Campylobacter jejuni toestablish its robustness and general applicability.

(146) Structural Characterisation of Burkholderiapseudomallei 576 O-Antigen by Mass Spectrometry

Valeria Ventura1, Ngugi Sarah2, Paul Hitchen1, Prior Joann2,Anne Dell1

1Imperial College London, London, United Kingdom; 2DefenceScience and Technology Laboratory [Dstl], Salisbury, UK

Burkholderia pseudomallei is the causative agent of melioidosis.The bacterium expresses endotoxic lipopolysaccharide (LPS) mol-ecules, vital for its serum resistance. These are composed of thehydrophobic Lipid A embedded in the outer membrane and thecore and O-antigen polysaccharides displayed on the surface of thebacterial cell. Currently there is no vaccine available for protectionagainst B. pseudomallei and little is known about the virulencemechanism of the pathogen.Reportedly 3% of strains possess an LPS structure that is immu-

nologically distinct from that of the typical strain. LPS wasextracted from cells of B. pseudomallei 576 strain (atypical) orB. pseudomallei K96243 strain (typical) by an adapted phenolextraction procedure, analysed on SDS-PAGE gels and visualisedby silver staining, revealing two distinct O-antigen profiles. Thecharacterisation of the O-antigen polysaccharide employed highsensitivity mass spectrometry techniques including GC-MS,MALDI-MS and MS/MS, to determine the composition and struc-ture of the B. pseudomallei LPS 576 O-antigen.Compositional and linkage analysis by GC-MS, showed a

higher monosaccharide complexity as well as branching in the 576strain as compared to the K96243 strain. Furthermore, Lipidremoval in the 576 strain needed more stringent conditions thanacid hydrolysis used to solubilise the K96243 strain, for whichHydrazinolysis techniques were explored. These in combinationwith O-antigen hydrolysis and analysis by MALDI-MS andMS/MS gave insights into the O-antigen repeat units of theheteropolymer.

The differences in O-antigen structure are reflected in differ-ences in the O-antigen biosynthesis genes, where the phenotypicversatility of B. pseudomallei is likely to be driven by the presenceof a highly dynamic and plastic genome. Whilst genes at the startand end of the typical and atypical LPS clusters share highsequence identity, the central region contains genes unique to eachcluster. This genetic difference will likely explain the structuraldifferences observed.

(147) Mutagensis of Conserved Cysteines in HyaluronanSynthase Can Uncouple Polymerizing Activity and Product

Size Control, Two Discrete Enzyme FunctionsBruce A. Baggenstoss, Paul H. Weigel

Univ of Oklahoma Health Sciences Center, Oklahoma City, OK

The Streptococcus equisimilis hyaluronan (HA) synthase (SeHAS)contains four Cys residues (C226, C262, C281 and C367) that areconserved in the Class I HAS family, including the three humanisozymes. In previous studies of single Cys-to Ser and multipleCys-to-Ala SeHAS mutants, we found that: (i) a Cys-null mutant isactive, (ii) Cys modification inhibits HAS activity, and (iii) the con-served Cys residues are clustered at the membrane-enzyme interfacein or near substrate binding sites (Kumari and Weigel, Glycobiology15, 529, 2005). Based on growing interest in the size-dependencefor many biological functions of HA, we examined this panel ofCys mutants using a single technique (size exclusion chromato-graphy coupled to multi-angle laser light scattering) that allowssimultaneous assays on the same sample for both HA synthesisactivity and HA product size. Among 18 mutants compared to wild-type, three showed no change in either function and three showedchanges in both (decreased activity and HA size). However, onlyone function was altered in the 12 other mutants, which showedeither decreased HA polymerizing activity or decreased productsize. No mutants made larger HA, eight made smaller HA, and 10showed no change in size. About half the mutants showed nochange in activity, and half were less active. The mutants fell into 4of 9 possible groups in terms of changes in HA size and/or syn-thesis rate (i.e. no change, increased, or decreased). Changes inspecific Cys residues were associated with each group. We concludethat: (1) the four conserved Cys residues, individually and inspecific combinations, control both the rate of HA polymer assem-bly by HAS and the HA product size, and (2) their participation inone enzyme function can be uncoupled from, and is thus indepen-dent of, the other function. (Supported by NIH grant GM35978)

(148) A Novel UDP-GlcNAc/GalNAc 4-epimerase, MMP1090,from the Archaeon Methanococcus Maripaludis

Yan Ding, Ken Jarrell, Inka BrockhausenQueen’s University, Kingston, Canada

Methanococcus maripaludis are methane-producing anaerobiccocci that move through the action of flagella. These archaeaassemble flagella and pili composed of flagellin and pilin glyco-proteins, respectively. The flagella are composed of three differentflagellins, all modified at multiple positions with an N-linkedtetrasaccharide, based on GalNAc beta-linked to an Asn residue.The Mmp1090 gene is part of a cotranscribed unit encompassing


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MMP1089-1094, and has significant similarity to 4-epimerasegenes from other bacteria. In vitro assays using a purifiedHis-tagged version of MMP1090 expressed in E. coli showed thatthe enzyme catalyzes the reversible conversion betweenUDP-GlcNAc and UDP-GalNAc, indicating that this enzyme is aUDP-GlcNAc/GalNAc 4-epimerase. The enzyme products wereisolated using a GSL I lectin column. MMP1090 favored the con-version of UDP-GalNAc to UDP-GlcNAc, and the equilibratedmolar ratio of UDP-GalNAc: UDP-GlcNAc was 1: 2.5. Theenzyme also converted UDP-Glc/UDP-Gal with a preference forUDP-Gal, but did not act on GlcNAc/GalNAc-pyrophosphate-lipids. MMP1090 did not require divalent metal ions but didrequire NAD+ as a cofactor. The optimum pH for activity was pH9. The combined results indicate that MMP1090 is a novel proteinwith 4-epimerase activity that might be involved in the synthesisof flagellin glycans. This work is supported by Discovery Grantsfrom the Natural Sciences and Engineering Research Council ofCanada to K.F.J. and to I.B. Y.D. thanks the China ScholarshipCouncil (2010622028) for financial support.

(149) Subcomponent Analysis of MultivalentPolysaccharide-Based Meningococcal Vaccines from ChinaMatthew C. Cook1, Sabrina Gibeault2, Vasilisa Filippenko1,

Qiang Ye3, Junzhi Wang3, Jeremy P. Kunkel11Centre for Vaccine Evaluation, Health Canada, Ottawa, ON,Canada; 2La Cité Collégiale, Ottawa, ON, Canada; 3NationalInstitutes for Food and Drug Control, Beijing, People’s Republic

of China

The active components of most meningococcal (Neisseria menin-gitidis) vaccines are four antigenic serogroup polysaccharides(ACYW135) derived from the bacterial capsule. The vaccines,monovalent or multivalent mixtures of either free polysaccharidesor polysaccharides conjugated to antigenic carrier peptides, maybe in liquid or lyophilized formulations with or without excipients.International collaborations between the Centre for Vaccine

Evaluation of Health Canada and the National Institutes for Food andDrug Control of the State Food and Drug Administration (People’sRepublic of China) allow for unprecedented access to biologicaltherapeutics and vaccines available in China. Methods for serogrouppolysaccharide analysis of meningococcal vaccines, which had beendeveloped and qualified at Health Canada with standards and anarrow range of commercial vaccines, were challenged to measurethe subcomponents present in a broad collection of previously inac-cessible multivalent vaccines provided by the NIFDC.Previously optimized hydrolysis and chromatographic methods

were used to analyze serogroup subcomponents in 23 lots from 8different polysaccharide-based meningococcal vaccine products forinterlot and interproduct comparisons. Replicate operations by threedifferent analysts further challenged the methods. Analysis of YandW135 serogroups required the removal of interfering lactose excipi-ent prior to hydrolysis. One particular brand of non-cellulosic cen-trifugal filter proved far superior to all other dialysis and filtrationoptions tested for this purpose.Centrifugal filtration successfully removed lactose excipient

without loss of polysaccharides to allow for the determination of Y

and W135 serogroups. Statistical analysis indicated that the methodswere remarkably reproducible when challenged by a greater varietyof vaccines and multiple analysts (CV <5%). Results indicated someinterlot and interproduct variations. However, all vaccines werewithin acceptable specifications for each serogroup polysaccharide,with the exception of all lots of one vaccine – which were deficientin the serogroup A polysaccharide subcomponent.

(150) Synthesis of 2-FL and LDFT by metabolicallyengineered E. coli through the fkp gene from Bacteroides

fragilisFernando J. Arteaga-Cabello1, Miriam T. Arciniega-Fuentes1,

David S. Newburg1, John McCoy3, Guillermo M. Ruiz-Palacios11INCMNSZ, Mexico City, Mexico; 2Boston College, Chestnut Hill,

MA; 3Glycosyn, Inc., Medford, MA

Recently, the interest to synthesize different fucosylated oligosac-charides has increased. In several processes reported in E. coli,the main drawback is the low intracellular levels of GDP-fucoseused by the fucosyltransferases, producing unacceptable yields.Authors have increased yields by improving their synthesisthrough modification of the de novo pathway with excellentresults. Here we propose an alternative synthetic route forGDP-fucose, the “salvage pathway” reported in Bacteroidesfragilis, where it contributes to capsule construction, and similarto the “salvage pathway” from mammalians. We used an E. coliunable to use the D-lactose and L-fucose (LacZ− and FucI−) assource of energy, subcloned with genes futC from H. pylori,which encodes the α1,2-fucosyltransferase; fkp from B. fragilis,which encodes the bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase; and fucR, a transcriptional acti-vator of the fuc operon. Using this construct, we standardized ahigh-cell density fermentation process (OD600 = 110–120),where 2-FL and Lactodifucotetraose (LDFT) were synthesized(confirmed by NMR spectroscopy) at concentrations up to 10 g/Land 0.5 g/L, respectively, starting from concentrations of 10 g/Llactose and 5 g/L of L-fucose, with complete consumption oflactose. By modifying the initial concentrations of these sugarswe improved ratio up to 5:1 (2-FL:LDFT). Although the pro-duction of LDFT as a byproduct of the reaction was unexpected,instead of being a problem, it adds value to the processbecause, with 2-FL, LDFT is one of the major fucosylatedoligosaccharide constituents of milk from secretor mothers, andits structure is of great importance in the study of this group ofoligosaccharides. This process is highly efficient and possible toescalate, for the massive production for clinical use of variousoligosaccharides using different fucosyltransferases.

(151) Methanosarcina mazei Sweet Uniqueness at its CellSurface Protein Glycans

Deborah Francoleon1, Rachel Ogorzalek Loo2, Joseph Loo2,A. Jimmy Ytterberg2, Unmi Kim2, Robert Gunsalus2,

Catherine Costello11Boston University School of Medicine, Boston, MA; 2UCLA, Los

Angeles, CA


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Little is known about glycosylation in archaea, the least under-stood kingdom of life, and it may differ substantially from thatof mammals. The versatile archaeon Methanosarcina mazei(M. mazei) produces methane via all known methanogenesispathways. Lacking cell walls, this organism is surrounded by aproteinaceous surface layer structure (S-layer). We had previouslydescribed MM1976 as the S-layer protein of M. mazei, as a gly-coprotein with irregular SDS-PAGE migration and possibly con-taining α-D-mannose or α-D-glucose. Here we use ConcanavalinA (ConA) lectin affinity chromatography to enrich the S-layerprotein. MALDI-TOF MS was employed to measure the intactmass of MM1976 from ConA eluate and yielded three majorresolved glycoforms with averaged glycan mass contributions of�1,612 Da, �2,819 Da, and �4,337 Da. Trypsin-digestedConA eluate was enriched for glycopeptides by ERLIC(Electrostatic Repulsion Hydrophilic Interaction) and the fractionswere analyzed by direct infusion nanoESI-MS/MS.Uncharacteristic of Archaea, tandem MS revealed a unique,novel core structure common to all the identified glycopeptides.Attached to the peptide, arranged in a linear fashion, the core iscomposed of 4 Hex with a dHex residue at the non-reducingend (dHex Hex4). Most observed glycopetide structures con-tained a tri-branched substituted octasaccharide structure of com-position Hex5dHex2HexNAc with molecular mass 1,305 Da.Glycopeptides from four different surface glycoproteins con-tained the same tri-substituted octasaccharide structure, hintingthat this glycan motif might be conserved among M. mazeisurface proteins. We are currently working to determine thepeptide glycosylation sites through multistage tandem MS/MSemploying CID, HCD and in some cases ETD fragmentation.Also, in order to account for all the glycan mass contributionspresent in MM1976, the ERLIC fractions are being subjected toreversed phase and HILIC chromatography coupled to MS/MSanalysis. This research is supported by NIH-NCRR grants P41RR010888, S10 RR020946 and S10 RR025082.

(152) Glycoinositolphospholipids from Leishmania braziliensisand Leishmania infantum: Modulation of Innate Immune

System and Variations in Carbohydrate StructureRodrigo Soares1, Rafael Assis1,3, Izabela Ibraim1,

Fátima Noronha3, Salvatore J. Turco21Oswaldo Cruz Foundation, Belo Horizonte, Brazil; 2University ofKentucky, Lexington, KY; 3Department of Microbiology, UFMG,

Belo Horizonte, MG, Brazil

The essential role of the lipophosphoglycan (LPG) ofLeishmania in innate immune response has been extensivelyreported. However, information about the role of the LPG-relatedglycoinositolphospholipids (GIPLs) is limited, especially withrespect to the New World species of Leishmania. GIPLs are lowmolecular weight molecules covering the parasite surface and aresimilar to LPG in sharing a common lipid backbone and aglycan motif containing up to 7 sugars. Critical aspects of theirstructure and functions are still obscure in the interaction withthe vertebrate host. In this study, we evaluated the role of thosemolecules in two medically important South American species

Leishmania infantum and Leishmania braziliensis, causativeagents of visceral (VL) and cutaneous Leishmaniasis (CL),respectively.

GIPLs derived from both species did not induce NO orTNF-α production by non-primed murine macrophages.Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2 -/- and TLR4 -/-) exposed to GIPLs from both species,with exception to TNF-α, did not induce the production ofdifferent cytokines (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40,IFN-γ) or p38 activation. GIPLs induced the production ofTNF-α and NO, primarily via TLR4. Pre incubation of macro-phages with GIPLs reduced significantly the amount of NO andIL-12 even in the presence of IFN-γ and lipopolysaccharide(LPS), which was more pronounced with L. braziliensis GIPLs.A structural analysis of the GIPLs showed that L. infantum hasType I GIPLs while L. braziliensis has Type II.

The structural differences in the GIPLs are responsible forthe differential macrophage modulation during the early steps ofinfection. Thus, GIPLs are important inhibitory molecules duringthe interaction with innate immune compartment and likely to becritical in enabling Leishmania survival and proliferation in thehost.

(153) Leishmania amazonensis Glycosphingolipids,Glycosylinositolphospholipids and InositolPhosphorylceramide Expression duringAmastigote-Promastigote Differentiation

Anita Paula Ortiz De Godoy, Helio K. Takahashi, Anita H. StrausUniversidade Federal de Sao Paulo, São Paulo, Brazil

This study was focused in the analysis of the expression of glyco-sphingolipids (GSLs), glycosylinositolphospholipids (GIPLs) andinositol phosphorylceramide (IPC) in promastigote and amastigoteforms of Leishmania (Leishmania) amazonensis, the main causa-tive agent of cutaneous leishmaniasis in South America. Parasite(glyco)(sphingo)lipids expression were analyzed by high perform-ance thin layer chromatography, ESI/MS, indirect immunofluores-cence and flow cytometry. Levels of IPC synthase (ipcs) geneand inositol phosphosphingolipid phospholipase C-Like (iscl)gene were analyzed by real-time PCR. Two mAbs, LST-1 andLST-2 were used to identify promastigote IPC and GIPLs. LST-1detected IPC preferentially located at the inner leaf of plasmamembrane, whereas LST-2 detected GIPLs at promastigotesurface. Both LST-1 and LST-2 reacted only with promastigotes,indicating that IPC and GIPLs are promastigote-stage-specificantigens. On the other hand, mAb ST-4, directed to L. amazonen-sis glycosphingolipids only recognized amastigote forms. Whenamastigotes were cultured at 23°C, differentiation to promasti-gotes is observed by morphological changes, decrease of parasitereactivity with mAb ST-4, and a progressive increase of parasitereactivity with mAbs LST-1 and LST-2. In amastigotes theexpression of ipcs gene was similar to that observed in promasti-gotes. Thus the low expression level or absence of IPC in amasti-gotes may result from the high levels of iscl expression. Asalready described for L. amazonensis GSLs (Straus et al, 1993),and now for GIPLs and IPC, these lipids are expressed


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differentially in promastigotes and amastigotes, i.e. IPC isexpressed preferentially in promastigotes and it is modulatedmainly by the iscl expression whereas, GIPLs and neutral glyco-sphingolipids are expressed mainly in amastigotes and promasti-gotes, respectively. The steps involved in the L. amazonensisgenetic control responsible for switching GSLs to GIPLs onamastigote–promastigote differentiation are under investigation,and may contribute for the development of new therapeuticalstrategies in Leishmaniasis.Supported by FAPESP, CNPq and CAPES.

(154) N-glycan Specific Lectin (RVL) from Remusatia viviparawith Potent Insecticidal Activity

Murugesh Swamy BaleKarnatak University, Dharwad, India

Complex high mannose type glycan binding lectin (RVL) purifiedfrom the edible tubers of Remusatia vivipara from araceae familyshowed potent nematicidal activity against Meloidogyne incognita.RVL showed amino acid sequence similarity with other highmannose binding lectins of this family (Bhat et al., 2010) and itsrecent X- ray crystallography studies determined at 2.4 Å revealedβ-prism II (BP2) fold structure. Glycan array analysis revealed thatRVL recognizes N-linked complex glycans but not O- linkedsugar chains. Tri-mannosyl pentasacharide core is absolute necess-ary for binding to N linked complex glycans and it show greateraffinity towards the complex fucosylated core structures howeverfucosylation is not mandatory for binding. Highest binding affinitywas shown for Fucα1-2Galβ1-4(Fucα1-3) GlcNAcβ1-2Manα1-6(Fucα1-2Galβ1-4(Fucα1-3) GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4(Fucα1-6) GlcNAcβ-6AA, where as its nonfucosylated counterpart Galβ1-4GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 showednegligible binding, indicating the importance of fucosylation for theRVL binding. Interestingly the lectin binds to GP120 HIV viral coatprotein. In the present study we determined its effect on the larvaeof Spodoptera litura, a major economic threat for many of the agri-cultural crops worldwide. S. litura larvae maintained on artificialdiet containing purified lectin showed remarkable larval mortalityand was dose dependent. Two RVL binding receptors with Mr. 66and 39 kDa were identified from the gut epithelial cells of S. lituralarvae by lectin blot studies. Considering its interaction with theglycans of epithelial gut cells of nematodes and insects RVL can bea valuable tool to understand the nature of these glycans from lowerorganisms.

(155) Toxoplasma Skp1 is Modified by aHydroxyproline-Dependent Cytoplasmic Glycosylation

Pathway Similar to that Which Modulates Oxygen-Sensingin Dictyostelium

Yuechi Xu, Zhuo Wang, Hanke van der Wel, Kevin Brown,Ira Blader, Christopher West

University of Oklahoma Health Sciences Center, Oklahoma City,OK

HIFα-type prolyl 4-hydroxylases mediate responses to hypoxia inanimals via stabilization of HIFα, a major transcriptional factor.

Recent studies have shown that a related gene (DdP4H1) contrib-utes to oxygen regulation of development in the social amoebaDictyostelium, by a divergent mechanism involving hydroxylationand subsequent glycosylation of Skp1, an adaptor subunit of theSCF class of E3 ubiquitin ligases. Sequences related to DdSkp1,DdP4H1, and glycosyltransferases that form a chain that capsSkp1-hydroxyproline, occur in the genomes of Toxoplasma gondiiand other protists, suggesting that this mechanism of oxygen-sensing may be widespread. Here we show that recombinantTgSkp1 and DdSkp1 are each excellent substrates for recombinantTgP4Hb and DdP4H1. Western blot analysis of tachyzoitesrevealed an apparent Mr larger than that of recombinant TgSkp1and absence of a proline epitope present on unmodified Skp1, inparallel with findings in Dictyostelium. To test the contribution ofTgP4Hb to the Mr difference, its locus in the RH-strain wasknocked out and disruption confirmed by PCR. The apparent Mr

of tachyzoite Skp1 was reduced, confirming the suspected enzy-matic function of TgP4Hb in vivo. The extent of the Mr shiftimplicates the previously detected αGlcNAc-transferase-likeactivity and another glycosyltransferase-like gene in contributingto the Mr of wild-type TgSkp1, and furthermore suggests the exist-ence of a second modification. A possible cellular function issuggested by the ability of overexpressed TgP4Hb to partiallycompensate deletion of P4H1 in Dictyostelium oxygen-sensing,whereas an enzyme-dead mutant TgP4Hb did not. These findingsindicate conservation of the Skp1 hydroxylation/glycosylationpathway across divergent protist phyla, and offer a molecularapproach to investigate oxygen and metabolic sensing duringToxoplasma life cycle progression.

(156) Selective Mild Acid Hydrolysis and Structure of a NovelFucan from Sea Cucumber, as well as Antithrombotic Activity

Shiguo Chen1, Xingqian Ye1, Changhu Xue3, Guoyun Li3,Jingfeng Wang3, Guangli Yu3, Li’ang Yin3, Wengang Chai21Zhejiang University, Hangzhou, China; 2Imperial College

London, Harrow, United Kingdom; 3Ocean University of China,Qingdao, China

A novel sulfated fucan was isolated from sea cucumberIsostichopus badionotus. No specific enzymatic or chemicalmethod is available for the preparation of tailored oligosaccharidesfrom this novel sulfated fucans, we employed an apparently non-specific approach to cleave this polysaccharide based on mildhydrolysis with acid. Surprisingly, the linear sulfated fucan wascleaved by mild acid hydrolysis on an ordered sequence. Thestructural of the novel fucan was assigned by negative-ion electro-spray tandem mass spectrometry with collision-induced dis-sociation (ESI-CID-MS/MS) followed with1D and 2D NMRanalysis of the oligosaccharide fractions produced by mild acidhydrolysis. It were finally identified as a regularly repeatingsequence of [3-α-L-Fucp-(2, 4SO4)-(1→ 3)- α-L-Fucp-(2SO4)-(1→ 3)-α-L-Fucp-(2SO4)-(1→ 3)- α-L-Fucp-(1→ ]n. Acidchooses a preferential cleavage of the glycosidic linkage formedbetween the nonsulfated residue (unit D in Figure 1) and thesecond 2, 4-sulfated unit (unit A in Figure 1), no obvious loss ofsulfate group happened. Bioactivity assay indicated the fucan


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possessed much lower anticoagulant activity than the fucosylatedchondroitin sulfate (fCS-Ib) in I. badionotus, but had better effectto inhibit both thrombin and factor Xa when mediated byantithrombin-III. Further antithrombotic assay in vitro surprisinglyfound fucan had almost the same effect on inhibiting the gener-ation of thrombus in vitro compared with the fCS-Ib and LWMH.Those results indicated that the linear fucan in I. badionotus mightbe a better antithrombotic drug, with high-efficiency but lower riskof bleeding.

(157) Redesigning the Carbohydrate Recognition Site of HenLysozyme

Gabriel Gutierrez-MagdalenoMexico, Df, Mexico

In recent years, our research group has been involved in the studyof protein-carbohydrate interactions, trying to clarify the under-lying energetic-structural principles of the phenomenon. As a newstage in our investigation, we have aimed to redesign the protein’srecognition site, changing its specificity towards other saccharidicstructures. The primary target of the present project is to redesignthe recognition site of hen’s egg lysozyme, to make it specific forglucose β(1-4) oligomers. Using the crystal structure of lysozymewith chitotriose (GlcNAcβ(1-4)GlcNAcβ(1-4)GlcNAc) as a tem-plate, a model of lysozyme-binding cellotriose (Glcβ(1-4)Glcβ(1-4)Glc) was built. After several cycles of mutations andenergy minimizations using Rosetta, a construct was obtainedwhose binding energy was comparable to that of the wildlysozyme-chitotriose complex. Mutations Ile98Gln, Ile58Gln andLeu75Arg optimized interactions with OH(2) groups in cellotriose.The optimal conformation of the two new Gln residues was stabil-ized by mutations Leu56Ser, Trp108Tyr, which make no directcontact with the ligand. Furthermore, mutations Ala107Asn andTrp63Tyr yielded increased affinity by improving interactions withcommon groups in cellotriose and chitotriose. Recombinant wildlysozyme was obtained in Aspergillus niger. Recombinant proteinwas characterized by means of circular dichroism and fluorescencespectroscopy, and its binding to chitotriose was measured calori-metrically. Overall, the secondary and tertiary structures, thethermal stability and the binding properties of recombinant lyso-zyme were identical to those of lysozyme purchased from SIGMA.Advances in the expression, purification and structural and ener-getic characterization of different lysozyme mutants are presentedhere.Key words: Lysozyme, desing, recognition site.

(158) The Anti-angiogenic Activity of the Extractsand Polysaccharides from Karenia mikimotoi

Chengyu Tan, Ying Zhao, Di Wu, Qian Li, Haibei Hu,Minhui Ye, Wei Li

Dalian Ocean University, Dalian, China

Karenia mikimotoi is a kind of bloom microalgae which can oftenresult in serious ocean harm in the world. More studies focused onits toxin about K. mikimotoi and its control. The article was on the

anti-angiogenic activity of the extracts and polysaccharides fromK. mikimotoi. The two extracts were obtained by use of ethanol-water (v/v 70:30) and water under boiling. The water extract wasadded to 3% trichloroacetic acid to get rid of protein, then ethanolwas added up to 80% to precipitate polysaccharide. The content ofsoluble sugar may reach 22.4%. The effect of two extracts andpolysaccharide on proliferation and migration of human umbilicalvein endothelial cells (HUVECs) induced by 10% MCF-7 culturefluid was measured. The results showed that two extracts had notoxicity to normal HUVECs, but 70% ethanol extract might inhibitthe proliferation of induced HUVECs under the concentration of0.2, 0.5 mg·mL−1, the inhibition rate might be 40.2, 38.0%respectively, while the water extract and its polysaccharide mightinhibit the migration of induced HUVECs under the concentrationof 100, 50 µg·mL−1 respectively. All of these results indicated thatthe different extracts from K. mikimotoi have potential anti-angiogenic activities via different actions. The experiments wassupported by Education Department Project from LiaoningProvince (No. 2009169).

(159) Comparison of Structure and Bioactivity of Two SeaCucumber Fucosylated Chondroitin Sulfates with Slight

Difference in Sulfation of Fucose BranchesShiguo Chen1, Donghong Liu1, Guoyun Li2, Changhu Xue2,

Wengang Chai31Department of Food Science and Nutrition,Zhejiang, Hangzhou,China; 2Ocean University of China, Qingdao, China; 3Imperial

College London, Harrow, United Kingdom

Abstract: The structure and bioactivity of the two fucosylatedchondroitin sulfate (fCS-Pg) from sea cucumber P. graeffei andI. badionotus were compared. The structure investigated by 2DNMR and mass spectrometry indicated that that the two polysac-charides have identical monosaccharide structure( Fuc: GlcA :GalNAc = 1:1:1), almost the same size chain and sulfate content,as well as the same chondroitin sulfate E backbone. However, thesulfation pattern of their fucose branches were different, FCS-Pgwas mainly 3,4-O-sulfated whereas fCS-Ib was mainly with 2,4–O-SO4. As a consequence of the differences, the two fCSs differedgreatly in their bioactivity. FCS-Pg has much lower in vitro antic-oagulant activity then FCS-Ib, and both the two polysaccharidesexhibited anticoagulant activity by potentiation of antithrombin IIIand heparin cofactor II activity on thrombin and FXa, but low IC50 was needed when mediated by Heparin cofactor II. In con-trasted with the FCS-Ib, FCS-Pg has much lower acuity in bothinhibition the thrombin and FXa. Further antithrombotic assay invitro surprisingly detected that they had almost the same effect oninhibiting the generation of thrombus, also the same level asLWMH. This results indicated that not only the location of2,4-disulfated fucose units in the polysaccharide chains dictates tothe inhibiton of thrombosis, but also 3,4-disulfate-fucose branches.Our experimental indicated that the FCS-Pg from P. graeffei mightbe a better antithrombotic drug, with high-efficiency but lower riskof bleeding.


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(160) Optimized Linkage Analysis of Oligo- and PolysialicAcids by Fluorescence Labeling and PermethylationHildegard Geyer1, Sebastian P. Galuska1, Werner Mink1,Peter Kaese1, Rita Gerardy-Schahn2, Rudolf Geyer1

1Institute of Biochemistry, Justus-Liebig-Universit, Giessen,Germany; 2Hannover Medical School, Hannover, Germany

Oligo- and polysialic acid (oligo- and polySia) chains can be syn-thesized by many prokaryotic as well as eukaryotic organisms andare involved in a number of distinct biological processes depend-ing on their chain lengths, the type of sialic acids present and theirglycosidic linkages. For the determination of the length of the oli-gomer/polymer chains and the sialic acid constituents a wide panelof highly sensitive techniques is available, but no microscalemethod exists for linkage analysis. Here, we describe a highly sen-sitive procedure for the analysis of the glycosidic linkages presentwithin oligo- and polySia chains by permethylation after precedingfluorescence labeling of the reducing end. Using α2,8- andα2,9-linked sialic acid polymers the type of linkage could bedetermined employing less than 10 ng of each polymer. Moreover,a mixture of different oligo- and polySia species can be separatedby anion-exchange chromatography after fluorescence labeling andpeaks of interest can be collected individually for methylationanalysis. The described strategy offers a very sensitive and effi-cient detection of internal sialic acid residues and their glycosidiclinkages.

(161) Production of an Antibody against Long-FormN-acetylglucosamine-6-O-sulfotransferase 1 that is Expressed

in Human High Endothelial VenulesMaiko Fujiwara1, Motohiro Kobayashi1, Hitomi Hoshino2,Kenji Uchimura2, Yasuhiro Sakai1, Tsutomu Nakada3,

Minoru Fukuda4, Jun Nakayama11Shinshu University Graduate School of Medicine, Matsumoto,Japan; 2National Center for Geriatrics and Gerontology, Obu,Japan; 3Shinshu University, Matsumoto, Japan; 4Burnham

Institute for Medical Research, La Jolla, CA

Lymphocyte homing is mediated by an adhesive interactionbetween L-selectin expressed on lymphocytes and its carbohydrateligands 6-sulfo sialyl Lewis X (sLeX)-capped glycoproteins, col-lectively called peripheral lymph node addressin (PNAd),expressed on high endothelial venules (HEVs) in the lymph nodes.N-Acetylglucosamine (GlcNAc)-6-O-sulfation in the sLeX oligo-saccharides is critical for L-selectin binding, which is catalyzed byGlcNAc-6-O-sulfotransferases (GlcNAc6STs). Among fiveGlcNAc6STs cloned in humans, GlcNAc6ST-1 and GlcNAc6ST-2have been confirmed to be expressed in HEVs, and play a criticalrole in the biosynthesis of L-selectin ligand. Human GlcNAc6ST-1has been cloned as a 1,593-bp open reading frame at the 5′ endwith two in-frame methionine codons, apart from 141 bp, thatagree with the consensus sequence for translation initiation. Itcould be speculated that both long and short forms ofGlcNAc6ST-1 may present; however, most studies so far employedexpression vectors harboring cDNA encoding a short form of theenzyme. Moreover, expression of the long-form GlcNAc6ST-1

protein has not been confirmed. In the present study, we havedeveloped an antibody recognizing the amino acid residuesbetween the first two methionines of human GlcNAc6ST-1,which specifically recognizes the long-form. Western blot analy-sis and double immunofluorescence staining with this antibodyand Golgi markers, we found that the long-form protein isendogenously expressed in humans, predominantly intrans-Golgi network (TGN) of HEV-composing endothelialcells. We further evaluated possible difference of subcellularlocalization between the two forms of GlcNAc6ST-1 by dualimmunofluorescence staining of Hela cells doubly transfectedwith cDNAs encoding long and short forms, but no apparentdifference was observed. In addition, we compared intracellularGlcNAc-6-O-sulfation activity between the two forms, but noapparent difference was observed. This work was supported byGrants-in-Aid for Young Scientists B-2079027 and B-22790343from the MEXT Japan and in part by a grant PO1 CA71932from the NIH.

(162) Glycoengineering Insect Cells for a2,3-sialylationof Recombinant Glycoproteins

Hideaki Mabashi-Asazuma, Donald L. JarvisUniversity of Wyoming, Laramie, WY

Sf9 is an insect cell line that is widely used as a host for recombi-nant glycoprotein expression by baculovirus expression vectors.We previously glycoengineered these insect cells to produceα2,6-sialylated glycoproteins by transformation with mammaliangenes including rat ST6GalI. More recently, we have focused onengineering α2,3-sialylation using two native α2,3-sialyltransferasegenes, mouse ST3GalIII and ST3GalIV, and two engineeredα2,3-sialyltransferase genes, ST3fix and hST3GalIVb. ST3fix is amouse ST3GalIII construct in which the N-terminal cytoplasmicanchor/transmembrane domains were replaced by the correspond-ing domains from rat ST6GalI. hST3GalIVb is a humanST3GalIV construct engineered to have two additional dibasicmotifs proximal to its transmembrane domain to enhance its ER toGolgi transport. Expression of GFP-tagged versions of these fourST3 constructs in Sf9 cells showed that hST3GalIVb was mostclosely colocalized with a Golgi marker. Interestingly, however,hST3GalIVb-transfected Sf9 cell extracts had no ST3 activitywhen assayed in vitro. Assessment of their in vivo functions byusing a baculovirus to co-express human erythropoietin as anacceptor molecule showed that all four ST3 constructs inducedα2,3-sialylation of human erythropoietin, but hST3GalIVb pro-vided the highest efficiency. Thus, the hST3GalIVb construct,which had extra dibasic motifs proximal to its transmembranedomain, induced the highest level of Golgi localization and in vivofunction and this construct was therefore used to isolate the firstinsect cell line capable of producing α2,3-sialylated recombinantglycoproteins.

(163) Examining the Sialic Acid Salvaging Pathwayin Lepidopteran Insect CellsAnn M. Toth, Donald L. Jarvis

University of Wyoming, Laramie, WY


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Previously, we reported evidence for a sialic acid salvagingpathway in insect cells glycoengineered to express mammalianglycosyltransferases, but no enzymes for CMP-sialic acid biosyn-thesis or transport. Growth media containing fetal bovine serum orfetuin, a serum sialoglycoprotein, supported de novo glycoproteinsialylation by these engineered cells. Our interpretation was thatinsect cells can take up extracellular sialoglycoproteins, removetheir sialic acids, and reutilize them in an endogenous pathwaythat yields CMP-sialic acid for glycoprotein sialylation. In thisstudy, we further examined this model. Both parental (Sf9) andglycoengineered (SfSWT-1) insect cells cultivated with [3H]-sialicacid-labeled fetuin took up radioactivity in a saturable manner.Low temperature and ammonium chloride inhibited uptake, indi-cating that the mechanism involved receptor-mediated endocytosis.Fetuin or asialofetuin, but not BSA, also inhibited uptake, indicat-ing that fetuin was specifically recognized independently of its sia-lylation status. The incorporated radioactivity was almost entirelyconverted to an acid-soluble form, suggesting that endogenous sia-lidases removed sialic acid from the radiolabeled fetuin.Furthermore, in SfSWT-1 cells, which express functional mamma-lian glycosyltransferases, a small percentage of the incorporatedradioactivity was acid-insoluble and neuraminidase-sensititive,suggesting that the released sialic acid had been converted toCMP-sialic acid and utilized by the ectopic sialyltransferase for denovo glycoprotein sialylation. Finally, SfSWT-1 and SfSWT-6cells cultivated with [3H]-sialic acid-labeled fetuin containedsimilar percentages of acid-precipitable radioactivity. Thus, bothcell types salvaged similar levels of sialic acid, despite the factthat SfSWT-6 cells express a functional mammalian CMP-sialicacid synthetase and CMP-sialic acid transporter in addition tomammalian glycosyltransferases. These results support the ideathat Sf9 cells have a sialic acid salvaging pathway that can convertfree sialic acids to CMP-sialic acids, which ultimately appear inthe Golgi, where they can be utilized as donor substrates byectopically expressed sialyltransferases.

(164) Phosphatase-coupled Sulfotransferase AssayBrittany Prather, Cheryl Ethen, Miranda Machacek,

Zhengliang WuR&D Systems Inc, Minneapolis, MN

Sulfotransferases are a large group of enzymes that transfer sulfatefrom the donor substrate, 3′-phosphoadenosine-5′-phosphosulfate(PAPS), to various acceptor substrates, generating 3′-phosphoade-nosine-5′-phosphate (PAP) as a by-product. A universalphosphatase-coupled sulfotransferase assay is described here. Inthis method, Golgi-resident PAP-specific 3′-phosphatase is used torelease the 3′-phosphate from PAP and generate 5′-adenosine mono-phosphate (5′-AMP). In addition, CD73, a 5′- nucleotidase, can beused to release the 5′-phosphate from 5′-AMP to increase the assaysensitivity by two-fold. The released phosphate is then detectedusing malachite green phosphate detection reagents. This assayeliminates the need for both radioisotope labeling and substrate-product separation, and is high-throughput compatible. Using themethod, we measured the enzyme kinetics of well studied

SULT1A1 and SULT2A1. The measured Km values are in therange of the reported values. In addition, we measured the enzymekinetics of SULT1C4, CHST3 and CHST10. The kinetic data mayallow us to better understand the biological functions of theseenzymes.

(165) A Role for Endothelial Mannose Residues inInflammation Dependent Monocyte Adhesion Under Flow

David W. Scott, Balu K. Chacko, Rakesh P. PatelUniversity of Alabama at Birmingham, Birmingham, AL

Cardiovascular disease is leading causes of morbidity and mor-tality in the Western world and is often initiated by atherosclerosis,a progressive disease of the vasculature. A key event in athero-sclerosis is monocyte adhesion and extravasation at sites of vascu-lar inflammation. These sites are characterized by the expressionof adhesion molecules, which are known to be N-glycosylated, yetfew studies have examined if this modification plays a role in med-iating endothelial-monocyte interactions. Using a lectin screeningassay we found that TNFalpha stimulated high-mannose/hybridN-glycans expression on the apical surface of endothelial cells,without affecting complex N-glycans or O-glycan species A glyco-sylation specific PCR array demonstrated that TNFalpha stimu-lation decreased alpha-mannosidase transcripts (MAN1A2,MAN1C1), which resulted in a decrease in alpha-mannosidaseactivity. These TNFalpha dependent effects were inhibited by pre-incubating cells with the PPARgamma agonist rosiglitazone.Furthermore, rosiglitazone attenuated monocyte adhesion duringflow without altering protein expression of canonical adhesionmolecules (ICAM-1, VCAM-1, CD62e) responsible for monocyterolling and adhesion, thereby suggesting that adhesion moleculesalone are insufficient for mediating adhesion. To determine ifmannose residues could be participating in monocyte adhesion,flow dependent monocyte adhesion was determined in the pres-ence and absence of alpha-mannopyranoside. Providing competingmannose epitopes in the flow media attenuated TNFalpha inducedmonocyte adhesion under flow, but not under static conditions.Finally, immunoprecipitation studies using the high mannose/hybrid selective lectin, ConA, coupled with LC-MS based proteinidentification revealed both ICAM1 and VCAM1 as potentialadhesion molecules whose N-glycosylation is regulated byTNFalpha. Collectively, these data suggest that TNFalpha treat-ment inhibits early N-glycan processing leading to the presentationof high-mannose/hybrid N-glycans on the endothelial cell surfaceand that these mannose residues participate in monocyte adhesionduring vascular inflammatory stress.

(166) Glycoengineered Insect Cells Can Use a BacterialN-acetylglucosamine-6-phosphate 2′-epimerase to Produce

Sialic Acid without Exogenous PrecursorsChristoph Geisler, Donald L. JarvisUniversity of Wyoming, Laramie, WY

Lower eukaryotic glycoprotein production systems such as thebaculovirus/insect cell system are unable to routinely produce sia-lylated glycoproteins. We previously glycoengineered the


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baculovirus/insect cell system by transformation with highereukaryotic genes encoding the biosynthetic machinery required forsialic acid biosynthesis and utilization. However, even the mostadvanced of our previously engineered systems required the use ofa growth medium supplemented with N-acetylmannosamine, anexpensive sialic acid precursor. To avoid this requirement, we havenow glycoengineered insect cells with an E. coli gene encodingN-acetylglucosamine-6-phosphate 2′-epimerase. This bacterialenzyme normally functions in sialic acid degradation by convert-ing N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate in a single, reversible step. As insect cells have thelatter, but lack the former, we hypothesized that the bacterialenzyme expressed in insect cells would function in reverse andproduce N-acetylmannosamine-6-phosphate from endogenousN-acetylglucosamine-6-phosphate. We tested this hypothesis innew insect cells lines glycoengineered to express the bacterialN-acetylglucosamine-6-phospate 2′-epimerase together with a suiteof higher eukaryotic genes required for downstream sialic acid bio-synthesis and utilization. Sialic acid assays showed that these celllines produced sialic acid without any N-acetylmannosamine sup-plementation. Importantly, the new glycoengineered insect celllines also were able to produce sialylated glycoconjugates withoutany supplementation. Our data indicate that the reaction catalyzedby N-acetylglucosamine-6-phospate 2′-epimerase in bacteria iseffectively reversed in insect cells to yield the sialic acid precursorrequired for glycoprotein sialylation. In conclusion, this new gly-coengineering strategy expression circumvents the need to addN-acetylmannosamine to the growth medium and provides aviable alternative to UDP-N-acetylglucosamine 2′-epimerase/N-acetylmannosamine kinase expression for the endogenousproduction of sialic acid precursor pools.

(167) A Key Role for Mgat1 and Complex N-glycans duringSpermatogenesis

Frank Batista, Pamela StanleyAlbert Einstein College of Medicine, Bronx, NY

To investigate a requirement for Mgat1 and complex N-glycansduring spermatogenesis, conditional deletion of the Mgat1 gene inspermatogonia was performed using Stra8-iCre recombinase.Deletion efficiency was determined by PCR genotyping of tail andtestis genomic DNA, and by germline transmission of deletedalleles. Stra8-iCre deleted floxed Mgat1 alleles with 100% effi-ciency. However, no progeny were obtained from Mgat1[F/F]:Stra8-iCre males, and no sperm were found in mutant epidydimi at50 days post-partum (dpp), although some epididymi containedround spermatids. Importantly, Mgat1[F/ + ]:Stra8-iCre heterozy-gous males were fertile. Thus, the Mgat1+ allele rescued comple-tely, and the Stra8-iCre transgene had no effect on fertility. Testisweight in Mgat1[F/F]:Stra8-iCre males was markedly reduced(�34% at 50 dpp and �50% at 152 dpp). Apoptosis was increased�3.5-fold in Mgat1[F/F]:Stra8-iCre tubules at 50 dpp. In controlmales, binding of the lectin L-PHA to complex N-glycans, wasmost intense in spermatogonia, weaker in spermatocytes and

stronger again in elongated spermatids. There was essentially noL-PHA binding to germ cells in Mgat1[F/F]:Stra8-iCre males,showing that by 28 dpp, complex N-glycans were not expressed inspermatogonia or their derivatives. At 28 dpp and later, all tubuleswere aberrant and contained multinuclear giant cells (MNC), somewith elongated nuclei. Thus, complex N-glycans appear to be dis-pensable for the differentiation of spermatogonia to spermatocytesbut required for the maintenance of spermatid/Sertoli cell contact.It is of interest that transcription and translation of the novelinhibitor of Mgat1 activity termed GnT1IP are increased in sper-matocytes. Identifying the nature of the block to spermatogenesiswhen Mgat1 is deleted in germ cells, together with the elucidationof the mechanism underlying how complex and high mannoseN-glycans affect spermatogenesis, will reveal Mgat1 glycoproteintargets necessary for spermatogenesis, and novel roles forN-glycans in regulating germ cell maturation.

Supported by NIH grant RO130645 to PS.

(168) Detection of LacNAc and GlcNAc Moieties on the Cell’sSurface with Glycosyltransferases

Natalia Mercer1, Boopathy Ramakrishnan1,2, Marta Pasek1,Elizabeth Boeggeman1,2, Luke Verdi1, Pradman K. Qasba1

1SGS, CCR-NP, NCI-Frederick, Frederick, MD; 2BSP,SAIC-Frederick, Inc., SGS, CCR-NP, Frederick, MD

In the present work, we have used mutant galactosyltransferasespreviously developed in our lab to detect GlcNAc and LacNAc onthe surface of human cervical cancer cells (HeLa). The mutantenzymes have a cavity that has been carved in the donor site toaccommodate UDP-Gal with a chemical handle at C2, such asazide (GalNAz) or keto group (C2-keto-Gal). The chemicalhandles are used for conjugation with fluoroprobes or biotin carry-ing bioorthogonal group to detect the acceptor GlcNAc orLacNAc. A double mutant of b1,4-galactosyltransferase(b1,4GalT), Y289L-M344H, is used for detecting GlcNAcresidue. The Tyr289Leu (Y289L) mutation allows the carving ofthe cavity to accommodate UDP-GalNAz, whereas the secondmutation, Met344His (M344H), located in the enzyme’s metalbinding site, changes the metal cofactor requirement from Mn2+to Mg2+. Detection was investigated using confocal microscopyand flow cytometry. Green membrane fluorescent signal (corre-sponding to DIBO-Alexa 488) was detected on the HeLa cellsonly when cells were pre-treated with sialidase and galactosidaseenzymes, indicating that glycans with free GlcNAc residues arenot abundant on the surface of HeLa cells. The LacNAc moiety onthe cell surface was detected using a1,3-galactosyltransferase(a1,3GalT) mutant enzyme, which transfers GalNAz orC2-keto-Gal to N-acetyl-lactosamine (LacNAc). The GalNAzor C2-keto-Gal labeled glycans were coupled with alkyne- oraminooxy-biotin, respectively. On fixed cells, coupled biotin wasdetected with streptavidin-Alexa 488. On extracts, coupled biotinwas detected with streptavidin-HRP. Fluorescent signal could bedetected on cell membranes, as opposed to control (noUDP-GalNAz). Cells that were pre-treated with sialidase, increased


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the signal intensity, indicating that the density of exposed LacNAcresidues was augmented by the removal of sialic acid. Similarresults were obtained by Western Blot. In conclusion, the use ofb1,4GalT-Y289L-M344H and a1,3GalT-280AGG282 enzymescould be a powerful tool to study the cell’s glycophenotype.

(169) O-GalNAc Glycosylation of alpha DystroglycanDuy Tran1, Jae-Min Lim2, Mian Liu2, Lance Wells2,

Kelly Ten Hagen1, David Live21Developmental Glycobiology Unit, NIDCR, NIH, Bethesda, MD;

2University of Georgia/CCRC, Athens, GA

Alpha-Dystroglycan (aDG) is an important functional componentof the dystrophin glycoprotein complex, presenting unusual fea-tures and a unique opportunity to study aspects of O-linked glyco-sylation. The high degree of sequence conservation for the aDGcentral mucin-like region of numerous vertebrate species indicatesthe significance of this domain and specific sequence elements init. Defects in glycosylation of its more unusual O-Man glycans areassociated with forms of muscular dystrophy, but the conventionalO-GalNAc modifications are also present. The latter may beimportant in the mechanical properties of aDG, in extending fromthe muscle cell surface to the extracellular matrix. aDG is the first,and still the only well-characterized example of a mammalianprotein with O-Man glycosylation. Glycosylation site-mappingdata lays the groundwork to address factors that regulate its patternon aDG in a quantitative way, particularly how O-mannosylation,the initial step, impacts the sites of O-GalNAc addition. A majorobstacle to this, however, has been that intermediate processingforms are not available from natural sources, therefore, a series ofaDG-based O-Man glycopeptides have been prepared and theirproperties as acceptors for recombinant polypeptide GalNAc trans-ferase enzymes have been investigated. This provides direct infor-mation on how the pattern of preexisting O-Man modificationseffects GalNAc addition, and with mapping by MS, exactly wherethe GalNAc is added. Isoforms ppGalNAc-T1 and T3 are the oneswe have found to be active on the aDG mannosyl glycopeptides,and we have been able to recapitulate the patterns of glycosylationfound on native aDG. qPCR of rat skeletal muscle indicatesppGalNAc-T1 is the most prominent member of this enzymefamily present. This, and our in vitro results with this isoformcorrelates well with what is found in native aDG.

(170) Does POMGnT1 Activity Influence O-Man-6-PO4Glycans of alpha-Dystroglycan

Kai-For Mo, Stephanie H. Stalnaker, Pam Kirby, Mian Liu,Michael Pierce, Lance Wells, Geert-Jan Boons, David Live

CCRC, Univ. of Georgia, Athens, GA

Alpha-dystroglycan (aDG) studies have enhanced appreciationfor the diversity of protein O-glycosylation, particularlyO-mannosylation, and its impact on disease. Attention has beenfocused on the tetrasaccharide Neu5Ac-α2-3Gal-β1-4GlcNAc-β1-2Man-α-O-S/T, found on aDG. Defects in its assembly due tocompromised activity of glycosyltransferases are associated withloss of aDG function. POMGnT1 is responsible for the initialextension of the O-Man with a GalNAc. When this activity is

compromised, severe neurological symptoms, muscle-eye-braindisease (MEB), result. Now a new O-linked aDG modificationwith the core carbohydrate structure, GalNAc-β1-3GlcNAc-β1-4Man(6-PO4)α-O-T has been reported. The Man(6-P)-O-Tlinkage is unprecedented, and this trisaccharide is a component ofthe glycan participating in the functionally important lamininbinding of aDG, mediated through an additional structure, still notidentified, and linked as a phosphodiester to the Man-6-P. The gly-cosidic linkage of the GlcNAc in the latter case is 1-4 rather than1-2 to Man in the tetrasaccharide. Chemical removal of the diesterresults in aDG with similar reduced molecular weight to that ofaDG from an MEB patient. These observations imply somerelationship between the glycans and raise questions about regu-lation of O-Man glycan elongation at different sites, includingthe nature of the activity making the GlcNAc-β1-4 linkage, andthe relationship of POMGnT1 activity directly or indirectly to thephosphorylation of mannose and/or formation of the phosphodie-ster. We investigated this with synthetic Man and Man-6-P glyco-peptides evaluated as acceptors for recombinant POMGnT1 action,contributing insights into the site preferences and events that affectthe divergent elongation paths of the O-Man glycans. Resultsshow that the O-Man modified peptide is an acceptor forPOMGnT1 action, but the O-Man-6-P form is refractory. Theseand other data help in understanding the factors that determine thelocalization of glycans to specific sites, and the order of eventsneeded to generate this pattern.

(171) Differentiation of Oligosaccharide Linkage Isomersby Electron Activated Dissociation

Xiang Yu, Cheng Lin, Catherine E. CostelloBoston University School of Medicine, Boston, MA

The electron activated dissociation (ExD) behavior of oligosac-charides was determined by the electron energies and the type ofcharge carriers. Depending on the electron energy, metal-adductedoligosaccharides can undergo several fragmentation processes,including electron capture dissociation (ECD) at low energies,hot-ECD at intermediate energies, and electronic excitation dis-sociation (EED) at high energies. In this study, these ExD tech-niques were applied to the differentiation of oligosaccharidelinkage isomers. Three pairs of permethylated oligosaccharidelinkage isomers were investigated, including milk oligosaccharideisomers LNT and LNnT, LNFP I and LNFP III, and blood groupantigens Sialyl Lewisa (SLea) and Sialyl Lewisx (SLex). AlthoughECD produced extensive fragmentations, the cross-ring fragmentsgenerated were insufficient for determination of the linkage differ-ences and were often accompanied by neutral losses. In the caseof SLea and SLex, owing to the preferred binding of metal cationto the sialic acid residue, ECD fragmentation was dominated bythe loss of the sialic acid, and the cross-ring cleavages at the redu-cing end were suppressed. By contrast, EED produced abundantcross-ring fragments for definitive differentiation of all three pairsof oligosaccharide linkage isomers

Because the metal cation is usually the preferred electroncapture site of metal-adducted oligosaccharides, the sites of ECDfragmentation are often limited by the binding positions of the


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metal cation. Such limitation is not as significant in EED, as it isinitiated by ionization of a ring or glycosidic oxygen. Thus, EEDgenerally produces a broader range of fragment ions than ECD.EED also generates cross-ring fragments with fewer neutral lossesand therefore with less ambiguity. These results suggest that EEDis a valuable tool for oligosaccharide linkage determination Thisresearch is supported by NIH-NCRR Grant Nos. P41 RR010888and S10 RR025082.

(172) Study of Corneal Keratan Sulfate Biosynthesis in vivousing Sulfotransferase Gene Knockout Mice

Tomoya O. Akama1,2, Minoru Fukuda2, Michiko N. Fukuda2,Tomoyuki Nakamura1

1Kansai Medical University, Moriguchi, Japan;2Sanford-Burnham Medical Research Institute, La Jolla, CA

Keratan sulfate proteoglycans are one of the major components ofthe corneal extracellular matrix, and suggested to have an impor-tant role for function of the cornea. Our study on biosynthesis ofkeratan sulfate glycosaminoglycan (KS-GAG), which consists ofrepeating disaccharide units of -3Galβ1-4GlcNAcβ1- with sulfateson 6-O position of GlcNAc and galactose, identified essentialenzymes for highly sulfated KS-GAG production in vitro. Toinvestigate a biological function of the KS-GAG producingenzymes in vivo, we obtained two sulfotransferase mutant mouselines, Chst1 knockout mice and Chst5 knockout mice, and ana-lyzed carbohydrate structure of corneal KS-GAG. Chst5 knockoutmice lack an equivalent enzyme of human corneal GlcNAc 6-Osulfotransferase (CGn6ST, also known as GlcNAc6ST-5 andGST4β), and Chst1 knockout mouse is deficient in keratan sulfategalactose 6-O sulfotransferase (KSG6ST). Previous studies demon-strated that GlcNAc sulfation catalyzed by CGn6ST must becoupled to elongation of the carbohydrate backbone synthesizedby β1,3 N-acetylglucosaminyltransferase-7 (β3GnT7) and β1,4galactosyltransferase-4 (β4GalT4). From this fact, we speculatedthat alteration of GlcNAc sulfation during KS-GAG synthesis mayaffect efficient elongation of the carbohydrate chain. Indeed, wefound shorter KS-GAG chain on lumican KS-PG, one of threecorneal KS-PGs, in Chst5 knockout mouse corneas. On the otherhand, we detected normal length of KS-GAG on lumican KS-PGin Chst1 knockout mouse corneas. These results confirmed anindependent reaction step of galactose sulfation fromGlcNAc-sulfated polyLacNAc production, which is catalyzed byβ3GnT7, β4GalT4 and CGn6ST in a cooperative manner.Interestingly, we found that Chst1 heterozygous mutant mousecorneas have significantly less sulfation on corneal KS-GAG thanthat from wild type or Chst5 heterozygote mice, and Chst1 homo-zygous mutant mice have no highly sulfated KS-GAG as found inChst5 KO mice. These results found on mutant mice establishedKS-GAG biosynthetic pathway in the corneal extracellular matrixin vivo.

(173) LC-MS and LC-MS/MS Study of Heparan SulfateOligosaccharides Processed by Human Sulf-2

Yu Huang, Xiaofeng Shi, Joseph ZaiaBoston University School of Medicine, Boston, MA

Sulf1 and Sulf2 are extracellular heparan sulfate (HS)-specific6O-endosulfatases that selectively remove 6O-sulfate groups fromHS chains. By remodeling the structural arrangement of6O-sulfation pattern of cell surface HS chains, Sulfs are involvedin cell growth, proliferation, migration and differentiation by mod-ulating HSPG-dependent extracellular signaling pathways includ-ing FGF, Wnt and etc.

Our previously study of human Sulf-2 (HSulf2) treatmentfollowed by exhaustive heparin lyase depolymerization has shownthat HSulf2 cleaves 6O-sulfate in the non-reducing end of HSchains. In the present study, we used a recently developedadditive pulsed make-up flow HPLC-chip based liquidchromatography-mass spectrometry (LC-MS) and LC-MS/MSplatform to discern the substrate structural preferences of HSulf2on HS oligosaccharides.

The results showed that HSulf2 acts preferentially on highly sul-fated HS dp (degree of depolymerization) 6-8 with 0-1 acetategroup. The abundances of low sulfated dp6-8 increased resultingfrom the activity of HSulf2. The average sulfation decreased morefor dp6-8 with zero acetate than for those with one acetate group.Since only 6O-sulfation is cleaved by HSulf2, only certainisomers were affected and this was reflected by the chromato-graphic peak shape change of the extracted ion chromatograms(EICs). Those dp6-8 with EIC changes were chosen for tandemMS. A customized make-up flow HPLC-chip was used to pulsesulfolane during the elution time of the target precursors toenhance their charge states for more informative tandem massspectra. Although these tandem mass spectra seem to look similarfor the entire EIC peak range and entire collision energy range, bycareful selection of retention time windows and collision energy,the spectra differences were made more evident, suggesting asubset of the oligosaccharides have been processed and HSulf2.

(174) Structural Investigation of 13C Labeled N-linked Glycansfrom Trichomonas vaginalis by Electron Transfer Dissociation

Liang Han1, Catherine E. Costello21Boston University, Boston, MA; 2Boston University School of

Medicine, Boston, MA

Trichomonas vaginalis, which is sexually transmitted, causes vagi-nitis in women and urethritis in men and increases the risk of HIVtransmission. Trichomonas makes a truncated Asn-linked glycan(N-glycan) composed of seven sugars (Man5GlcNAc2), which isused for quality control of protein folding in the lumen of the ER.For the samples studied here, metabolic labeling with13C1-mannose served to efficiently distinguish TrichomonasN-glycans from contaminating N-glycans originating from thefetal bovine serum used to culture the parasites.

The classic fragmentation technique for structural identificationof glycans is collision-induced dissociation (CID). However, CIDgenerates product ions primarily by the rupture of glycosidicbonds, and thus provides information only on monomer residuesand their sequences. The peaks from cross-ring fragmentationoften have very low abundances in such spectra. We are applyinga newly developed mass spectrometry fragmentation technique—–


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electron transfer dissociation (ETD) to structural determinations ofglycans.The N-linked glycans labeled with 13C at the C1 of the Man resi-

dues were released from the Trichomonas samples with PNGase Fand were permethylated. The magnesium-adducted permethylatedglycans were analyzed on a Bruker AmaZon quadrupole ion trap.ETD generated both glycosidic bond cleavage and cross-ring clea-vage ions. The cross-ring cleavages clarified the different linkagetypes and branching patterns of the N-linked glycans. The utiliz-ation of ETD-CID MS3 techniques and the presence of the13C1-labels in the mannose residues made it possible to verify peakassignments and to distinguish the overlapping peaks. These resultspredict that ETD should contribute to confident structural analysisof a wide variety of oligosaccharides.This research is supported by NIH grant P41 RR10888. The

authors are grateful to Bruker Daltonics for loan of the AmaZoninstrument and to our collaborators A. Carpentieri, P. W. Robbinsand J. Samuelson for generation of the 13C-labeled Trichomonasglycans.

(175) Photocrosslinking of O-GlcNAc-modified Proteinsto Neighboring MoleculesSeok-Ho Yu, Jennifer Kohler

UT Southwest Medical Ctr, Dallas, TX

O-GlcNAc is a common and essential post-translational modifi-cation in metazoa and higher plants. Hundreds of proteins havebeen demonstrated to be O-GlcNAc-modified, yet, in most cases,the functional significance of the modification remains poorlyunderstood. We reasoned that by introducing a photocrosslinkinggroup directly onto O-GlcNAc residues, we could covalentlycapture complexes between O-GlcNAc-modified proteins andneighboring molecules. We report a metabolic labeling strategy toinsert a diazirine functional group onto O-GlcNAc modified pro-teins, relying on a protected, diazirine-modified analog ofGlcNAc-1-P and mutations to the UDP-GlcNAc pyrophosphory-lase 1 gene. We show that diazirine-modified O-GlcNAcylatedproteins can be produced in mammalian cells and crosslinked toneighboring molecules. We demonstrate the utility of this technol-ogy by using it to capture interactions between FG-repeatnucleoporins and nuclear transport factors.

(176) Screening of Complex-Fucoidans from Different Speciesof Brown Algae as Procoagulant Drug Candidates Based

on Their Activity, Structural Properties, ImpuritiesZhenqing Zhang1, Sabine Knappe2, Susanne Till2,

Inanova Nadia1, James Catarello1, Catherine Quinn1, Navik Julia1,Joseph Ray1, Thang Tran1, Friedrich Scheiflinger2,

Christina Szabo1, Michael Dockal21Baxter Healthcare Corporation, Round Lake, IL; 2Baxter

Innovations GmbH, Vienna, Austria

Fucoidan is a mixture of sulfated polysaccharides extracted frombrown seaweeds. Fucoidan has a wide variety of biological effects,including pro- and anticoagulant activities. Based on its procoagu-lant activity, fucoidans are described as non-anticoagulant sulfated

polysaccharides (NASP). To select the most active fucoidan candi-dates for development of a hemophilia treatment, we screenedextracts from four brown algae species. Assessment of the pro-and anticoagulant activities of a selected set of fucoidan prep-arations showed improvement of clotting parameters by severalfucoidans mainly based on calibrated automated thrombography(CAT), and activated partial thromboplastin time (aPTT) assays. Inaddition, some fucoidan species were able to activate the contactpathway, an undesired property that has been reported for sulfatedglycosaminoglycans. Fucoidans have complex structures due totheir large molecular weight (Mw), wide Mw distribution, dispersedegree of sulfation, variable monosaccharide composition, anddifferent linkages. Because of this complexity, screening for thehighest quality candidates includes additional considerations withrespect to chemical structure and heterogeneity. Chemical evalu-ation of fucoidan candidates was based on the in-depth structuralanalyses of size, polydispersity, chemical heterogeneity, organicand inorganic impurities with different techniques. This workdescribes the extensive screening of a variety of fucoidan prep-arations for desired coagulation activity and window, structuralproperties, and impurity profile. Toward this end, new fucoidan-specific analytical assays were developed.

(177) Sulfotransferase and Fucosyltransferase that RegulateExpression of the 5D4 Keratan Sulfate Epitope in Early

Postnatal Mouse BrainHitomi Hoshino1, Shiori Niimi1, Tomomi Hosono1,

Makoto Michikawa1, Reiji Kannagi2, Kenji Uchimura11National Center for Geriatrics and Gerontology, Obu, Japan;

2Aichi Med Univ, Res Complex for the Med Frontiers, Nagakute,Japan

Keratan sulfate (KS) comprises repeating disaccharide units ofgalactose and N-acetylglucosamine (GlcNAc) that are potentiallymodified with C-6 sulfation. Alpha1,3-linked fucose is known tobe present on the C-3 of sulfated GlcNAc residues within thechain. Sulfated poly-N-acetyllactosamine structure, GlcNAc(6S)-Gal(6S)- GlcNAc(6S)-Gal(6S)- GlcNAc(6S)-Gal, is specifi-cally recognized by the 5D4 monoclonal antibody, which has beenused most extensively to evaluate KS expression in mammalianbrains. We have previously shown that GlcNAc6-O-sulfotransferase-1 (GlcNAc6ST-1) is an enzyme responsiblefor synthesis of the 5D4 epitope in the adult mouse brain afterinjury. On the other hand, the expression of the 5D4 epitope isspatiotemporally regulated in the developing brain. It has beenobscure that GlcNAc6ST-1 is also involved in synthesis of the5D4 epitope in early postnatal mouse brain. We therefore analyzedthe expression of GlcNAc6ST-1 and the 5D4 epitope in brains ofpostnatal days (P) 1-21 mice. Western blotting and immunohisto-chemical analyses have shown that the highest level ofGlcNAc6ST-1 expression was observed in the P1 cortex and thatthe expression declined with postnatal days. The expression levelof the 5D4 epitope correlated with that of the enzyme. The 5D4epitope was not detected in postnatal brains of mice deficient inthe GlcNAc6ST-1 gene. Interestingly, the 5D4 epitope was


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upregulated in the P1 cortex of mice deficient in the fucosyltrans-ferase IV gene. These results indicated that GlcNAc6ST-1 isinvolved in synthesis of the 5D4 KS epitope in early postnatalmouse brain, and that the competitive reaction between biosyn-thesis of 5D4 epitope and alpha1,3-fucosylation by fucosyltrans-ferase IV may occur in the developing brain.

(178) Improvement in Production of Secreted Proteinsby Use of the Glycosylation TagShou Takashima, Junko Amano

The Noguchi Institute, Itabashi, Japan

Glycans attached to proteins often play an important role inprotein folding, secretion, stabilization of protein, and expressionof protein activity. In the production of recombinant glycoproteinshaving such functional glycans, it is important how effectivelyglycans attach to the protein. In this study, we used humaninterleukin-6 (IL-6) as a model glycoprotein and examined itsglycan function. IL-6 has two N-glycosylation potential sites atN73 and N172, and their consensus sequences forN-glycosylation are NKS and NAS, respectively. But the N172site is hardly glycosylated. When the NKS sequence of the N73site of IL-6 was changed to NKT (S75T) and this IL-6 mutantwas over-expressed in HEK293 cells, the glycosylation on theN73 site was greatly increased and secretion of this IL-6 was pro-moted. So we thought that N-glycans of IL-6 should play animportant role in secretion. Then we added various peptidesequences containing the consensus sequence for N-glycosylation,which we termed the glycosylation tags, to the C-terminal regionof IL-6 and examined their effects on secretion. As a result,some tags were successfully glycosylated and the amount ofsecreted IL-6 increased. It seems that in the production of recom-binant proteins, yields of some proteins could increase by usingthe proper glycosylation tag. The glycosylation tag can bedesigned to contain the cleavage site for the specific protease sothat the glycosylated tag is removed from the target protein aftersecretion by the treatment with the specific protease if the glyco-sylated tag is undesirable for the expression of protein function.The glycosylation tag may be a powerful tool for the efficientproduction of some recombinant proteins.

(179) Developmental Roles of Putative PolypeptideGalNAc-Transferases in Zebrafish

Naosuke Nakamura, Eiichi Kaneda, Yoshiaki Nakayama,Akira Kurosaka

Kyoto Sangyo University, Kyoto, Japan

Mucin-type O-glycosylation is one of the most common post-translational modifications of proteins and is catalyzed by a familyof UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases(ppGalNAc-Ts). To date, 16 isozyme genes of this family havebeen cloned from mammals. Their analyses indicate that they havedistinct tissue expression patterns and partially overlapping butunique substrate specificities. We previously cloned mammalianppGalNAc-T9 and WBSCR17, a highly homologous gene toppGalNAc-T9, and demonstrated that both are predominantlyexpressed in the brain.

To investigate the developmental roles of ppGalNAc-Ts in vivo,we have been using zebrafish as a model organism. We found bysearching the zebrafish genome database that zebrafish has most ofmammalian orthologue genes. We then cloned the brain-specificorthologue genes (ppGalNAc-T9 and WBSCR17) from zebrafish anddemonstrated the functional roles of WBSCR17 in the brain develop-ment (2010 Annual Conference of the Society for Glycobiology).Recently, we have cloned a human ppGalNAc-TL4 gene and its zeb-rafish orthologue genes, ppGalNAc-TL4a and -TL4b, which are hom-ologous to mammalian brain-specific isozyme genes. The analysis ofppGalNAc-TL4a expression by whole mount in situ hybridization inzebrafish indicated that it was expressed throughout the embryos withthe stronger expression in the brain and the tail at 24 and 48 hpf.When the expression of either of ppGalNAc-TL4a or -TL4b wasknocked down with morpholino antisense oligonucleotides, both ofthe knockdown embryos exhibited similar morphologic changes,such as short and curled tails. These findings indicated thatppGalNAc-TL4a and -TL4b are involved in the normal tail develop-ment in the zebrafish embryos. We are investigating the molecularmechanism that leads to the altered tail phenotypes associated withthe ppGalNAc-TL4 knockdown.

(180) Exploration of Disease-Specific Epitopes by using MUC1Glycopeptide Microarray

Wataru Takada1, Takahiko Matsushita2, Hiroshi Hinou2,Shin-Ichiro Nishimura2

1Sumitomo Bakelite Co., Ltd., Kobe, Japan; 2Hokkaido University,Sapporo, Japan

We have developed the plastic substrate for the glycan array wherevarious free sugar chains could be immobilized handily. The redu-cing end of sugar chains can be immobilized in the free by‘Glycoblotting method’, trapping their hemiacetal group selec-tively. Here we present a MUC1 glycopeptide microarray as one ofthe application of this array.

MUC1 is a highly glycosylated membrane-bound glycoproteindistributing on the apical surface of normal epithelial cells. In tumorcells, aberrantly O-glycosylated MUC1 is overexpressed on thewhole-cell surface, which is due to MUC1 shedding or cell surfacerelease to bloodstream. Therefore, MUC1 is considered an attractivetarget as serum tumor biomarker. Recently, we have revealed theMUC1 heptapeptide PDTRPAP having alpha 2,3-sialylated Tantigen at Thr residue as an essential epitope of anti-KL-6 mono-clonal antibody, which is a beneficial tool for diagnosis and moni-toring patients with interstitial pneumonia, by focused MUC1glycopeptide library and common ELISA protocol. [1]

For effective epitope analysis with less sample, we fabricatedMUC1 glycopeptide microarray. These microarray plastic slideshave the surface coated by aminooxy-functionalized polymer.Aminooxy groups allow glycopeptide library introduced 5-oxohex-anoic acid at N-terminus in the end of solid-phase synthesis todisplay via oxime bond formation in mild acidic aqueous condition.So glycopeptides can be immobilized on this microarray easily andregioselectively.

The results of epitope identification by utilizing our developingmicroarray displayed overlapping MUC1 tandem repeat peptides


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and glycopeptide library focused on the site and structure inO-glycosylation will be discussed.

Reference[1] N. Ohyabu, H. Hinou, T. Matsushita, R. Izumi, H. Shimizu,K. Kawamoto, Y. Numata, H. Togame, H. Takemoto, H. Kondo,S.-I. Nishimura, J. Am. Chem. Soc., 2009, 131, 17102–17109.

(181) Development and Application of Versatile GlycanMicroarray

Kohta Igarashi1, Wataru Takada1, Hiroki Abe1,Takahiko Matsushita2, Shin-Ichiro Nishimura2

1Sumitomo Bakelite Co., Ltd., Tokyo, Japan; 2Hokkaido UniversityGraduate School of Advanced Li, Sapporo, Japan

Glycan-Protein interactions play significant roles in biological pro-cesses, including intracellular traffic, cell differentiation, recog-nition, signaling processes, adherence, etc. The glycan microarrayis gaining a lot of attention as a tool to analyze the function of thesugar chain, and it is expected that this array is useful for thedevelopment of the new diagnostics and treatment method of asickness[1].We developed a unique plastic substrate for the glycan microar-

ray by using special surface treatment technology. It enables toimmobilize various free sugar chains very easily and also enablesto reduce non-specific adsorption of biomolecules. It became poss-ible to immobilize reducing end of sugar chains directly onto theplastic surface by using ‘Glycoblotting method’, that is the methodfor trapping their hemiacetal group without complicated procedureto introduce the functional linker for their immobilization. Andalso, it became possible to detect glycan-protein interaction easilyand sensitively because back-ground noise was reduced.It introduces the glycolipid sugar chain microarray as one of the

application of this array. When the reactivity with several kinds ofcommercially available lectins was evaluated on the array wherethe sugar chain part of the ganglioside that appeared in the biosyn-thetic pathway of the ganglioside was immobilized, each lectinshowed reactive specificity, and it was possible to perform thelectin screening. In addition, various experiment examples that usethe glycan microarray are announced.

Reference[1] O. Blixt, et al., Proc. Natl. Acad. Sci. 101, 17033 (2004).

(182) Recognition of Specific Heparan Sulfate OligosaccharidesInvolved in Tumor Development. Application of the Molecular

Imprinting TechnologyMouna Mothere

Laboratoire CRRET, Creteil, France

Heparan sulfates (HS) are complex polysaccharides belonging tothe glycosaminoglycans (GAGs) family. HS are generally locatedon the cell surface and in the extracellular matrix from where theyinfluence cells functions. GAGs are known to bind and regulatethe function of a number of distinct proteins known as ‘heparinbinding proteins’ (HBP) including chemokines, growth factors,enzymes, etc. It is known that the activity of HS depends on the

presence in their chains of specifically sulfated sequences that canspecifically regulate the activity of HBP. In the case of tumordevelopment, an enhanced over-expression of heparanase, theenzyme that depolymerize HS, generates a variety of oligosacchar-ides that are released in the tumor matrix. At present, the structureand biological roles of these HS oligosaccharides are unknown.

Although many tools to characterize GAGs exist, there is still agreat need to develop new technologies allowing rapid and selec-tive isolation of particular HS fragments. Here, we used theMolecular Imprinting Technology (MIT) to synthesize polymerswith cavities capable to recognize a particular HS related pentasac-charide, used as template molecule. After synthesizing a library ofpolymers designed for template recognition, we selected a particu-lar polymer able to specifically recognize the template oligosac-charide and validated its reproducible chemical synthesis. Studiesto selectively extract the template oligosaccharide from HS extractsprepared from tumor tissue or tumor cells that over-express hepara-nase are in progress. The MIT technology is a promising tool forthe specific selection, extraction and identification of endogenousHS oligosaccharides produced during tumor growth.

(183) Examining Targets of Protein O-Fucosyltransferase 2Christina Leonhard-Melief, Robert S. Haltiwanger

Stony Brook University, Stony Brook, NY

Protien O-fucosyltransferase 2 (Pofut2) is an ER resident glycosyl-transferase that adds fucose to serines or threonines in properlyfolded Thrombospondin type 1 repeats (TSRs) with the putativeconsensus sequence CX2-3 (S/T)CX2G. The O-fucose can beelongated to a disaccharide by a b1-3glucosyltransferase. Searchesof the mouse genome for this consensus sequence within a TSRyielded 52 potential target proteins. Using mass spectral methods,site mapping in our lab as well as others have confirmed the pres-ence of the Glcβ1-3Fuc disaccharide on eleven of these targets.Pofut2 knockout mice show defective gastrulation resulting inembryonic lethality (Du, J., Takeuchi, H., Leonhard-Melief, C.,Shroyer, K.R., Dlugosz, M., Haltiwanger, R.S. and Holdener, B.C.(2010) O-Fucosylation of thrombospondin type 1 repeats restrictsepithelial to mesenchymal transition (EMT) and maintains epiblastpluripotency during mouse gastrulation. Dev. Biol. 346, 25-38).We predict this lethality is due to loss of O-fucosylation of one ormore targets. In order to further refine the list of targets that maycontribute to the embryonic lethality, we are examining details ofthe putative consensus sequence. We performed site directed muta-genesis on the third TSR from human thrombospondin 1, testingthe importance of the invariant residues (cysteines and glycine) aswell as altering the “X” positions by inserting sequences of pre-dicted targets with characteristics distinct from those found inO-fucose sites already confirmed. Constructs encoding the mutantsin TSR3 are being expressed in HEK293T cells and evaluated forexpression, secretion, and glycosylation. We are using 6-alkynylfucose (6AF) and click chemistry to monitor the level ofO-fucosylation, and the results are being confirmed using massspectrometry. Our goal is to be able to more accurately predictwhich proteins will be targets for Pofut2. Supported by NCI grantCA12307171.


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(184) Regulation of Colon Cancer Stem Cells and ColonAdenoma Progression of ApcMin/+ mice by GnT-V Expression

LevelHuabei Guo1, Heather Johnson1, Tamas Nagy2, Michael Pierce11CCRC, UGA, Athens, GA; 2Department of Pathology, UGA,

Athens, GA

There is increasing evidence that a variety of cancers are initiatedand maintained by a small proportion of cells called cancer stemcells (CSCs). Deletion of GnT-V, which catalyzes a specific modi-fication of N-glycans with β(1,6) branching, reduced the size ofthe compartment of CSCs in the her-2 mouse model, leading to ainhibited tumor onset. In the present study, the regulation of colonCSCs and adenoma development were investigated.Overexpression of GnT-V in human colon cancer LS180 cellsenhanced both anchorage-independent cell growth and tumor for-mation by injection of tumor cells into NOD/SCID mice, indicat-ing increased cell proliferation and tumorigenicity caused byoverexpression of GnT-V. CSCs population was significantlyincreased after GnT-V expression in LS180 cells, which was con-sistent with a reduced proportion of CSCs observed for controltumor cells after treatment with swainsonine, an inhibitor ofN-linked β(1,6) branching, indicating the regulation of CSCs byGnT-V expression level. To further confirm this regulation,Apcmin/+ mice with different GnT-V backgrounds were then gen-erated. We found that knockout of GnT-V had no significant effecton adenoma formation (number of adenoma/mouse), but the sizeof adenoma was remarkably reduced, which was accompanied byincreased survival of ApcMin/+ mice with GnT-V deletion(p < 0.01), suggesting an inhibition in the progression of colonadenoma caused by deletion of GnT-V. Furthermore, the pro-portion of CSCs in colon adenoma from GnT-V null mice was sig-nificantly reduced compared with controls, consistent with reducedadenoma progression observed in GnT-V null ApcMin/+ mice.These results demonstrate that GnT-V expression and its branchedN-glycan products effectively modulate relative proportion ofcolon cancer stem cells that, most likely in turn, regulate colontumor (adenoma) progression and provide a potential therapeutictarget for the treatment of colon cancer.Key words: Cancer stem cells (CSCs), GnT-V, colon cancer,ApcMin/+ mice

(185) Investigation of Glycan Catabolism and Biosynthesisin Human Embryonic Stem Cells

Alison Nairn1, Mitche dela Rosa1, Kazuhiro Aoki1,Mindy Porterfield1, Michael Kulik2, Stephen Dalton2,

J. Michael Pierce1,2, Michael Tiemeyer1,2, Kelley Moremen1,21Complex Carbohydrate Research Center, U of GA, Athens, GA;2Dept. of Biochemistry and Mol Biology, U of GA, Athens, GA

Glycan structures on cell surface and secreted glycoconjugates areknown to change during development and contribute to numerousbiological recognition events during embryogenesis and in adulttissues. The diversity of glycan structures displayed on cell surfaceglycoproteins and glycolipids results from an interplay betweenbiosynthetic, catabolic, and remodeling processes that can be

highly regulated in animal systems. Regulation of glycan structurescan also modulate cell surface retention versus endocytosis andrecycling of cell surface glycoconjugates to influence their steadystate levels or intracellular localization. Previously, we used quanti-tative real-time PCR (qRT-PCR) to monitor steady-state transcriptlevels for biosynthetic glycan-related genes during differentiationof embryonic stem cells (ESC) into cardiac progenitor Islet-1+cells in an effort to predict changes in glycan structures during EScell differentiation. In the present study, we extend these analysesto genes encoding enzymes involved in glycan degradation inthree different human ES cell lines (BG02, H7 and H9) and aninduced pluripotent stem cell (iPSC) line in comparison to theequivalent lines differentiated into Islet-1+ cells. These transcriptabundance data for catabolic enzymes combined with the priorstudies on the transcripts for biosynthetic enzymes have been com-pared to the glycan structures determined by NSI-MSn(LTQ-Orbitrap MS) from the corresponding cell populations.Distinct changes in several glycan structures were found to corre-late with changes in transcripts encoding corresponding biosyn-thetic or catabolic enzymes suggesting at least partial regulation ofglycan structures at the transcriptional level. These data provide amore global picture of the interplay between glycan biosynthesisand catabolism in the elaboration of glycan structures in differen-tiating human ES cells. (Supported by NIH grant RR018502)

(186) Structural Comparison of Plant GlycosyltransferasesSara Fasmer Hansen, Ryan McAndrew, Andy DeGiovanni,Peter McInerney, Jose Henrique Pereira, Masood Hadi,

Paul Adams, Henrik Vibe SchellerJoint BioEnergy Institute, LBNL, Berkeley, CA

Plant cell walls are composed primarily of structural polysacchar-ides including cellulose, hemicelluloses and pectins. Thesecomplex polysaccharides are synthesized by glycosyltransferases(GTs) – a family of enzymes that transfer a sugar residue from anactivated donor substrate, usually a nucleotide sugar, to an accep-tor such as a growing oligosaccharide. GTs generally have narrowsubstrate specificity, and are highly stereo- and regiospecific. TheGTs involved in hemicellulose and pectin biosynthesis are mem-brane proteins located in the Golgi apparatus. Plants have a largenumber of such proteins, e.g. more than 300 in Arabidopsis, mostof which have an unknown function.

Predicting the function of a putative GT based on sequencesimilarities is problematic and closely related sequences havedifferent catalytic activities. GTs appear to share limited number ofprotein fold types and only two structural folds, GT-A and GT-B,have been identified to date. However, for many GT families –

particularly those specific to plants – no structure has been solved,so it is not clear if other fold types exist.

Crystallization and structural comparison of the catalyticdomains could help to find conserved motifs involved in substraterecognition of the many GTs in plants. We have selected a diversegroup of rice and Arabidopsis GTs potentially involved in cellwall biosynthesis. Using bioinformatics and modeling, secondarystructures were predicted for optimal construction of truncationvariants suitable for crystallization. The protein variants were


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expressed in E.coli with fusion-protein tags for improvement ofsolubility and expression and for purification. More than 20 pro-teins were expressed at high levels as soluble proteins and somewere selected for initial crystallization efforts. Crystals have beenobtained and results of the analysis will be reported.Funding is provided by The Carlsberg Foundation and by the

US Department of Energy, Office of Science, through contractDE-AC02-05CH11231 with LBNL.

(187) Assessing the Conformational Distribution and Exposureof Immunoglobulin G N-glycans using Paramagnetic Tags

and NMRAdam Barb1, James Prestegard2

1CCRC, UGA, Athens, GA; 2University of Georgia, Athens, GA

The complex-type biantennary N-glycan on the ImmunoglobulinG Fc fragment modulates cellular responses of the adaptiveimmune system by tuning the strength of the Fc – Fcγ receptorinteraction. However, the underlying mechanism remains unde-fined as existing structural data suggest the glycan resides deeplyburied between the two Fc polypeptide chains and cannot directlyengage the Fcγ receptor. We recently characterized the dynamicsof the galactose residues at the glycan termini using solutionnuclear magnetic resonance (NMR) spectroscopy and found that,contrary to previous conclusions based on x-ray crystallographyand limited NMR data, the terminal galactose residues of bothglycan branches were highly dynamic. While this study providedevidence for alternate exposed conformations of the N-glycan,these data failed to provide a structural model of glycan confor-mation. To guide structural calculations of the glycan motions wenow report a conformational ensemble restrained by long rangedistance (10-40Å) and orientation restraints relative to an ectopiclanthanide ion. To position the ion a lanthanide-binding tag wasincorporated into the three-helix Z-domain of protein A and theresulting construct was shown to bind the Fc fragment with highaffinity. Glycan-ion distances estimated using complexes of the Fcfragment with this designed lanthanide-binding Z-domain anddifferent lanthanide ions (Dy3 + , Yb3 + , and Gd3+) are consistentwith the galactose termini occupying a solvent exposed confor-mation a significant proportion of time. These findings suggestglycan conformation and dynamics, in addition to composition,are important components in the Fc signaling function

(188) Endothelial Heparan Sulfate is Essentially Requiredfor Vascular Development

Siyuan Zhang1, Junlin Jiang1, Yu Yamaguchi2, Lianchun Wang11University of Georgia, Athens, GA; 2The Burnham Inst., La Jolla,

CA

Angiogenesis is a process in which the primitive vascular plexus orcapillaries remodel into a mature vasculature. Angiogenesis isessential for embryonic development. Many of the events that occurduring normal angiogenesis are recapitulated in pathological pro-cesses, such as tumor angiogenesis, ischemic coronary arterydisease and stroke. Therefore, new insights into normal angiogen-esis will not only advance our understanding of this fundamentalprocess, but will also likely lead to the development of rational

therapeutics for both prevention of pathological angiogenesis andpromotion of new blood-vessel growth in ischemic tissues. In thisstudy, we have examined the role and its underlying mechanisms ofheparan sulfate, a highly sulfated polysaccharide, in vascular devel-opment by specifically ablating the heparan sulfate biosyntheticgene Ext1 in mouse endothelium (denoted as T-Ext1-/- mice). TheT-Ext-/- mice are embryonic lethality between embryonic day 12.5– 15.5. Phenotype characterization observed that the Ext1 ablationdiminished heparan sulfate expression by endothelial cell, leadingto vascular pattern defect, abnormal filopodia formation, disruptedvascular maturation and mural cell recruitment accompanying withincreased vascular regression. Molecular mechanism studiesobserved that the vascular development essential signaling path-ways including VEGFR2, PDGFRβ and TGFβ were disrupted inthe T-Ext1-/- mice. Together, our studies revealed that endothelialheparan sulfate is essentially required for vascular development byfacilitating VEGF and PDGFb/TGFβ-mediated endothelial cellfunctions and mural cell recruitment, respectively.

(189) Analysis of Bovine Cervical Mucin O-glycansTharmala Tharmalingam1,2, Katarzyna Pluta2, Paul McGettigan2,

Ronan Gough2, Weston Struwe1, Eamonn Fitzpatrick2,Mary E. Gallagher2, Pauline M. Rudd1, Niclas G. Karlsson3,

Stephen D. Carrington21NIBRT, Dublin, Ireland; 2Veterinary Medicine, UCD, Belfield,

Dublin, Ireland; 3Medical Biochemistry, University of Gothenburg,Gothenburg, Sweden

Fertility in dairy cows has declined over the past five decades asmilk production per cow has increased. We are interested in theidentification of biomarkers of key biological processes in thereproductive tract, which may act as targets for intervention to haltthis decline. As part of this integrated programme we have charac-terised the bovine cervical transcriptome and mucin glycome overthe perioestus period. The oestrus cycle was synchronised in 30beef heifers using intravaginal progesterone releasing devices fol-lowed by prostaglandin injection. Preliminary histological stainingfrom the cervical tissue of cattle identified some changes in sugarexpression levels in stored cervical mucins throughout the oestrouscycle. Mucus was harvested from cervical tissues by extraction inGuanidine HCl, and mucins subsequently purified by density gra-dient centrifugation and size exclusion chromatography. Here wepresent a detailed structural analysis of O-glycans released frompurified cervical mucins by LC-MSn. Predominant structures werefucosylated, sialylated, and sulphated extended core 2 O-glycanstructures. We present data to support our hypothesis that cervicaltissue and secretions undergo oestrous cycle related changes thatmay be associated with changes in mucus structure, sperm trans-port and mucosal barrier function.

(190) Sialylated Glycoproteins of Drosophila melanogasterToshihiko Katoh1, Vladislav Panin2, Michael Tiemeyer11CCRC, University of Georgia, Athens, GA; 2Texas A&M

University, Collge Station, TX

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The addition of sialic acid onto N-linked glycans is a dynamic andprominent modification in these species but is exceedingly rare inDrosophila and other protostomes. The presence of sialylatedN-linked glycans in Drosophila has been directly demonstrated byglycomic profiling and the functional importance of sialylation hasbeen shown by characterization and mutagenesis of the only sialyl-transferase (dSiaT) encoded within the organism’s genome.However, a lack of information about protein substrates for thedSiaT has hampered the interpretation of phenotypes associatedwith decreased sialylation. Therefore, we undertook the identifi-cation of sialylated glycoproteins extracted from Drosophila headsusing a robust, chemo-affinity approach (periodate oxidation andaniline-catalyzed oxime ligation, PAL), which incorporates abiotin tag specifically onto sialylated glycopeptides (Zeng, Y.,et al., 2009, Nat Methods 6, 207-209). Following trypsin diges-tion, glycopeptides were enriched by removal of non-glycosylatedpeptides and then divided into two equal portions for treatmentwith or without neuraminidase. After biotin tagging by PAL, bioti-nylated sialoglycopeptides were captured on streptavidin-agarose,extensively washed, and released by PNGaseF. LC-MS/MS of thematerial released from the sample not treated with neuraminidaseidentified 15 sialylated glycopeptides, belonging to 10 glyco-proteins. Two of these peptides were also detected following neur-aminidase treatment but at significantly lower abundance. Thesialylated glycoproteins include cell adhesion molecules, extra-cellular matrix components, and neural-specific transporters.Further genetic and biochemical analysis will elucidate the role ofsialylation in regulating the function of these glycoproteins.

(191) Induction of Epithelial-Mesenchymal Transition inHuman Lung Epithelial Cells with O-glycosylated FibronectinYao Ding1,2, Kirill Gelfenbeyn1,2, Leonardo Freire-de-Lima1,2,

Kazuko Handa1,2, Sen-itiroh Hakomori1,21Pacific Northwest Diabetes Research Institute, Seattle, WA;

2University of Washington, Seattle, WA

Epithelial-mesenchymal transition (EMT) was originally observedin early embryogenic development, and has been indicated to playa key role in cancer progression to metastasis. Although glycansexpressed as glycospingolipids (GSL), or glycoproteins, have beenknown to regulate cell phenotypes, the role of glycans in EMT hasnot been fully investigated. Our previous studies in this areashowed the involvement of i) certain GSLs in mouse mammarycells (Feng G., et al, PNAS 106: 7461-6, 2009) and ii) oncofetalfibronectin (onfFN), which is defined by mouse monoclonal anti-body (mAb) FDC6, whose reactivity requires the addition ofO-α-GalNAc to the Thr residue of peptide VTHPGY, at the IIICSdomain of FN (Matsuura H., et al, JBC 264: 10472-6, 1989) inhuman prostate cells (Freire-de-Lima L., et al, in preparation). Tofurther analyze the role of onfFN in EMT induction, we estab-lished i) mouse mAb, YKH1, which reacts with normal FN(norFN) that carries the peptide without O-glycan, by immunizingwith KLH-bound KTPFVTHPGYDTGNTCQC and ii) humanhepatoma HUH7 cells, which overexpresses the gene of polypep-tide N-acetylgalactosaminyltransferase 6 (GALNT6), in order toprepare more onfFN. The transfectant, HUH7/GALNT6, produced

and secreted more onfFN compared to the parent cells. Total FNprepared from HUH7/GALNT6 cells was separated with mAbYKH1, and FDC6-bound protein A/G column, to isolate onfFNand norFN, respectively. We compared onfFN and norFN for theirability to induce EMT using human lung cells A549, and foundthat onfFN significantly induced EMT. This was assessed by achange in morphology, reduced expression of typical epithelialmarker, E-cadherin, enhanced expression of mesenchymal marker,vimentin, and enhanced cell motility. norFN did not show suchactivity. Further characterization of onfFN and norFN in term ofcell adhesion and cell signaling is ongoing. Supported by fundsfrom The Biomembrane Institute.

(192) Characterization of Three Novel Heparinases Clonedfrom Bacteroides eggerthii

Alicia M. Bielik1, Elizabeth McLeod1, David Landry1,Vanessa Mendoza2, Joseph Zaia2, Ellen P. Guthrie1

1New England BioLabs, Inc., Ipswich, MA; 2Boston UniversitySchool of Medicine, Boston, MA

Heparin and heparan Sulfate (HS) are linear sulfated polysaccharideslocated on cell-surface membranes and in extracellular matrices invirtually all animal tissues. Heparin/HS chains consist of repeatingdisaccharide units of [(GlcAβ(1-4)GlcNAcα(1-4)] with poly-dispersesulfation, N-acetylation and uronosyl epimerization. The structuralelucidation of such complex and diverse polysaccharides is an excep-tionally challenging task and cannot be accomplished withoutenzyme tools. Three heparinase enzymes are known to cleaveheparin and HS chains selectively, via an elimination mechanism.Heparinase I cleaves highly sulfated heparin/HS chains, heparinaseIII cleaves less sulfated HS chains, while heparinase II cleavesdomains of both high and low sulfation on both heparin and HS.Currently, heparinases are commercially available as recombinantand non-recombinant proteins from Flavobacterium heparinum.

This poster describes biochemical methods used to clone, purifyand characterize the function and substrate specificity of heparinaseI, II and III from Bacteroides eggerthii. The purification of crudecell extracts of each heparinase was performed using a streamlinedseries of standard column chromatography techniques. Porcineintestinal mucosa heparin/HS was used to assay for enzymaticactivity by reading absorbance at 232nm. Enzyme purity wasjudged by gradient polyacrylamide gel electrophoresis and MassSpectrometry analysis. The purified heparinases show no detectablesulfatase or uronidase activities with 7-amino methylcoumarinlabeled heparin oligosaccharides as substrates. A comparison ofsubstrate specificities between Heparinase I, II and III fromFlavobacterium heparinum versus Bacteroides eggerthii is demon-strated using heparin/HS substrates in conjunction with disaccharideanalysis with online Size Exclusion LC/MS techniques.

(193) Comparative Glycomics of Human and Bovine MilkGlycosaminoglycans

Yang Mao1, David S. Newburg2, Catherine E. Costello1,Joseph Zaia1

1Boston University School of Medicine, Boston, MA; 2BostonCollege, Chestnut Hill, MA


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The human milk glycans, including glycoconjugates and oligosac-charides, constitute a major innate immunologic mechanism bywhich human milk protects breast-fed infants against infections.Although numerous efforts have been carried out to study thenutrient composition of human milk, little is known about its gly-cosaminoglycan (GAG) components. Previous studies byNewburg et al show that the human milk GAGs inhibit HIV gly-coprotein (gp120) binding to its host cell CD4 receptor. Studyingthe structure-activity relationship of the human milk GAGs mayidentify novel therapeutic agents against HIV. Toward this end, wepurified GAGs from human and bovine milk. The GAG compo-sitions released by specific bacterial polysaccharide lyases wereanalyzed by size exclusion chromatography (SEC) and hydrophilicinteraction chromatography (HILIC)-coupled online mass spec-trometry.The heparan sulfate (HS) disaccharide composition of human

milk is very different from that of bovine milk. The human milk HSfraction contains 17% ΔHexA-GlcNAc, 41% ΔHexA(2S)-GlcNAc(6S), 0% ΔHexA2S-GlcNAc6S, 13% ΔHexA- GlcNS, 27%ΔHexA2S-GlcNS6S, and 2% ΔHexA2S-GlcNS6S. The bovinemilk HS fraction contains 5% ΔHexA-GlcNAc, 14% ΔHexA(2S)-GlcNAc(6S), 0% ΔHexA2S-GlcNAc6S, 23% ΔHexA- GlcNS,48.5% ΔHexA2S-GlcNS6S, and 9.5% ΔHexA2S-GlcNS6S. In con-trast, the chondroitin sulfate/dermatan sulfate (CS/DS) disaccharidecomposition of human milk is similar to that of bovine milk. TheCS/DS fraction of human milk contains 30% ΔHexA-GalNAc, 70%ΔHexA-GalNAc(4S/6S), and undetectable amounts of ΔHexA2S-GalNAc6S or ΔHexA-GalNAc4S6S. The CS/DS fraction of bovinemilk contains 23% ΔHexA-GalNAc, 75% ΔHexA-GalNAc(4S/6S),and 2% ΔHexA2S-GalNAc6S or ΔHexA-GalNAc4S6S.Differences in the HS components of human and bovine milk werefurther studied on the oligosaccharide level. To our knowledge, thisis the first time that extensive mass spectrometry guided glycomicsanalysis has been carried out on milk GAGs.

(194) Identification of Protein Glycosylation Associatedwith Aggressive Prostate Cancer using Glycoproteomics

and Glycomics ApproachesXiangchun Wang, Hui Zhang

Johns Hopkins University, Baltimore, MD

Glycan biosynthesis is highly controlled by specific glycosyltrans-ferases and the substrates in different biological processes.Alterations of glycosylation have been shown to correlate with thedevelopment and progression of cancer. However, the glyco-proteins and the glycans that are altered by the enzymatic or sub-strate changes in glycan biosynthesis and responsible for diseasedevelopment have not been characterized. Here, different aggres-sive and non-aggressive prostate cell lines were treated with globalN-linked and O-linked glycosylation inhibitors, which dramaticallyaffected both non-aggressive and aggressive prostate cancer cellmigration. Glycoproteomic and glycomic analyses were used toidentify glycoproteins and glycans associated with the treatments.These glycoproteins and their glycans associated with aggressiveprostate cancer may be useful as prognostic markers for aggressiveprostate cancer.

(195) Repository of Recombinant Expression Constructs forMammalian Glycosylation Enzymes: Production of

Glycosyltransferases and Glycoside Hydrolases in MammalianCells

Kelley W. Moremen1, Lu Meng1, Heather Strachan1,Anna Poorani Ramiah1, Zhongwei Gao1, Roy Johnson1,

Alison Nairn1, Yong Xiang1, Mitche de la Rosa1,Sheng-Cheng Wu1, Harry J. Gilbert1, Donald L. Jarvis1,

Jason Steel3, Joshua LaBaer31University of Georgia, Athens, GA; 2University of Wyoming,

Laramie, WY; 3Arizona State University, Tempe, AZ

Glycan biosynthetic and catabolic enzymes play critical roles inencoding the diverse collection of carbohydrate structures involvedin biological recognition events in animal systems. Mammalianglycosyltransferases (GTs) (�204 members) and glycoside hydro-lases (GHs) (�75 members) are responsible for the assembly anddegradation of glycan structures attached to intracellularand secreted proteins and lipids, yet the enzymatic, biochemical,and structural characteristics of many of these enzymes are notwell understood and most are not available in sufficient quantitiesfor biochemical studies or enzymatic synthesis of glycan struc-tures. Challenges for recombinant production of the glycosylationenzymes arise from general requirements for eukaryotic expressionto provide chaperone systems and post-translational modificationsnecessary for effective folding and function. To address the needfor these recombinant products, we established a repository ofexpression constructs encoding the catalytic domains of all humanGTs and GHs for production in HEK293 cells, baculovirus/insectcells, or in E. coli hosts. In mammalian cells, we developed anefficient transient transfection strategy for serum-free HEK293 sus-pension cultures providing secretion of the recombinant productsinto the conditioned media as affinity tagged forms. Alternativevector platforms with either short epitope and affinity tags orlarger GFP fusions provide strategies for protein production, affi-nity purification, and protein detection and quantitation. Strategiesfor tag removal and glycan cleavage via endoglycosidase digestionhave also been developed to produce enzymes compatible withbiochemical and structural studies. The poster will summarize thestatus of construct generation and protein expression in mamma-lian cells as well as availability of the constructs from the reposi-tory. The ultimate goals are to provide recombinant platforms forproduction of active glycosylation enzymes for use in biochemical,chemo-enzymatic, and structural studies on these critical humanproteins. Supported by NIH Grant P41 RR-005351-20S1.

(196) Enzyme-substrate Complexes of Human ERMannosidase I and Golgi Mannosidase IA Demonstrate theStructural Basis for Differences in Substrate SpecificityYong Xiang, Khanita Karaveg, Lirong Chen, B. C. Wang,

Kelley MoremenUniversity of Georgia, Athens, GA

Family 47 glycohydrolases cleave α1,2-mannose linkages onAsn-linked oligomannose structures by an inverting mechanismthat involves a substrate interaction with an enzyme-bound Ca2+


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ion for substrate distortion into the transition state. Amongenzymes in this family, ER α-mannosidase I (ERManI) and Golgiα-mannosidase IA (GMIA) are key enzymes involved in N-linkedglycan biosynthesis in the endoplasmic reticulum (ER) and Golgicomplex. The structures of ERManI and GMIA are similar inoverall protein fold, but have complementary activities in theircleavage of the natural Man9GlcNAc2 substrate. ERManI preferen-tially cleaves a single α1,2-Man residue from the central B-branchof the tri-branched oligomannose substrate, whereas GMIA prefer-entially cleaves three α1,2Man residues from branches A and C,but poorly cleaves the B branch α1,2-Man residue. In order todetermine the structural basis for their respective substrate speci-ficity differences, human ERManI and murine GMIA were eachinactivated by replacing the enzyme-bound Ca2+ with La3+, andco-crystallized with the substrate analog Man9GlcNAc2-PA. Thecrystal structures were resolved by molecular replacement at resol-utions of 1.65 Å for ERManI and 1.77 Å for GMIA. The enzyme-substrate complex structures reveal that the two enzymes bind theirnatural glycan substrates quite differently, consistent with the top-ology differences in the clefts leading to the respective active sites.GMIA binds the non-reducing terminal α1,2-Man of branch A inthe -1 enzyme subsite, while the remainder of the glycan hasextended interactions in the cleft leading to the active site. In con-trast, ERManI binds to the non-reducing terminal α1,2-Man ofbranch B with a distinctive set of interactions between the glycansubstrate and the active site cleft. The comparison of the crystalstructures provides insights into the structural basis of substratespecificity for members of this enzyme family. Supported by NIHGrants R01GM047533 and R01DK075322.

(197) On the Path to Biobetter Therapeutic Glycoproteins:Simple and Rapid Domain-Specific Screening to Target and

Control Optimal Glycan ProfilesSteven Mast1, Bopha Sun1, Scott Fulton2, Michael Kimzey1,

Shiva Pourkaveh1, Abdel Minalla1, Ted Haxo1, Craig Nishida1,Jo Wegstein1

1ProZyme, Inc, Hayward, CA; 2BioSystem Development, LLC,Madison, WI

A strategy for “Target-directed Product Development” has beendescribed, where multiple glycosylated forms of the protein weregenerated and evaluated for Critical Quality Attributes (1). In thecited case, a follow-on biological candidate was selected forfurther development, whose N-glycan profile demonstratedimproved efficacy compared to the innovator product.Subsequently this optimal profile was controlled within tight speci-fications throughout clone selection, cell-culture optimization andformulation in order to speed regulatory approval. Yet significantanalytical challenges were reported due to the tedious, manualmethods employed and the variability of the results, requiring asignificant investment of resources to accomplish.This poster presents methods for simple and rapid screening of

N-glycan profiles suitable for every aspect of drug developmentand manufacturing, including biocomparability studies and bio-reactor monitoring and control (PAT). Use of the GlykoPrep™platform to dramatically streamline glycoprotein sample

preparation, coupled with rapid analysis using a Waters UPLC®,allows the generation of high-quality results overnight.

For some glycoproteins, the information presented in the totalN-glycan profile may be insufficiently detailed for Target-directedProduct Development. Here we show a domain-specific analysis ofthe N-glycans of a commercially available Fc-fusion protein.Analysis of the individual glycosylation profiles may be a betterindicator to investigate the role glycosylation plays in the efficacyof the protein.

Reference(1) Toll, Hansjörg. Analytical Challenges for MultipleGlycosylated Proteins. Paper presented at: WCBP 2011: 15thSymposium on the Interface of Regulatory and AnalyticalSciences for Biotechnology Health Products; 2011 January 10 -12; Washington, DC.

(198) Characterization of Cellulose in Developing CottonFibers

Allen K. Murray1, Robert L. Nichols21Glycozyme, Inc., Irvine, CA; 2Cotton Incorporated, Cary, NC

During our investigation of cotton fiber development and cellulosethe cellulose fraction of DP-50 fibers at ages 21, 25, 30, 38, 44 and56 days post anthesis (DPA) has been subjected to sequential 6NHCl degradation. Long accepted data demonstrates the initial releaseof a large amount of glucose which then slows down over time. Thestated explanation is that the non-crystalline material is releasedrapidly but the crystalline material takes much longer to be released.That would imply a difference in the material remaining. Lightmicroscopy of the remaining residue of each successive hydrolysisdoes not appear to support that conclusion. In each case there justappears to be less material but microscopically it looks the same. Thecellulose particles appear to either be present or to disappear but novisible change has been observed. There do appear to be two signifi-cant differences between the cellulose of developing fibers andmature cotton fibers. Two groups of glycans which elute after about12 minutes on HPAEC chromatograms of the developing fibersappear to have a much greater relative abundance at all ages than thecorresponding glycans from the cellulose of mature fibers. Theseglycans have been found to yield several amino acids on hydrolysis.Another difference between the cellulose of developing fibers andthat of mature fibers is that at the end of the sequential hydrolysesthere is simply nothing left of mature fibers while the developingfibers yield a series of cello-oligosaccharides in their final hydrolysis.The significance of these differences is not known at this time butthere are apparent differences between developing fibers and fullymature cotton which involve even the cellulose which was syn-thesized much earlier. Although 56DPA cotton is often considered tobe mature, changes are still occurring in the fibers.

(199) β-1,4-galactosyltransferase 1 Expression Is Requiredfor Platelet Production in vitro and in vivo

Silvia Giannini, Renata Grozovsky, Antonjia Jurak Begonja,Karin M. Hoffmeister

Brigham and Women’s Hospital and HMS, Boston, MA


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Platelet turnover requires correct glycan presentation andexpression. Deficiency in β-1,4-galactosyltransferase 1 (β4GalT1),the major enzyme that transfers galactose (Gal) from UDP-Gal toterminal N-acetylglucosamine (GlcNAc) to form type-2lactosaminyl structures, is lethal in embryonic mice. However,E14.5 β4GalT1−/- embryos are grossly indistinguishable from wildtype embryos. Here we sought to determine whether platelet for-mation and survival require β4GalT1. Hepatic stem cells wereobtained from embryonic livers at E14.5 and megakaryoctes weredifferentiated in vitro in the presence of thrombopoietin. β4GalT1−/-

megakaryocytes mature and differentiate normally in vitro asjudged by their number, morphology and expression of mainsurface glycoproteins specific for the megakaryocyte lineage(GPIbα/β, GPIX, αIIb and β3). However, following differentiation,β4GalT1−/- megakaryocytes were unable to produce proplateletsand platelets in vitro. Addition of exogenous β4GalT1 and thedonor substrate UDP-Galactose did not improve in vitro proplateletand platelet formation. Importantly, platelet numbers weredecreased by� 75% in β4GalT1−/- E14.5 embryos when comparedto wild type embryos. Platelet size was increased by� 50% inβ4GalT1−/- embryos, indicating that β4GalT1−/- mice haveincreased platelet clearance and/or that larger platelets are producedin vivo. Our data strongly support the notion that glycosylationmediated by β4GalT1 activity is crucial for platelet production invitro and in vivo. Our findings demonstrate for the first time a rolefor posttranslational glycan modification in platelet production.

(200) Novel Function of HNK-1 Sulfotransferase as a HormoneRegulator

Misa Suzuki-Anekoji1, Atsushi Suzuki1, Shin-Yi Yu2,Kiyohiko Angata1, Kay-Hooi Khoo2, Jun Nakayama3,

Michiko N. Fukuda1, Minoru Fukuda11Sanford-Burnham Medical Research Institute, La Jolla, CA; 2Inst.

Biol. Chem., Academia Sinica, Taipei, Taiwan; 3ShinshuUniversity School of Medicine, Matsumoto, Japan

HNK-1 sulfotrasnferase is known to sulfate glucuronic acidresidue of glycoproteins and glycolipids, forming HNK-1 antigen;SO3-GlcAβ1-3Galβ1-4GlcNAc-R. This structure is known to beabundant in brain. To determine the role of HNK-1 antigen invivo, we generated HNK-1 sulfotransferase (Chst10) deficient miceby homologous recombination. The Chst10 nulls showed noHNK-1 antigen in the brain. The gross anatomy of Chst10 nullswere normal. However, Chst10 nulls bred poorly, which requiredin vitro fertilization to maintain the line. Since steroid hormonesare often conjugated by GlcA in vivo, we hypothesized thatSO3-GlcA plays a role in reproduction in the mouse. Thus wetested activity of recombinant Chst10 toward glucuronide-conjugated sex steroid hormones, and found that Chst10 indeedtransfers sulfate to many of glucuronidated hormones includingestrogen and testosterone. The structures of in vitro sulfated hor-mones were confirmed by HPLC and tandem ion-mass/mass spec-trometry analyses. We also identified an HPLC peak comparableto Chst10 modified estradiol from serum of wild type mice, whichwas absent in Chst10 nulls. Cell binding assay and estrogen orandrogen response element reporter assays revealed that Chst10

modified estrogen or testosterone did not bind to each hormonereceptor, suggesting that Chst10-modified steroids are inactive invivo. Histologies of Chst10 null male mice revealed the absence ofventral prostate luminal cell layers, which are thought to rise fromprostate stem cells in response to androgen stimulation, suggestingthat regulation of androgen by SO3-GlcA is essential for develop-ment of luminal cell layer in the prostate. Chst10 null femalesshowed thick uterine endometrial epithelia, suggesting that theywere proliferated by un-regulated estrogens. These results suggestthat a failure in regulating steroid hormones by Chst10 caused sub-fertility in both male and female in the mutant mice. Takentogether, HNK-1 sulfotransferase regulates steroid hormone func-tions by sulfation of glucuronide-conjugated hormones.

(201) O-methyl Phosphoramidate Modifications on theCapsular Polysaccharide of Campylobacter jejuni are Involved

in Serum Resistance, Infection, and Insecticidal ActivityLieke van Alphen1,2, Christopher Fodor1,2, Cory Wenzel1,2,

Roger Ashmus1,2, William Miller3, Martin Stahl4,5,Alain Stintzi4,5, Todd Lowary1,2, Christine Szymanski1,2

1Alberta Innovates Centre for Carbohydrate Science, Edmonton,AB, Canada; 2University of Alberta, Edmonton, AB, Canada; 3U.S. Department of Agriculture, Albany, CA; 4Ottawa Institute ofSystems Biology, Ottawa, ON, Canada; 5University of Ottawa,

Ottawa, ON, Canada

Campylobacter jejuni is the most commonly reported cause ofbacterial foodborne illness in North America. C. jejuni decoratesits surface polysaccharides with a variety of variable phosphory-lated structures, including O-methyl phosphoramidate (MeOPN)modifications on the capsular polysaccharide. Although MeOPNmoieties are present in �70% of C. jejuni isolates as well asseveral other members of the epsilonproteobacteria, little is knownabout their biological role. The C. jejuni genes involved inMeOPN biosynthesis and transfer to capsule have previously beenidentified in strain 11168, and our group has recently confirmedvia site-directed mutagenesis that homologous genes in the highlyvirulent strain 81-176 are required for the biosynthesis and transferof MeOPN.

To determine the biological significance of MeOPN expressionin C. jejuni, a variety of in vitro and in vivo assays were performedcomparing the wild-type 81-176 strain to knockout mutantsdeficient in biosynthesis (Δcj1416) or transfer (Δorf7/orf20) ofMeOPN. Mutants lacking MeOPN were more sensitive to serumand exhibited 10-fold greater invasion of Caco-2 epithelial cells.To investigate the potential insecticidal effects of the MeOPNstructure in vivo, we used the Galleria mellonella (wax mothlarvae) model system in which live C. jejuni bacterial cells areinjected into the hemolymph of the larvae. In this model, wild-type C. jejuni 81-176 efficiently killed G. mellonella in a dose-dependent manner and the Δcj1416 mutant displayed reducedkilling, while complementation of this mutant resulted in wild-typekilling. Interestingly, the Δorf7/orf20 mutant killed G. mellonellaas efficiently as wild-type, suggesting that accumulation ofMeOPN within the bacterial cells might be killing the insects.Infection experiments with piglets demonstrated that the Δcj1416


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mutant was outcompeted by the wild-type strain. In contrast, theΔorf7/orf20 mutant showed similar levels of colonization aswild-type.

(202) Analysis of Human Milk Lactose by HPLC-MSCeng Chen, Gherman Wiederschain, David S. Newburg

Boston College, Chestnut Hill, MA

Lactose (lactobiose; 4-O-β-D-galactopyranosyl-D-glucopyranose)is found almost exclusively in milk, and is the principal sugar ofmost types of milk. In human milk, lactose is the solid componentfound in greatest abundance. Although numerous methods forlactose analysis have existed for over 100 years, they have primar-ily been validated and used to measure the lactose of bovine milkand milk products. Human milk contains high amounts of otherglycans whose functional groups resemble lactose, falsely elevat-ing lactose measurements obtained by many of the most commonanalytical methods. In human milk research, measuring lactoselevels precisely allows studies on lactogenesis, and individual vari-ation in lactose levels as a function of stage of lactation, geneticvariation, and ethnic grouping. Herein, a tandem LC-MSinstrumental method is described that directly quantifies lactose inhuman milk without any derivatization and interference from othermilk components. In this method, a porous graphite carboncolumn baseline resolves α- and β-anomers of lactose in a 5-minrun. The sum of the areas of the two anomer peaks detected byMS in negative mode compared with the nominal concentration oflactose standard produces a linear curve with a limit of quantifi-cation of 0.039 µg/mL. Validation of this method by addingknown amounts of lactose to the human milk matrix gives pre-cision of 2-3%, accuracy of 95-99%, and recovery of 91-104%.This rapid straightforward method of lactose analysis can providemore accurate data for human milk studies on understanding thebiochemical dynamics of lactation and lactogenesis, and in provid-ing accurately defined milk preparations for premature and otherinfants whose consumption needs careful regulation.

(203) Automated Glycan Structural Isomer Differentiationusing Bioinformatics Tool

Julian Saba1, Amy Zumwalt1, Ningombam Sanjib Meitei2,Arun Apte2, Rosa Viner1

1Thermo Fisher Scientific, San Jose, CA; 2PREMIER BiosoftInternational, Palo Alto, CA

Mass spectrometry (MS) has emerged as a powerful tool for thestructural elucidation of glycans. The use of permethylaytion incombination with multistage fragmentation (MSn) is a criticalaspect for glycan structural characterization. Only MSn trulycharacterizes a glycan structure as it allows identification ofbranching patterns, linkages and resolution of isobaric structureswhich are otherwise indistinguishable in MS/MS spectra.However, MSn analysis is complicated by large number of spectragenerated for a single structure. It is very common that one mustacquire MS6 or MS7 level of fragmentation to differentiate poten-tial glycan structural isomers. Here we present the use of a bioin-formatics tool (SimGlycan) for glycan structural isomerdifferentiation from MSn data.

SimGlycan software was used in combination with MSn andpermethylation to differentiate structural isomers present in oval-bumin glycans. The ion trap MS profile was acquired for per-methylated ovalbumin glycans and specific precursors weretargeted for MSn to differentiate structural isomers. SequentialMSn data were imported into SimGlycan software and variousstructural isomers of ovalbumin glycans were differentiated.SimGlycan differentiated structural isomers were verified usingmanual assignment and previously published data.

(204) The Development of Tools for the Affinity-BasedProteomic Profiling of exo-α-glycosidase ActivityMichael Gandy, Aleksandra Debowski, Keith StubbsUniversity of Western Australia, Perth, Australia

The development of activity-based proteomics probes (ABPPs) isa topic of wide general interest owing to the potential use of suchprobes in the identification of enzymes of biomedical interest orthose having desirable biochemical properties, the profiling ofthese enzymes in organisms, and the evaluation of inhibitor speci-ficity in tissues and cells. Unfortunately, relative to proteases andesterases, the development of ABPPs for carbohydrate-processingenzymes is greatly lagging. There is a clear need for such probes;there are over 120 families of glycan degrading enzymes known,each of which contain many annotated genes encoding known orputative enzymes. These enzymes are widely used in biotechnol-ogy and several are putative drug targets, known virulence factors,or critically important for cellular processes. Despite the wideinterest in these enzymes, no rigourous ABPP strategy has yetbeen developed to identify and purify exo-α-glycosidases.

Here we present a strategy for labelling exo-α-glycosidasesusing an iminosugar scaffold and a bifunctional photoaffinityprobe equipped with a highly photosensitive aromatic azide group.A biochemical reporter group can be appended to this complex viaa Staudinger ligation, thereby enabling subsequent proteomic pro-filing and purification. We detail the expedient synthesis of theseABPPs and demonstrate their detection limits using pure recombi-nant enzymes. We also show that the probes can selectively labelglycosidases in a complex proteome. We anticipate that thisgeneral strategy will find utility in the analyses of glycoside hydro-lases from various proteomes for both biotechnological and thera-peutic purposes.

(205) Genetic and Functional Mechanisms of DrosophilaSialylation

Hilary Witzenman, Dheeraj Pandey, Elena Repnikova,Michiko Nakamura, Rafique Islam, Niraj Kc, Courtney Caster,

Vladislav PaninTexas A&M University, Collge Station, TX

Sialylation is a ubiquitous glycan modification playing numerousimportant biological roles in vertebrate organisms. Yet, sialic acid-containing glycans are exceedingly rare in invertebrates. InDrosophila, they are limited to a subset of cells in the nervoussystem. The important function of sialylation in fruit flies wasrecently revealed by targeted inactivation of the DSiaT gene thatencodes Drosophila sialyltransferase. DSiaT mutations result in


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locomotor defects, abnormal development and physiology of neu-romuscular junctions, and temperature-sensitive paralysis pheno-type (Repnikova at al., J. Neurosci. 2010). These experimentsindicated that sialylated N-glycans play a specific function in theregulation of neural excitability. However, the biosynthesis ofthese glycans remains poorly understood. Using a combination ofbehavioral, neurophysiological and genetic approaches, we investi-gated the function of genes that potentially participate in the sialy-lation pathway or collaborate with sialylation genes. We foundstrong genetic interactions between some of these genes in theregulation of the nervous system. These results shed light on thegenetic and functional mechanisms of sialylation. Our experimentsalso revealed a complex relationship between genes involved insialylation, suggesting that some of them may have a dual functionin the nervous system that is not limited to the sialylation pathway.Further research will examine molecular mechanisms underlyingfunctional relationships between the genes involved in Drosophilasialylation. Supported by NIH/NIGMS grant GM069952 to VP

(206) Chemoenzymatic Labeling Strategy for Probing andVisualizing the Fucoseα(1-2)galactose Glycan Motif

Jean-Luc Chaubard1, Chithra Krishnamurthy1,Linda Hsieh-Wilson1,2

1California Institute of Technology, Pasadena, CA; 2HowardHughes Medical Institute, Pasadena, CA

Protein glycosylation plays a fundamental role in the control ofbiological systems. Recently, new technologies have afforded theability to further understand the function of these glycoconjugates.Metabolic labeling techniques have enabled the imaging and pro-teomic analysis of various glycans and glycoproteins while che-moenzymatic labeling techniques have been development to trackspecific glycan motifs, such as mono- and disaccharides. Here, wereport a robust chemoenzymatic strategy for the detection of thedisaccharide fucoseα(1-2)galactose (Fucα(1-2) Gal). TheFucα(1-2) Gal motif has been implicated in the molecular mechan-isms that underlie neuronal development, learning, and memory.The Fucα(1-2)-Gal motif is also overexpressed on many types ofepithelial cell tumors, representing a potential biomarker for thesecancers. We have exploited the substrate tolerance of the bacterialhomologue to blood group transferase A (BgtA), which transfersN-acetylgalactosamine (GalNAc) onto the galactose moiety ofterminal Fucα(1-2)-Gal glycans, to allow for the chemoselectiveinstallation of an azide functionality onto Fucα(1-2)Gal glycansusing the unnatural nucleotide sugar, UDP-N-azidoacetylgalactosa-mine. The bioorthogonal azide group allows chemoenzymaticallylabeled Fucα(1-2)Gal glycans to be selectively tagged with term-inal alkyne or strained alkyne reporters via Cu(I)-catalyzed orCu-free azide − alkyne cycloaddition (click chemistry) respectively,for fluorescent imaging or purification. BgtA specifically modifiesonly terminal Fucα(1-2)Gal glycans, as demonstrated by glycanmicroarrays. Using this chemoenzymatic method, we demonstratethe presence of Fucα(1,2)Gal glycosylation on neuronal proteinsimportant for synaptic vesicle release and cell adhesion while alsodetermining the relative expression levels of Fucα(1-2)Gal glycansacross several cancer cell lines.

(207) Identitying the Fucose Proteome in Rat Cortical NeuronsChithra KrishnamurthyCaltech, Pasadena, CA

Fucose, a terminal sugar found on N- and O-linked glycoproteins,has been implicated in cognitive processes such as learning andmemory. For example, both task specific learning and LTP havebeen shown to induce protein fucosylation at the synapse.However, very few fucosylated proteins have been characterized.In order to investigate fucosylated proteins in neurons, we metabo-lically label fucosylated glycans with unnatural fucose analogs.Both the azido and alkynyl fucose analogs can be successfullyincorporated into glycans in embryonic rat neurons. Fucosylatedglycoproteins can then be tagged with a biotin reporter for purifi-cation over streptavidin resin, and resolved by SDS-PAGE. LC/MSanalysis of tryptic gel digests allows us to identify fucosylated gly-coproteins in rat cortical neurons. We identify several novel pro-teins to be fucosylated, including MARCKS, Vdac1, NCAM L1and other proteins involved in neuronal communication and den-dritic spine morphology, suggesting important roles for fucose inthe molecular events that may underlie synaptic plasticity.Metabolic labeling of fucosylated glycans can also be achieved inhippocampal neurons to visualize the neuronal localization offucose glycoproteins and in vivo upon intracranial injection offucose analogs into rat pups. Identification of the fucosyl proteomein hand enables our investigation of fucose function in the nervoussystem. Future studies will focus on labeling of fucose in livinganimals to investigate the roles thee individual protein may play inmemory consolidation, development, and synaptic plasticity.

(208) Enzymatic Synthesis of Poly-N-acetyllactosamine (pLN)N- and O-Glycans for Galectin Recognition

Wenjie Peng1, Ryan Mcbride1, Jennifer Pranskevich1,Janani Rangarajan1, Michel Gilbert2, Warren Wakarchuk2,

Nahid Razi1, James C. Paulson11The Scripps Research Institute, San Diego, CA; 2National

Research Council Canada, Ottawa, ON

N- and O-glycans are biologically important post-translational gly-coproteins on the cell surface, which play a fundamental role inmany processes, including cell adhension, signal transduction, andimmune response. Extended poly-N-acetyllactosamine (pLN)glycans found on glycoproteins as well as glycolipids of somecell, have been characterized as special ligands for different lectinssuch as selectins and galectins as well as being tumor-associatedantigens. Extended LN structures on glycans are also shown toinfluence the receptor-binding specificity in different studies ongalectins and influenza-hemagglutinin receptor (ChandrasekaranA. et al. Nature Biotechnology 2008, 26, 107). Due to the com-plexity of natural glycans containing extended pLN, the avail-ability synthetic structures for biological studies has been limiting.Here we reported the enzymatic synthesis of a library of pLN N-and O-glycans for galectin binding detection, containing differentLN repeat unit.

We had previous synthesized pLNs on N-glycans using a newHelicobacter beta3GlcNAc transferase (JHP1032) (Sauerzapfe


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B. et al. Glycoconj J. 2009, 26, 141) and beta4GalT1 startingfrom the egg yolk extraction, for expansion of the Consortium forFunctional glycomics microarray library. This series included bian-tennary and triantennary N-glycans with or without core fucose,with each arm extended to penta- or hexa-LN (http://www.functio-nalglycomics.org). We have now used the same stratedy onO-glycans, producing extended pLN on O-glycan cores 1, 2, 3,and 4, with each arm extended to penta-LN. The elongated mol-ecules also can be further sialylated and fucosylated at the non-reducing end. A focused microarray analysis shows diferentialspecificity of galectins for the pLN glycans on N-linked andO-linked cores. (Supported in part by NIH grants AI058113 andGM62116).

(209) Rapid Sample Preparation of Biologics to SupportHigh-Throughput/High-Resolution Glycan Analysis

by Capillary ElectrophoresisAndras Guttman1, Zoltan Szabo1, Barry Karger1, Jeff Chapman2,

Craig Nishida3, Jo Wegstein31Barnett Institute, Boston, MA; 2Beckman Coulter, Inc, Brea, CA;

3ProZyme, Inc, Hayward, CA

Structural characterization of the glycan moieties of biologics,especially recombinant monoclonal antibody (rMAb) therapeutics,is critical during clone selection, cell-culture optimization andproduct characterization. High-throughput screening methods arerequired in order to return results promptly to allow multiple iter-ations for selection of optimal candidates. This poster gives anoverview of a state-of-the-art screening protocol with rapid samplepreparation coupled with capillary electrophoresis (CE)-basedglycan analysis that includes: automatable sample preparation withoptional purification modules to allow direct screening of cell-culture samples; glycan labeling for laser-induced fluorescent(LIF) detection; clean-up and desalting to reduce excess reagentpeaks; and oligosaccharide profiling, carbohydrate sequencing andglucose unit (GU) value-based structural prediction.Glycans released from subnanomolar amounts of rMAbs are

quickly and accurately prepared and analyzed, e.g., rapid (<7 min)CE separation of twelve, key, IgG glycans can be obtained alongwith 96-well format operation for convenient overnight processing.Particular attention is paid to full separation of core-fucosylatedand afucosylated forms, as the presence or absence of this modifi-cation is of high importance in regulating the effector function ofrMAbs. The excellent relative migration time reproducibility of theoptimized CE separation method (RSD <0.09%) facilitates high-fidelity peak assignments for the individual components in theglycan pools, and consequently allows high-precision, structuralpredictions using GU-value databases. Exoglycosidase, array-based, sequence verification of the predicted glycans is alsopresented.

(210) Glycosubstrates for Discovery of Selective Inhibitorsof Cell Surface Proteolysis by Adam Proteases

Anais Chavaroche, Nina Bionda, Mare Cudic, Gregg Fields,Dmitriy Minond

Torrey Pines Institute, Post Saint Lucie, FL

ADAM proteases are implicated in a variety of pathologies,including cancer, due to their ability to “shed” multiple glycosy-lated cell surface molecules (e.g., TNFalpha, L-selectin, CD44).Multiple clinical trials of zinc-binding inhibitors of ADAM17were discontinued due to toxicity as a result of the lack of inhibi-tor selectivity.

Our work is directed towards discovery of secondary bindingsite (exosite) inhibitors of ADAM proteases, which should providea viable alternative to active site inhibitors. Exosites withinADAM protease structure have been hypothesized to play role insubstrate recognition, but there are no reports addressing substratesequence, secondary structure, or post-translational modificationsresponsible for interactions with ADAM exosites.

We developed a potentially exosite-binding substrate for ADAMproteases based on a cleavage site of TNFalpha which contains aglycosylated serine residue. Specificity was significantly improvedfor the hydrolysis of glycosylated substrate by ADAM17 and8. Interestingly, ADAM10 exhibited decreased specificity with gly-cosylated substrate as compared to the non-glycosylated control.

Our data suggest that (1) glycosylation plays role in regulatingthe activity of ADAM proteases and thus might influence therepertoire of cleaved substrates in different diseases and (2)ADAM8 and 17, but not ADAM10, have an exosite that interactswith glycosylated residues. Based on these data, we hypothesizethat a glycosylated substrate should be superior to non-glycosylated one for a discovery of exosite inhibitors of ADAMproteases. We have constructed additional glycosylated and non-glycosylated substrates modeled after shed proteins, and are usingthese substrates to further refine the role of glycosylation in thespecificity of ADAM action.

(211) P1 Antigen is Present on the Serous Ovarian Cancer CellLine, IGROV1, Correlating with A4GALT Overexpression and

Altered Cell BehaviourFrancis Jacob1, Brian WC Tse1, Rea Guertler1, Sheri Nixdorf1,

Nicolai V. Bovin2, Neville F. Hacker3,Viola Heinzelmann-Schwarz1,3

1ACP, Lowy Cancer Research Centre, UNSW, Kensington,Australia; 2Institute of Bioorganic Chemistry, RAS, Moscow,Russian Federation; 3Gynaecological Cancer Centre, RHW,

Randwick, Australia

The P blood group system consists of three major antigens; Pk, Pand P1. They have been described in autoimmune haemolyticanaemia, urinary tract and parvovirus infections. We previouslydemonstrated that plasma levels of antibodies to P1 and Pk coulddiscriminate healthy controls from ovarian cancer samples (Jacobet al., 2011, Int J Cancer, epub). In this study, we investigated theexpression of P1 on ovarian cancer cells and its function in tumorbiology.

The expression of P1 on normal and ovarian cancer cell lineswas determined by fluorescence-activated cell sorting (FACS)using a human anti-P1 (IgM) antibody. Gene and proteinexpression of enzymes which synthesizes P1 and are involved inthe glycosphingolipid globo series pathway were examined.P1-positive subpopulations were sorted into high and low


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P1-expressing cells. Investigations on the influence of P1 on cellproliferation, migration and adhesion were performed usingin vitro scratch assay and xCELLigence System.Flow-cytometric analysis revealed that the high grade serous

ovarian cancer cell line IGROV1 contained a significant fractionthat was P1-positive. No other cell line investigated using FACSwas positive for P1. IGROV1 cells had an overexpression of up to80-fold of α 1,4-galactosyl transferase (A4GALT) compared toother cell lines, corroborating our FACS results. In contrast, theterminal galactose cleaving enzyme, α galactosidase A, was down-regulated in IGROV1 cells but was highly expressed in endome-trioid (TOV112D) and clear cell (TOV21G) ovarian cancer cells.IGROV1 was further characterized following cell sorting intoP1-high and negative fractions. Interestingly, these two populationshad different growth characteristics.We demonstrated that P1 is present on the ovarian cancer cells

IGROV1. The genes involved in its synthesis are aberrantlyexpressed and this potentially influences the growth behavior. Thisstudy provides further evidence that members of the P bloodgroup may play a role in ovarian cancer.

(212) Mechanism for Discrimination between Neu5Acand Neu5Gc IncorporationFan Yang, Jennifer J. Kohler

University of Texas Southwestern Medical Center, Dallas, TX

Humans are unable to produce the sialic acidN-glycolylneuraminic acids(Neu5Gc), duo to deletion of humangene coding for CMP-Neu5Ac hydroxylase (CMAH). ButNeu5Gc can be metabolically incorporated to glycoconjugatesbyhumans from dietary sources. These Neu5Gc-containing glycocon-jugates have been found in certain human tumors. BecauseNeu5Gc carries negative charge for human as incorporation ofNeu5Ac, we proposed a method to track them in both human androdent cells to discover mechanism for discrimination betweenNeu5Ac and Neu5Gc in corporation.

(213) Engineered GFP as a Sensitive Sensor of GlycosylationSite Occupancy

Marie-Estelle Losfeld1, Bobby Ng2, Hudson H. Freeze31Sanford Burnham Medical research institute, La Jolla, CA;

2Burnham Institute, La Jolla, CA; 3Sanford-Burnham MedicalResearch Institute, La Jolla, CA

Genetic defects in the glycosylation pathway lead to a family ofinherited disorders called congenital disorders of glycosylation(CDG). CDG patients are usually diagnosed by abnormal isoelec-tric focusing (IEF) of serum transferrin, possessing either impairedglycosylation or N-glycosylation sites unoccupied.However, no universal marker is available to test glycosylation in

patient cells such as fibroblasts. To address this problem, wedesigned an ER-retained GFP biomarker whose fluorescence is lostwhen it is glycosylated due to steric hindrance by the N-glycan. Inthis study, we demonstrate this new marker efficiency in HeLa cells,and in normal or patients’ fibroblasts to assess glycosylationdeficiency. We also present some applications including the possi-bility to screen compounds that rescue impaired glycosylation.

(214) Quantitative Proteomics Identifies ICAM-1as an N-glycosylation Deficiency MarkerPing He, Bobby Ng, Hudson H. Freeze

Sanford-Burnham Medical Research Institute, La Jolla, CA

Protein glycosylation, a very common post-translational modifi-cation, is a universal feature found in all cell types. The sugars areattached to asparagine residues (N-linked glycosylation) or serineor threonine residues (O-linked glycosylation). Many human inher-ited disorders are characterized by changes in glycosylation status,most of which involve N-glycosylation defects that include conge-nital disorders of glycosylation (CDGs). However, there are fewcellular markers to use for indicating glycosylation status withinthe cells. Plasma membrane glycoproteins are potential bio-markers. Stable isotope labeling by amino acids in cell culture(SILAC) is a quantitative proteomic platform to compare proteinlevels in different samples. Herein, we combined SILAC withlinear ion trap mass spectrometry (LTQ-Orbitrap) to identify andquantify membrane proteins from wild type CHO cells (Pro5) andCHO cells (Lec9) with a defect in dolichol synthesis. We ident-ified 48 N-glycosylated plasma membrane proteins under-represented in Lec9 cells as potential hypoglycosylated markers.Among those candidates, intercellular cell adhesion molecule 1(ICAM-1) was further verified to be reduced on the cell surface inLec9 cells and in CDG patient-derived fibroblasts (CDG-Ia, -Ib,-Ik, -DK,-OST, -PGM-1 and CDG with unknown mutation) com-pared with normal controls. Further functional assays showed thatN-glycosylation deficiency in some CDG fibroblasts resulted in 1)protease inhibitor (MG132)-induced accumulation of misfoldedICAM-1 due to the inhibition of degradation of misfoldedICAM-1, and 2)greater discrepancy in ICAM-1 level betweenCDG and control cells when upregulating ICAM-1 expression byTNF α. More importantly, mannose supplementation partially cor-rected hypoglycosylation in CDG Ib fibroblast as reflected byincreased ICAM-1 presence on plasma membrane. All of thesedata indicate that ICAM-1 could be a useful marker for under-standing N-glycosylation deficiency in CDGs and even for moni-toring clinical management of such disorders.

(215) Tumor Gangliosides Promote Tumor Infiltrationby Myeloid Suppressor Cells

Assefa Wondimu, Yihui Liu, Yi Zhang, Yan Su, Stephan LadischChildren’s National Medical Center, Washington, DC

Tumor gangliosides impact tumor formation and progression. Manytumors, such as neuroblastoma, medulloblastoma, and renal cellcarcinoma, shed membrane gangliosides into the tumor microenvir-onment where they may contribute to suppression of anti-tumorimmune responses. Myeloid derived suppressor cells (MDSC), aheterogeneous population of bone marrow-derived cells(CD11b+CD11c−Gr1+), play an important role during tumor growthand metastasis by suppressing tumor immunity, but factors causingMDSC accumulation in tumors remain to be fully identified. Herewe explored the influence of tumor gangliosides on MDSC accumu-lation in tumors in vivo, in the unique syngeneic constitutiveganglioside-depleted tumor cell model, DKO, developed by


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oncogenic transformation of murine embryonic fibroblasts lackingkey ganglioside synthetic genes (Oncogene 29:3297-306, 2010).Syngeneic C57Bl/6 mice were injected with 105 ganglioside-richwild type (WT) or ganglioside-depleted DKO cells, tumor growthmonitored, and tumor infiltrating cells analyzed by FACS. Theabsence tumor gangliosides (DKO tumors) significantly reducedtumor incidence and growth. Strikingly, whereas wild-type (WT)tumors had prominent MDSC infiltration, constitutive tumorganglioside depletion (DKO tumors) resulted in highly significantreduction of tumor infiltration by these cells (p < 0.0001), indepen-dent of tumor size. Both major MDSC subsets, granulocytic(Ly6G+Ly6Clow) and monocytic (Ly6G−Ly6C high), were reducedin the ganglioside-depleted DKO tumors, although the reduction ofthe granulocytic subset was more pronounced. Co-injection of pico-molar quantities of highly purified WT tumor gangliosides togetherwith the ganglioside-deficient DKO cells restored MDSC infiltra-tion in DKO tumors, and inhibition of tumor ganglioside synthesispharmacologically in a second tumor system (EL4 lymphoma) alsoresulted in reduced tumor accumulation of MDSC. Our findingsidentify tumor gangliosides as a tumor-derived factor enhancingtumor accumulation of MDSC, which in turn can potently inhibitcellular antitumor immune responses. Supported by NIH grantsR01CA42361 and R01CA61010.

(216) Engaging the Ashwell-Morell Receptor Elicits Protectionagainst Organ Damage and Disseminated Intravascular

Coagulopthy in Fatal SepsisPrabhjit Grewal1, Won Ho Yang2, Cheri Mann1, David Ditto3,Ricardo Lardone2, Dzung Le3, Nissi Varki3, Jamey Marth1,2

1UCSB, Santa Barbara, CA; 2Sanford-Burnham Med. Res. Inst.,UCSB, Santa Barbara, CA; 3UCSD, San Diego, CA

The Ashwell-Morell receptor (AMR) of hepatocytes rapidly clearsfrom blood circulation exogenously administered glycoproteinsbearing glycan ligands that include galactose andN-acetylgalactosamine. Although the AMR is conserved through-out mammalian radiation, endogenous ligands and a biologicalpurpose of this prototypical lectin have only recently been discov-ered. We previously identified circulating platelets and vonWillebrand factor (VWF) as endogenous AMR ligands and foundthat 70% of blood platelets are rapidly removed from circulation bythe AMR during sepsis induced by Streptococcus pneumoniae(SPN) infection. This AMR-dependent platelet clearance activity isinduced during SPN infection by the SPN-encoded NanA neurami-nidase. Hemostatic adaptation by the AMR reduces the onset of dis-seminated intravascular coagulation (DIC) - a lethal complication ofsepsis, and significantly improves the probability of host survival.Therefore our findings challenge the assumption that the earlythrombocytopenia of sepsis, which is typically a clinical hallmarkof infection, is due to the consumption of coagulation factors, andinstead suggest that AMR-mediated platelet clearance is a hostresponse to reduce the potential for DIC. We have now developedan approach to further discover novel AMR ligands in vivo and todetermine the role of platelet clearance in the therapeutic activity ofthe AMR during sepsis. Our findings will be presented and furtherindicate that engagement of the AMR early during infection by

intravenous administration of neuraminidase promotes a selectiveand transient hypo-thrombotic state that involves platelet clearanceand which minimizes disseminated intravascular coagulopathywhile increasing the frequency of host survival. At present, thereare no therapeutic agents in the clinic with similar anti-coagulantand therapeutic potency, and which can greatly inhibit organdamage and failure. Neuraminidase-induced AMR clearanceactivity appears to provide an effective anticoagulant therapy formultiple indications including preservation of organ health andfunction during the onset and progression of sepsis.

(217) General Patterns of Fibronectin Glycosylation Changein Pathological States

Anna Kulinich, Olga Kostjuk, Ganna Maslak,Iryna Pismenetskaya, Alla Shevtsova

Dnipropetrovsk State Medical Academy, Dnipropetrovsk, Ukraine

Fibronectin (FN) is an adhesive glycoprotein of extracellularmatrix and blood plasma that has several sites of O- andN-glycosylation. It takes part in cell migration, differentiation andsurvival. Carbohydrate determinants of this protein change underthe impact of different glycosidases and glycosyltransfereses acti-vated in pathological processes. Investigation of these changes canhave important theoretical and practical value. Objectives: toinvestigate fibronectin glycosylation in pathological states.Materials and methods: glycosylation of FN was investigated inplasma of patients with proloferative blood diseases (n = 25),chronic liver diseases (n = 25), heart diseases (n = 26) and healhydonors (n = 20) by lectin-enzyme analysis using deglycosylatedantibody to FN. Lectins specific to core α(1-6)-fucose ofN-glycanes (Aleuria aurantia lectin - AAL), terminal α(1-2)-fucoseof O-glycanes (Labornum anagyroides lectin – LABA), to sialicacid of bi- and polyantennary glycans (Sambucus nigra agglutinin– SNA, Maacia amurensis agglutinin - MAA) were used.

Glycosylation of FN changed in pathological states and dependson the type of the disease. Binding of FN with SNA decreased inmyocardial infarction and chronic hepatitis (63,8 ± 3,3% and 57,7± 2,1% accordingly) and did not reliably change in proliferativeblood diseases. Resembling results were obtained with MAA. FNbinding with AAL reliably increase in proliferative blood diseases(29,1 ± 29,9%) and with LABA in chronic hepatitis (414 ± 24,8%).

Glycan component of FN can influence its protease-resistanceand functional activity (binding with cells and matrix). Sialylationand fucosylation of FN change in investigated diseases and it canlead to alterations of cell functionality and development of patho-logical process. Results of this work can be useful in understand-ing of mechanisms causing diseases and diagnostics ofpathological states.

(218) Effective Enrichment of Cancer Stem Cellsby anti-CD133 Antibody and SSA Lectin

Shunsaku Takeishi1, Kumiko Okudo2, Kenta Moriwaki2,Naoko Terao2, Yoshihiro Kamada2, Shunichi Kuroda3,

Eiji Miyoshi21GP Biosciences Ltd., Yokohama, Japan; 2Osaka University,

Osaka, Japan; 3Nagoya University, Nagoya, Japan


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It is a universal principle that all cells in nature are covered with adense and complex array of glycans, which mediate many impor-tant biological phenomena, including structural and physical roles.Changes in glycosylation also occur in relation to both inner andouter cellular environmental changes such as cell activation, differ-entiation, inflammation, and malignant transformation. The changeof these phenomena can be captured by glycosylation patternsusing the recent novel technology called lectin microarray, whichis the powerful tool for differential analysis of glycan structuresand also for cell discrimination.Glycans exhibit characteristic changes in their structures during

development and have been used as markers for stem cells. It hasbeen reported that the differentiation of several cell types of stemcell were discriminated by the analysis using lectin microarray. Onthe other hands, though the glycan structures could be unique tocancer stem cells (CSCs), they have remained unknown. In thisstudy, we examined glycan structures in CD133 + CD13+ CSCsby means of lectin microarray and found that several lectins recog-nizing sialylated glycans, MAL-I, SNA, SSA, TJA-I, ACG, ABAand MAH, showed higher affinity to CD133 + CD13+ CSCs thanto CD133+ CSCs. CD133 + CD13+ CSCs were recently reportedto have higher CSC ability than CD133+ cells. In addition, wedemonstrated that CD133 + CD13-SSA+ cells isolated from Huh7cells had significantly higher ability to form tumors in NOD/SCIDmice and spheres under serum-free conditions than CD133 +CD13-SSA- cells. These results suggest that hepatic CSCs highlyexpress sialylated glycans and that SSA can be the candidate mol-ecule for isolating CSCs. Sialic acids are associated with manyphysiological and pathological events, including tumor malig-nancy. In particular, alteration of sialic acids is associated withcancer behavior, such as invasiveness and metastasis. From thispoint of view, it is very interesting that CSCs can be isolated usingsialic acid.

(219) Metabolic Studies of a Pathogenic Pathway to DiabetesCheri Mann1, Kazuaki Ohtsubo2, Jamey Marth1

1Center for Nanomedicine, Sanford-Burnham Institute, SantaBarbara, CA; 2RIKEN ASI Disease Glycomics, Ibaraki, Japan

As obesity continues to rise, so do the number of individuals withType 2 diabetes (T2D). The ongoing epidemic of human T2D hasincreased the need to understand the mechanistic origins of thislife-threatening disease. This laboratory recently discovered a mol-ecular and pathogenic pathway present in mice and humans that isresponsible for the onset of diet- and obesity-associated diabetes(Ohtsubo et al, 2011, Nature Medicine, in press). Increased fattyacid levels were found to induce the nuclear exclusion andimpaired function of transcription factors HNF1A and FOXA2,resulting in diminished GnT4a glycosyltransferase expression. TheGnT4a glycosyltransferase is encoded by the MGAT4A gene andproduces N-glycan linkages that are essential to retain glucosetransporter expression and glucose sensing at the beta cell surface.Correction of this defect in mice protected them from the onset ofdiabetes. While this mechanism may reflect an aberrant metabolicresponse to an abnormal modern-day diet, the unexpected conser-vation of this diabetogenic pathway in both mammalian species

suggests that its activation might be advantageous in some con-texts. We are investigating this latter hypothesis in the earlyresponses of mice undergoing fasting upon loss of access to food.Our studies incorporate measurements of beta cell biology includ-ing the regulation of transcription factors HNF1A and FOXA2, theexpression levels of the MGAT4A gene, the function of GnT4ameasured by the DSA lectin, and beta cell glucose transport andinsulin secretion responses. These studies further include litter-mates that are resistant to diet– and obesity–associated diabetesresulting from constitutive beta cell–specific expression ofMGAT4A or GLUT2 transgenes. Recent findings suggest an unex-pected role of this pathway to diabetes in the control of potentiallyadvantageous metabolic events normally induced during the earlyphases of fasting following food withdrawal.

(220) Multiorgan Toxicity Reviewed in Blood Toxicoprotomevia Glycocapture Assisted Global Quantitative Proteomics

(gagQP)Bingyun Sun

Institute for Systems Biology, Seattle, WA

Blood is an ideal window for diseases and health, especially forcomplex pathologies that multiple organs are enrolled simul-taneously. Tissue signature proteins leaked into the blood streamunder these pathological conditions are valuable and easily acces-sible biomarkers for diagnosis and prognosis of such complexity.To proof-of-concept, we systematically characterized serum pro-teins in an acetaminophen induced mouse toxicity model throughglycopeptide capture assisted global quantitative proteomics(gagQP) approach. Differential protein concentration in pooledsera of overdosed mice was quantified by an N-isotag labelingapproach together with glycopeptide capture aided front-endsample fractionation. Organ-specific genes derived from transcrip-tome analyses were used to delineate the insulted organs from theobtained blood proteome. Our results suggested a sever intrahepa-toxicity companioned by responses raised from several extrahepa-tic organs including kidney, heart/muscle, bone marrow, brain,intestine, fat, etc. Using western blot, we validated many of thesenon-hepatic injuries including kidney, heart/muscle, and bonemarrow as well as the organ specificity of transcriptome derivedgene signatures. Our study approved the first time the possibilityto discover multiple organ toxicity responses simultaneously fromblood proteome using organ-specific protein signatures. We hopeour effort could shine light on the network interactions at organand tissue level in complex diseases.

(221) Identification of Glycosylation on Specific Glycositesusing Lectin and Chemical Immobilizations and Mass

SpectrometryYan Li

Institute of Biophysics, Beijing, China

Glycosylation is the most common form of protein modifications.Each glycoprotein can be glycosylated at different glycosites andeach glycosite may be modified by different glycans. This struc-tural heterogeneity has complicated the study of the structure-function relationships of glycoproteins. In this study, we report a


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method for the rapid identification and quantification of the glyco-sylation on particular glycosites from complex biological mixturesusing glycan-lectin affinity and chemical immobilization, followedby mass spectrometry. We showed that upon the identification ofdisease-specific changes in glycosylation using our lectin microar-ray technique, glycosites containing aberrant glycans were readilyidentifiable and quantifiable using the developed method. Theapplication of this method may facilitate our understanding of howperturbed glycosylation impacts upon disease progression andcould be used to discover glycosylated proteins that could serve asbiomarkers or therapeutic targets to improve clinical outcomes ofpatients.

(222) A Physiological Approach to Assess the Affinity of LectinCarbohydrate Interactions using Cancer Cells Immobilised

on a Biosensor SurfaceDiluka Peiris, Anatoliy Markiv, Miriam Dwek

University of Westminster, London, United Kingdom

Alterations in protein glycosylation offer potential for cancer-specific biological targeting strategies. Studies to identify changesin glycosylation in cancer often utilise lectins but the affinity ofinteraction is weaker than those between proteins and theirligands. The studies so far do not, however, recapitulate the phys-iological setting and the affinity of a lectin to its ligand at the cellsurface is generally thought to be greater than reported.A physiological system for assessing lectin: glycan interactions,

using a cell-based biosensor, was evaluated. Cancer cells weregrown on the biosensor chip surface and fixed in formaldehyde.The kinetics of lectin: glycan interactions were tested with avariety of lectins. The interaction affinities were an order of magni-tude higher at the cell level than using conventional biosensorbased approaches. The system was used to assess a synthetic formof the lectin from Helix pomatia (HPA) which is of some interest,due to its ability to bind to aggressive metastatic cancer. The HPAwas produced with a red fluorescent protein (RFP) tag and thedata showed that, despite its bulk, RFP does not interfere with theHPA glycan binding site.This technology enables molecular interactions to be studied in

a relevant cellular environment. As one of the intrinsic problemsin developing biological therapies is a lack of understanding of theaffinity with which successful therapies interact with the cellsurface, this methodology will be of interest to researchers study-ing working on new biological approaches for targeting cell sur-faces in a wide range of diseases, including cancer.

(223) Lectin Microarray Glycoprofiling of Intact UrineExosomes and Tamm-Horsfall Protein

Jared Gerlach1, Anja Krüger1, Shirley Hanley1, Susan Gallogly1,Marie Hogan2, Christopher Ward2, Matthew Griffin1,

Lokesh Joshi11National University of Ireland Galway, Galway, Ireland;2Department of Medicine, Mayo Clinic, Rochester, MN

Background/Aims: Urine samples are readily used for diagnosisand monitoring of kidney diseases. Glycosylation changes of urineexosome-like vesicles (ELVs) may specifically reflect kidney

disease severity and treatment responses. Methods for characteriz-ation of intact ELV glycosylation are limited and masking byco-purified Tamm-Horsfall Protein (THP) may be problematic. Weadapted lectin microarrays to profile urine ELV surface glycosyla-tion characteristics and to compare ELV profiles with purified THP.Methods: 43 lectins were arrayed on Nexterion® H slides with anon-contact printer. ELVs, enriched from healthy urine by ultracen-trifugation (UC) or ultrafiltration (UF) were labeled with CM-DiI orPKH26, incubated with lectin arrays and imaged at 5 µm resolution.Carbohydrate-mediated binding was verified based on ≥50% inhi-bition with haptenic sugars. Some ELV samples were pre-treatedwith specific deglycosylation enzymes. Results for individuallectins were expressed as mean ±SD of the fluorescence intensitiesless background fluorescence. Identical protocols were appliedto AlexaFluor® 647-labeled THP. Results: CM-DiI andPKH26-lableled urine ELVs gave clear, reproducible fluorescencepatterns with lectin microarrays. The lectin profiles for UC- andUF-enriched ELVs were comparable and were both broadly similarto AF647-THP microarray profiles. However, several lectinsdemonstrated differences in relative fluorescence intensitiesbetween ELVs and THP. Highest intensities for ELVs were observedat GlcNAc-binding lectins LEL and WGA suggesting N-linkedglycosylation or terminal GlcNAc modifications. For Gal/GalNAc-binding lectins, ELVs bound to SNA-II with higher inten-sity than THP while the opposite was observed for ABL.Importantly, the lectin microarray profiles were responsive to enzy-matic deglycosylation of intact ELVs. Conclusions: Lectin microar-ray profiling provides a sensitive platform to screen urine ELVs forglycosylation abnormalities. Although microarray patterns forenriched ELVs and THP were broadly similar, differential affinitiesof individual lectins suggest that non-THP glycoproteins on thesurface of urine ELVs make distinct contributions to these profiles.

(224) High-Throughput HPLC-based N-Glycan Analysisof Human Plasma Proteins Identifies Potential Biomarkers

for Maturity Onset Diabetes of the Young (MODY)Barbara Adamczyk1, Gaya Thanabalasingham2,3,

Jennifer Huffman4, Jayesh Kattla1, Mislav Novokmet5,Igor Rudan6,7, Anna Gloyn2,3, Caroline Hayward4,

Rebecca Reynolds8, Torben Hansen9,10, Iwar Klimes11,Pal Njolstad12,13, Jim Wilson6, Nicholas Hastie4, Harry Campbell6,

Mark McCarthy2,3, Pauline Rudd1, Katharine Owen2,3,Gordan Lauc5,14, Alan Wright4

1NIBRT, Dublin, Ireland; 2Oxford Centre for Diabetes, Oxford,UK; 3Oxford NIHR Biomedical Research Centre, Oxford, UK;4MRC Human Genetics Unit, Edinburgh, UK; 5Genos, Zagreb,

Croatia; 6Centre for Population Health Sciences, Edinburgh, UK;7University of Split School of Medicine, Split, Croatia; 8Centre for

Cardiovascular Science, Edinburgh, UK; 9Novo NordiskFoundation Centre, Copenhagen, Denmark; 10Faculty of Health

Sciences, Odense, Denmark; 11DIABGENE and DiabetesLaboratory, Bratislava, Slovakia; 12Department of Clinical

Medicine, Bergen, Norway; 13Department of Pediatrics, Bergen,Norway; 14Faculty of Pharmacy and Biochemistry, Zagreb,

Croatia


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Maturity Onset Diabetes of the Young (MODY) due to hepatocytenuclear factor 1-α (HNF1A) mutations accounts for 2% of dia-betes, but is often misdiagnosed as Type 1 (T1DM) or Type 2 dia-betes (T2DM), resulting in suboptimal clinical management.Biomarkers that distinguish HNF1A-MODY from other types ofdiabetes would enable more effective molecular diagnostics. Arecent genome-wide association study identified HNF1A as themaster regulator of plasma protein fucosylation. Lower activity ofthe HNF1A transcription factor results in decreased levels ofplasma protein N-glycans with antennary fucose.In the current study, a high-throughput HPLC method was

employed for structural determination and quantification of plasmaN-linked glycans from diabetic patients. An initial study profiledplasma protein N-glycans from 33 HNF1A-MODY and 41 T2DMsubjects. Disease-related glycosylation alterations provided gooddiscrimination between diabetes subtypes and therefore werefurther examined in subjects with HNF1A-MODY (n = 189),GCK-MODY (n = 117), HNF4α-MODY (n = 40), T1DM, (n = 98),T2DM (n = 167) and non-diabetic controls (n = 98).The results showed that the levels of triantennary fucosylated

glycans (glycan peaks DG9/[DG9 + DG8]) were substantiallylower in HNF1A-MODY than in other diabetes subtypes in boththe initial and the validation groups (P < 5x10-7 for all pairwisecomparisons). C-statistic measures of discriminative accurancywere 0.94 for HNF1A-MODY vs T1DM and 0.90 forHNF1A-MODY vs T2DM. These findings confirm that theDG9-glycan index allows clinically-useful discrimination betweenHNF1α-MODY and common types of diabetes.In conclusion, glycan profiling is a promising diagnostic tool

for HNF1A-MODY that could improve diagnosis rates.

(225) AToolbox of Human Cell Lines for the Screeningand Production of Fully Human and Glycooptimized

BiotherapeuticsSteffen Goletz, Renate Stahn, Antje Danielczyk, Hans Baumeister,

Annett Hillemann, Anja Löffler, Lars Stöckl, Doreen Jahn,Sven Bahrke, Rainer Stahn, Anke Flechner, Marion Schlangstedt,

Uwe KarstenGlycotope, Berlin, Germany

Glycosylation is one of the major post-translational modificationsof biotherapeutics that depends on the cell line used for pro-duction. By establishment of the GlycoExpress toolbox (GEX) wehave generated a set of glycoengineered human cell lines for thehigh yield production of fully human glycoproteins to optimize theglycosylation of antibodies and non-antibody biotherapeutics.Amongst them e.g.: SialoMax with optimized sialylation (α2-3and α2-6) and branching, SialoFlex and FucoFlex which allowsadjustment of the sialylation and the fucosylation degree fromalmost 0 to max, respectively, MabExpress which is optimized togenerate highly active antibodies. The GlycoExpress technologyallows to screen for the optimal glycosylation of any protein andto produce the glycooptimized biopharmaceutical for clinical use.In a number of in vitro and in vivo assays the optimally

glycosylated protein is identified and the corresponding cell line isfurther developed for GMP-production of that glycooptimized bio-pharmaceutical. GlycoExpressTM and its production technologiesallow an unmet fully reproducible glycosylation and PTM patternirrespective of production scale, fermenter size and fermentationtime and between sites shown for antibodies and non-antibodyproteins which is not met by other production technologies. Inaddition, the productivity is superior to current systems.

Glycooptimization with GlycoExpress improved these fullyhuman glycosylated biologics up to several hundred folds inrespect to activity, bioavailability, immunogenicity and/or patientcoverage. More than 15 proteins have been successfully glycoopti-mized so far with three antibodies and one protein hormone cur-rently in clinical development.

(226) Commensal Bacteria Expressing the CarbohydrateHuman Tumour-Specific Antigen Galß1-3GalNAca-(Thomsen-Friedenreich) as Potential Tumour Vaccine

Steffen GoletzGlycotope, Berlin, Germany

The tumour-specific Thomsen-Friedenreich antigen (TFα) hasattracted much attention as a potential target for a cancer vaccine.However, several attempts to develop a clinically effective vaccineemploying synthetic TF did not succeed in overcoming its lowintrinsic immunogenicity. Natural anti-TF antibodies that are fre-quently found in sera of healthy adults are thought to be generatedin response to constituents of the gastrointestinal microbiota. Wetherefore examined a broad variety of commensal bacteria for theexpression of TFα. An in-depth analysis with a novel set of anti-bodies revealed that structures immunochemically identical to TFαat the surface of human intestinal bacteria are in fact extremelyrare and not present on known bacterial strains. We identifiedstructures immunochemically identical to the true TFα antigenexclusively on two novel Bacteroides ovatus strains isolated fromthe faeces of healthy human individuals after TF-immunoaffinityenrichment. An accurate glycoanalysis of one strain confirmed thepresence of the Galβ1-3GalNAc- structure in the capsular polysac-charide of the strain and demonstrated it to be α-anomericallylinked as a branching structure within a heptasaccharidic repeatingunit (G. Henderson et al. Glycobiology Advance AccessPublication on May 7, 2011). We further examined the ability ofpasteurized bacteria from this strain to generate a TFα-specifichumoral immune response in mice after intraperitoneal or oralapplication. By applying an analysis system that circumvents theinterfering effect of natural (polyreactive) antibodies, we demon-strated that specific anti-TFα IgM and IgG antibodies wereinduced in mice in response to oral or intraperitoneal vaccinationwith the pasteurized strain. Our data suggest that the TFα expres-sing strain combines a unique set of properties (high antigendensity, antigen clustering, presentation of TFα as side chains oncapsular polysaccharides, non-toxicity, and intrinsic adjuvant prop-erties), which makes it a unique candidate for the development ofan adjuvant-free TFα-specific anti-tumour vaccine.


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(227) Bacterial-Derived Thomsen-Friedenreich AntigenActivates Specific T Cells via Presentation on Dendritic Cells

Christoph Goletz, Sandra Mikolajczyk, Philippe Ulsemer,Steffen Goletz

Glycotope, Berlin, Germany

The Thomsen-Friedenreich antigen (TFα; Galβ1-3GalNAcα1-) isa tumor-specific carbohydrate antigen and an interesting epitopefor the design of novel cancer vaccines. Beside the occurrence ona broad variety of different cancer cells, we found TFα to beexpressed on two novel gastrointestinal species. We identified aBacteroides ovatus strain to possess TFα within the capsular poly-saccharide. In this study, we aimed to investigate whether TFαderived from our B.ovatus strain is presented on dendritic cells andwhether bacterial TFα-loaded dendritic cells have the potential toinduce TFα-specific T cells.The fully functional human dendritic cell line NemodDC was

used as a model system. Loading of NemodDCs with crude cellenvelope-preparations of our B.ovatus strain resulted in presen-tation of TFα, which was demonstrated by detection withTF-specific antibodies. Co-staining with antibodies towards theco-stimulatory molecule CD80 and the dendritic cell-markerCD209 revealed that bacterial TFα is mainly presented onCD80+CD209+ NemodDCs, which represent mature dendriticcells. In order to conclusively demonstrate the antigen presentationof bacterial TFα, NemodDCs loaded with bacterial TFα were usedto prime human PBMCs. Generation of TFα-specific T cells wasdemonstrated by specific restimulation with NemodDCs loadedwith lysates from the glycoengineered TFα-positive human tumorcell NM-D4. The only overlapping structure between our B.ovatusstrain and the NM-D4 cell line is the TFα structure. TF-negativecounterparts of our B.ovatus strain and of the NM-D4 cells wereused as negative controls. They induce no specific restimulation orpriming, respectively. Consistent with these findings, incubation ofNM-D4 cell lysate-loaded NemodDCs with TFα-specific anti-bodies prevented the specific restimulation of TFα-positiveB. ovatus-primed PBMCs.These findings show that bacterial TFα can activate specific T

cells via presentation on dendritic cells. It further indicates that ourB.ovatus strain is a promising candidate for the development of aTF-based cancer vaccine.

(228) Biochemical Characterization Of PMM2-depletedZebrafish Suggests An Unexpected Mechanism For

Glycosylation Deficiency In CDG-IaNingguo Gao1, Abigail Cline2, Heather Flanagan-Steet2, HudsonH. Freeze3, Kirsten C. Sadler4, Richard Steet2, Mark A. Lehrman1

1UT-Southwestern Medical Center, Dallas, TX; 2University ofGeorgia, Athens, GA; 3Sanford-Burnham Medical ResearchInstitute, La Jolla, CA; 4Mount Sinai School of Medicine,

New York, NY

Congenital Disorder of Glycosylation (CDG) Type Ia is caused bymutation of the pmm2 gene encoding phosphomannomutase,which converts mannose-6-P (M6P) to mannose-1-P (M1P).

Patients suffer from developmental, neurological, and metabolicabnormalities associated with impaired protein N-glycosylationattributed to diminished M1P, a precursor of GDP-mannose andthe lipid-linked oligosaccharide (LLO) needed for N-glycosylation.Previously we used pmm2-directed morpholinos to develop aCDG-Ia model using zebrafish, and showed the morphant embryoshad reduced pmm2 RNA, reduced phosphomannomutase activity,and developmental abnormalities consistent with CDG-Ia. Wehave now applied Fluorophore-Assisted CarbohydrateElectrophoresis to directly analyze molecules relevant to LLO bio-synthesis in pmm2 morphant embryos, as well as morphantsrescued by co-injecting pmm2 mRNA. In pmm2 morphants weobserved losses of LLOs (58% of control) and total N-glycans(67% of control), and restoration by mRNA rescue, as expectedfor CDG-Ia. However, the pmm2 depletion was insufficient toreduce M1P and GDP-mannose concentrations. This suggestedthat the CDG-Ia-like phenotype resulted from another aspect ofPmm2 depletion. Aside from M1P loss, Pmm2 deficiency mightalso increase the enzyme’s substrate, M6P. We reported recentlythat M6P behaves as a second messenger-like signaling moleculein mammalian cells in a novel pathway that leads to destruction ofLLO molecules, ostensibly in response to viral infection to hinderviral glycoprotein synthesis. In pmm2 morphants, M6P was indeedelevated (187% of control), and surprisingly free glycans (pro-ducts of LLO destruction) were increased (195% of control). Botheffects were rescued by co-injecting pmm2 mRNA. Thus, M6Pelevation may cause LLO depletion in our zebrafish CDG-Iamodel. Extrapolating to CDG-Ia patients, in whom Pmm2deficiency also likely causes M1P insufficiency, impaired N-linkedglycosylation might result from synergistic mechanisms that hinderLLO synthesis (M1P loss), and accelerate LLO destruction (M6Pelevation) perhaps in an antiviral mechanism gone awry.

Supported by NIH-ARRA RC1-HD064159.

(229) Colorectal Cancer Desmoplastic Reaction Up-RegulatesCollagen Synthesis and Restricts Cancer Cell Invasion

Vivien Jane Coulson-Thomas1,2, Yvette May Coulson-Thomas1,Tarsis F. Gesteira1, Ana M. Mader3, Jaques Waisberg4,

Maria A. Pinhal1, Andreas Friedl5, Leny Toma1, Helena B. Nader11Federal University of São Paulo - UNIFESP, Sao Paulo, Brazil;

2University of Cincinnati, Cincinnati, OH; 3Department ofPathology, Faculdade de Medicina AB, Sao Paulo, Brazil;4Department of Gastrosurgery, Faculdade de Medicina, Sao

Paulo, Brazil; 5University of Wisconsin-Madison, Madison, WI

During cancer cell growth many tumors exhibit various grades ofdesmoplasia, unorganized production of fibrous or connectivetissue, composed mainly of collagen fibers and myofibroblasts.The accumulation of extracellular matrix (ECM) surroundingtumors directly affects cancer cell proliferation, migration andspread, therefore the study of desmoplasia is of vital importance.Stromal fibroblasts surrounding tumors are activated to myofibro-blasts and become the primary producers of ECM during desmo-plasia. The composition, density and organization of this ECMaccumulation plays a major role on the influence desmoplasia has


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upon tumor cells. In this study, we analyzed desmoplasia in vivoin human colorectal carcinoma tissue, detecting an up-regulationof collagen I, collagen IV and collagen V in human colorectalcancer desmoplastic reaction. These components were then ana-lyzed in vitro co-cultivating colorectal cancer cells (Caco-2 andHCT116) and fibroblasts utilizing various co-culture techniques.Our findings demonstrate that direct cell-cell contact betweenfibroblasts and colorectal cancer cells evokes an increase in ECMdensity, composed of unorganized collagens (I, III, IV and V) andproteoglycans (biglycan, fibromodulin, perlecan and versican).The desmoplastic collagen fibers are thick, with an altered orien-tation, as well as, deposited as bundles. This increased ECMdensity inhibited the migration and invasion of the colorectaltumor cells in both 2-D and 3-D co-culture systems. Therefore thisstudy sheds light on a possible restricting role desmoplasia couldplay in colorectal cancer invasion.

(230) Visualizing Alterations in the Trafficking ofGlycoconjugates within Niemann-Pick Type C Cells using

Cu-Free Click TechnologyEric Ngalle Mbua, Steven Johnson, Heather Flanagan-Steet,

Margreet Wolfert, Geert-Jan Boons, Richard SteetUniversity of Georgia-Athens, Athens, GA

The bioorthogonal chemical reporter strategy is emerging as a ver-satile methodology for labeling biomolecules such as nucleicacids, lipids, carbohydrates and proteins. In particular, a biomole-cule modified by an azido group can be tagged by Staudinger lig-ation using modified phosphines, by a copper (I)-catalyzedcycloaddition with terminal alkynes (CuAAC), or by a strain-promoted alkyne-azide cycloaddition (SPAAC). Using the com-pound, 4-dibenzocyclooctynol (DIBO) and its derivatives, we pre-viously demonstrated differences in cell surface sialylation withina group of established CHO glycosylation mutant cells (Lec2, Lec13, Lec 32, Cog-1, and Cog-2). Here, we show that this technol-ogy can be used to investigate the trafficking defects associatedwith Niemann-Pick type C (NPC), a lysosomal storage disordercharacterized by impaired cholesterol efflux from late endosomesand lysosomes due to deficiency in the integral membrane proteinNPC1. Consistent with the endocytic transport defect in thesecells, accumulation of sialylated molecules was observed withinintracellular vesicles in NPC-null fibroblasts. These vesicles exhib-ited partial co-localization with markers of early and late enod-somes. Interestingly, unlike mucolipidosis type II fibroblasts, nodetectable labeling was observed in cholesterol-laden lysosomes inthe NPC cells, suggesting that the accumulation of cholesterolrenders these lysosomes incapable of receiving glycosylated mol-ecules for degradation. Our preliminary data indicated that treat-ment of NPC fibroblasts with cyclodextrin effectively reducedcholesterol accumulation but did not appear to impact the accumu-lation of labeled vesicles. This finding implies that NPC1 mayhave additional functions within the endocytic network. Together,our results provide a novel perspective on the trafficking defects inNPC cells and further highlight the utility of this methodology toanalyze cells with altered biosynthesis, trafficking and turnover ofglycoproteins.

(231) Strategies for Glycomics and Glycoproteomicsand Glycosaminoglycan Analysis by Mass Spectrometry

and HPLCParastoo Azadi1, Mayumi Ishihara1, Christian Heiss1,

Krajang Talabnin1, Zhirui Wang1, Sashka Dimitrievska2,Marjan Huizing3, Laura Niklason2, Roberto Sonon1

1Complex Carbohydrate Research Center, UGA, Athens, GA; 2YaleUniversity, New Haven, Connecticut; 3National Human Genome

Research Institute, NIH, Bethesda, MD

The Analytical Services Laboratory of the Complex CarbohydratesResearch Center (CCRC) of university of Georgia offers analyticalservices to universities, research institutes and companies. Due tothe increased in number of more challenging projects, we willpresent some of these research projects that we have been involvedin the past year that include the N-linked glycan site mapping ofthe SIV virus glycoprotein; N- and O-linked profiling of micekidney tissue; N-linked profiling using in-gel digestion of purifiedbiglycan glycoprotein.

In addition we will present data from our glycosaminoglycanresearch with decellularized arterial human grafts that are beingdeveloped as small-caliber vascular grafts. Currently their maindrawback is high thrombogenicity, which can be reduced by recel-lularizing the luminal side of vessels with host cells. Our collabor-ators have proposed glycomimicry on decellularized grafts, i.e.reconstruction of the cell glycocalyx. They have covalentlyattached a poly(vinyl amine) (PVA) polymer onto native decellular-ized arteries, which were then functionalized with a high concen-tration of heparin. The current study examines the quantitation ofheparin on the lumen sides of these vessels. We have analyzedsamples from, the vessel decellularized, vessel decellularized,EDC/NHS crosslinked in the absence of Heparin, vessel decellu-larized and EDC/NHS crosslinked with low Heparin and vesseldecellularized and EDC/NHS crosslinked with high Heparin usingfluorescent tag and HPLC after depolymerization.

(232) Characterization of O-glycosylation of the b-amyloidPrecursor Protein from Wild Type and CRND8 Transgenic

Mice Models of Alzheimer’s DiseaseIrina Perdivara1, Robert Petrovich1, Erik J. Tokar2, Mike Waalkes2,

Paul Fraser3, Ken Tomer11NIEHS, Research Triangle Park, NC; 2NTP/NIEHS, ResearchTriangle Park, NC; 3University of Toronto, Toronto, Canada

For more than a decade, amyloid precursor protein (APP), the keyprotein involved in Alzheimer’s disease (AD) pathogenesis hasbeen known to be modified by N- and O-linked glycosylation,O-GlcNAcylation, Tyr sulfation and phosphorylation. APP hasthree isoforms, APP695, APP751 and APP770, derived fromalternative splicing of a single gene located on chromosome 21.Although these post-translational modifications may affect thebalance between the amyloidogenic and non-amyloidogenic path-ways, there have been only few studies on APP O-linked glycosy-lation. Recently, using collision induced dissociation and electrontransfer dissociation mass spectrometry, we reported the firstidentification of three O-glycosylation sites in the extracellular


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domain of APP695 secreted from CHO cells, as Thr291, Thr292and Thr576. The Core 1 type O-glycans observed at these siteswere mostly short structures of type GalNAcα1-O-Thr,Galβ1-3GalNAcα1-O-Thr, Siaα2-3Galβ1-3GalNAcα1-O-Thr and(Siaα2-6)(Galβ1-3)GalNAcα1-O-Thr. The relevance of these siteswas further investigated by Kitazume et al by site directed muta-genesis (J Biol Chem, 2010, 285(51), 40097-103). They showedthat brain endothelial cells largely expressed APP770 and that theO-glycosylated form is preferentially secreted. Because it appearsthat O-glycosylation is relevant in the processing of both APP695and APP770, we are currently expanding our study to characteriz-ation of APP in CRND8 transgenic mice and their wild type litermates. CRND8 mice express an aggressive phenotype of AD, dueto simultaneous Swedish (K670M/N671L) and Indiana (V717F)mutations. We will report our study aimed at isolation, purificationand identification of glycosylated structures in APP isolated frombrain of CRND8 transgenic and wild type mice. Specific resultsinclude: (i) comparison of the mRNA levels of the APP genebetween CRND8 and wild type mice; (ii) identification of in vivosites of O-glycosylation on the extracellular APP domain, and iii)identification of O-GlcNAcylation sites on the cytoplasmicC-terminal tail of APP.

(233) Exogenous Mannose Supplementation RescuesMpi-deficient Zebrafish in a Model of Congenital Disorder

of GlycosylationJaime Chu1, Sabrina Rosa1, Alexander Mir1, Ningguo Gao2,

Mark Lehrman2, Richard Steet3, Hudson H. Freeze4,Kirsten Sadler1

1Mount Sinai School of Medicine, New York, NY;2UT-Southwestern Medical Center, Dallas, TX; 3University ofGeorgia-Athens, Athens, GA; 4Sanford-Burnham Medical

Research Institute, La Jolla, CA

Patients with congenital disorders of glycosylation (CDG) have arecessive mutation of one of the many genes needed for proteinN-glycosylation. Lipid-linked oligosaccharide (LLO) synthesis isnecessary for glycan transfer to selected asparagine residues ofnascent polypeptides and requires conversion of fructose-6-phosphate to mannose-6-phosphate via phosphomannose isomer-ase (MPI; also called PMI). MPI-CDG patients can be treated withoral mannose supplements to increase flux through a minor comp-lementary metabolic pathway that produces mannose-6-phosphate,enabling LLO synthesis, and augmenting protein glycosylation.MPI-CDG is the only type of CDG that can be treated as patientsreceiving mannose therapy are largely devoid of symptoms. Tostudy the cellular and genetic pathways that contribute to themulti-systemic disease of CDG patients, we established a zebrafishmodel of MPI-CDG using morpholino knockdown of Mpi in zeb-rafish embryos. mpi morphants have less than 15% residual Mpienzyme activity at 4 days post-fertilization and show decreasedlevels of LLO by fluorophore-assisted carbohydrate electrophoresisanalysis. These deficiencies result in 40% embryonic lethality with70% of surviving embryos displaying several morphological phe-notypes including small eyes, dysmorphic jaws, pericardial edema,and curled tails. Importantly, these morphological phenotypes are

rescued by mannose supplementation. With mannose, less than20% of mpi morphants are affected and those that are affectedpresent a milder phenotype. These findings not only validate zeb-rafish as a means to study MPI deficiency but also establish zebra-fish as a useful tool to study other glycosylation disorders.(Supported by NIH-ARRA RC1-HD064159).

(234) TSG-6 Irreversibly Transfers Heavy Chains fromthe Pathological Hyaluronan-Heavy Chain Complex

onto Hyaluronan OligosaccharidesMark Lauer, Vincent Hascall, Anthony Calabro

Cleveland Clinic, Cleveland, OH

Pathological changes to hyaluronan (HA) in the synovial fluid(SF) of synovial joints (SJ) is a hallmark feature of inflammationin rheumatoid arthritis (RA). Normally synthesized from the cellsurface of synoviocytes as a high molecular weight (>1600 kDa)structural carbohydrate, HA serves as a hydrodynamic lubricant,reducing friction at these joints by increasing the viscosity andelasticity of SF. During SJ inflammation, a pathologic change toHA occurs via a unique protein modification involving the heavychains (HCs) of the serum-derived proteoglycan inter-α-inhibitor(IαI). As serum exudates enter SF during inflammation, thusbathing “naked” HA with the HC donor IαI, the enzyme “tumornecrosis factor stimulated gene – 6” (TSG-6) covalently transfersHCs from IαI onto the HA backbone forming a HC modified HAmatrix (HC-HA). The HC-HA matrix promotes the retention ofinflammatory cells that produce a variety of tissue destructivefactors causing joint destruction. While current therapies targetinflammatory cell function, we discovered a novel upstreamapproach to disable the inflammatory cell retention mechanism byirreversibly transferring HCs from HC-HA matrices to HA oligo-saccharides 10 monosaccharides long (HA10). This discovery isbased upon the observation that the formation of the HC-HAcomplex is a dynamic process whereby TSG-6 “swaps” HCs fromplace-to-place on the HA backbone of large HA molecules untilthe density of HCs per HA molecule reaches an equilibrium.Although the much smaller HA10 serves as an acceptor for HCswapping between HA molecules, it is unable to serve as a HCdonor. Thus, the transfer of HCs onto HA10 is an irreversible reac-tion where subsequent HC swapping cannot occur. This irrevers-ible swapping of HCs can be exploited to remove HCs frompathological HC-HA matrices found in RA and other inflamma-tory diseases, thereby removing the retention signal for inflamma-tory cells, thus decreasing inflammation.

(235) TSG-6: Novel Regulator of Viral-Induced HyaluronanSynthesis in Inflamed Airways

Mark Lauer, Georgiana Cheng, Shadi Swaidani, Amina Abaddi,Mark Aronica, Vincent HascallCleveland Clinic, Cleveland, OH

Hyaluronan (HA) is a very large (>1500 kDa) structural glycosa-minoglycan found in extracellular matrices in every organ andtissue system in the body. Under normal conditions, it is syn-thesized at the cell surface without any protein modifications.However, during inflammation, HA is transformed into a specific


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ligand for inflammatory cell receptors via a unique protein modifi-cation mediated by the enzyme ‘tumor necrosis factor stimulatedgene 6′ (TSG-6). This ligand is the heavy chain (HC) – HAcomplex that is formed when TSG-6 transfers HCs from theserum-derived proteoglycan inter-α-inhibitor (IαI) onto the HAbackbone. In asthma and other respiratory diseases, HC-HApromotes the accumulation of inflammatory cells in the smoothmuscle cell region surrounding airway vasculature and bronchi. Byadding recombinant TSG-6 to the culturing media, we artificiallyadded HCs to HA “cable” structures produced by airway smoothmuscle cells (ASMCs) in response to the viral mimetic poly(I:C).This not only promoted inflammatory cell adhesion to the HAcables, but resulted in the formation of more pronounced cablestructures. This suggests that HC transfer promotes and/or stabil-izes HA cable structure during its synthesis. Importantly, weobserved that TSG-6 amplified HA synthesis by ASMCs up to 35fold above baseline, ranking this enzyme as one of the mostpotent stimulators of HA synthesis yet discovered. Interestingly,this induction of HA synthesis did not occur by TSG-6 alone, butonly in cooperation with poly(I:C). This induction did not occurby an increase in hyaluronan synthase (HAS) transcription, thoughincreased HAS2 translation was observed. These observationsdemonstrate that TSG-6 is an important regulator of both the syn-thesis and subsequent HC modification of HA matrices duringairway inflammation.

(236) Chemical Inhibition of O-GlcNAc Processing: Roleof O-GlcNAc in Tau-Driven Neurodegeneration

Scott Yuzwa1, Xiaoyang Shan1, Matthew Macauley1,Thomas Clark1, Yuliya Skorobogatko2, Keith Vosseller2,

David Vocadlo11Simon Fraser University, Burnaby, Canada; 2Drexel College of

Medicine, Philadelphia, Pennsylvania

The aggregation of the microtubule-associated protein tau is a keyprocess contributing to the progressive death of neurons inAlzheimer disease (AD). Tau is well known to be post-translationally phosphorylated, however, serine and threonine resi-dues of tau have also been found to bear N-acetyl-D-glucosamineresidues via a β-O-glycosidic linkage (O-GlcNAc). The functionof the O-GlcNAc modification on tau has remained elusive,although it has been shown that O-GlcNAc on tau exists in adynamic reciprocal relationship with phosphorylation. Tau hyper-phosphorylation is known to be a characteristic of AD and isthought to play a role in the aggregation of tau into paired helicalfilaments which give rise to neurofibrillary tangles in the brain.One potential mechanism to reduce tau phosphorylation is toinhibit O-GlcNAcase (the enzyme responsible for removingO-GlcNAc) thus preventing the removal of O-GlcNAc and poten-tially preventing the addition of phosphorylation. Previously, wehave shown that treatment with a very potent and selective inhibi-tor of O-GlcNAcase (Thiamet-G) increases cellular O-GlcNAclevels and reduces phosphorylation of tau at pathologically rel-evant sites in the healthy mammalian brain (1). Here, we describethe long-term treatment of a disease model of tau-driven

neurodegeneration with Thiamet-G. We have examined both histo-logical and biochemical outcomes as well as the impact on motorimpairment in these long-term treated animals. This study hasallowed us to postulate a potential role for the O-GlcNAc modifi-cation of tau in tau-driven disease.

Reference1. Yuzwa, S.A., et al. “A potent mechanism-inspiredO-GlcNAcase inhibitor that blocks phosphorylation of Tauin vivo.” Nat Chem. Biol. 4, 483-490 (2008).

(237) ER Stress Reduces Angiogenesis and the Breast TumorProgression

Aditi Banerjee1, Juan Martinez1, Krishna Baksi2,Dipak Banerjee1,3

1University of Puerto Rico, San Juan, PR; 2Universidad Centraldel Caribe, Bayamon, PR; 3Institute of Functional Nanomaterials,

Rio Piedras, Puerto Rico

N-acetylglucosaminyl 1-phosphate transferase (GPT) catalyzes thefirst step of asparagine-linked (N-linked) protein glycosylationprocess. Angiogenesis is the hallmark for solid tumor growth.Microvascular capillary endothelial cells when treated with a com-petitive inhibitor of GPT tunicamycin, angiogenesis is halted byarresting cells in late G1. The treatment also inhibited theMatrigel™ invasion and chemotaxis. Genomic analysis by cDNAmicroarray indicated that a large number of upregulated genes cor-respond to transcription and translation. Exogenous addition ofpro-angiogenic VEGF could not reverse the anti-angiogenic sig-naling of tunicamycin suggesting derangement of cell survival.Significant reduction of cell surface N-glycan expression supportedthe development of ER stress and induction of unfolded proteinresponse (upr)– mediated apoptotic cell death. Down regulation ofprotein tyrosine kinase activity as well as the phosphorylation ofVEGF receptors R1 and R2 within 3 hours of tunicamycin treat-ment strongly suggested that the action of tunicamycin starts muchearly.

Translating the in vitro knowledge into an in vivo model ofathymic (Blab/c) nude mice (nu/nu) indicated that tunicamycinblocks neo-vascularization almost quantitatively in Matrigel™implants. H & E staining of the Matrigel™ sections supportedreduced vascularization. Immunohistochemical analysis of the sec-tions also indicated reduced expression of CD34 and CD144.Intravenous injection of tunicamycin once a week slowed down adouble negative grade-III breast adenocarcinoma growth by�50-60% in three weeks. The effect was dose-dependent.Histolopathological analysis of the paraffin sections indicated sig-nificant reduction in vessel size, the microvascular density andtumor mitotic index. Ki-67 and VEGF expression in tumor tissuewere also reduced. A significant reduction of N-glycan expressionin tumor microvessel corroborated with the vessel size. Highexpression of GRP-78 in CD144 positive cells supported ER stressin tumor microvasculature. Supported in part by NIHU54-CA096297, Susan G. Komen for the Cure BCTR0600582(DKB), and NIH/NCRR/RCMI G12-RR03035 (KB) grants.


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(238) Inhibition of U937-Cell Adhesion to Human EndothelialCells by Glycosylated Lysozyme Mutants

Ralph Melcher1, Ina Kraus2, Dorothea Moeller1,Stephanie Demmig1, Dorothee Rogoll1, Theodor Kudlich1,Wolfgang Scheppach3, Michael Scheurlen1, Andrej Hasilik2

1Department of Medicine II, Wuerzburg, Germany; 2Institute ofPathological Chemistry, Marburg, Germany; 3Department of

Medicine, Juliusspital, Wuerzburg, Germany

The glycosylated human lysozyme mutant hLysII/IV1 has beenexpressed and characterized in CHO cells expressing fucosyltrans-ferases FUCIII, IV, V, VI, or VII. Digestional analysis with endo-and exoglycosidases showed that hLysII/IV-FUCTVI containspredominantly biantennary complex N-glycans with a variablenumber of lactosamine repeats; these were terminally sialylatedand fucosylated, suggesting the presence of the sLex antigen. Incontrast, hLysII/IV-FUCTIII and IV were not terminally sialylatedand hLysII/IV-FUCV and VII were not terminally fucosylated. In astatic adhesion assay, binding of U937 to activated HUVEC cellswas efficiently blocked in the presence of medium supernatantcontaining hLysII/IV-FUCTVI and by affinity-purified hLysII/IV-FUCTVI. The binding was not attenuated in the presence ofhLysII/IV-FUCTIII, IV or V secretions. Titration experimentsshowed that significant inhibition of adhesion is achieved at 1ng/ml hLysII/IV-FUCTVI (0.07 nM), making it one of the mostpotent known inhibitors of adhesion described to date.

(239) Polysialyltransferases STX and PST are Involved inGenerating Neuraminic-Acid-Containing Polysialic Acid(NeuPSA), a Modification Found Abundantly in Human

Cancer CellsLindsay Steirer, Joshua Lee, Gregory Moe

CHORI, Oakland, CA

Background: Neuraminic-acid containing PSA (NeuPSA), ade-N-acetyl derivative of PSA, can be found in most normal adulthuman tissues; however, expression is considerably elevated inprimary tumors. In humans, two polysialyltransferases, STX andPST, synthesize PSA. Few proteins, two of which are autopolysia-lylated STX and PST, are known to be modified with PSA. In thisstudy, we investigated the link between NeuPSA expression andSTX/PST expression in SK-MEL-28 melanoma cells. Methods:The subcellular distribution of NeuPSA-modified antigens wasdetermined using confocal microscopy with organelle specific anti-bodies and the anti-NeuPSA antibody, SEAM 3. mRNAexpression levels of STX and PST were determined in multiplecancer cell lines using quantitative PCR. STX and PST expressionwas knocked down in SK-MEL 28 cells using RNA interferenceand the change in NeuPSA expression was measured by flow cyto-metry. Results and Discussion: NeuPSA antigens marked bySEAM 3 were mainly intracellular, but �25% of SK-MEL-28cells expressed NeuPSA on the cell surface. Intracellular SEAM 3reactivity was co-localized with golgi, ER and cellular matrixmarkers. Since STX and PST are the only PSA-modified proteinsknown to have a similar cellular distribution, this suggests STXand/or PST may be the NeuPSA-modified antigens recognized by

SEAM 3. We confirmed STX and/or PST expression in multiplecancer cell lines, including SK-MEL-28 cells. Using RNA interfer-ence to knock-down STX and PST expression we confirmed thedependence of NeuPSA expression on STX and/or PST inSK-MEL-28 cells. We found NeuPSA reactivity decreased inconcert with decreased STX or PST expression. The findingssuggest the polysialyltransferases may be modified with NeuPSA.Additionally, NeuPSA, STX, PST and as of yet, unidentified PSAde-N-acetylase(s) have an unrecognized role in cancer cell biology.

(240) HPLC MS/MS Analyses of Neu5Ac, Neu5Gc and KDNin Head and Neck (H&N) Tumors of the Throat

Frederic A. Troy1,2, Fang Wang2, Baoying Xia2, Bing Wang2,31Univ of California Sch of Med, Davis, CA; 2Xiamen UniversityMedical College, Xiamen City, China; 3University of Sydney,

Sydney, Australia

An increased level of free deaminated Sia, KDN, was discoveredin human ovarian cancer (OVCA), suggesting that KDN may bean oncofetal antigen (JBC (1998) 273; 27199-). To follow up thisunexpected finding, we have used HPLC-MS-MS to quantify thelevel of free and bound KDN, Neu5Ac and Neu5Gc in H&Ntumors of the throat and regional lymph nodes, and correlatedthese findings with tumor and nodal involvement, and the differen-tiation status and location within the throat. The following newfindings are reported: (1) The level of free KDN accounted for94.3% and 93.5 % of the total amount of KDN in 49 throatcancers and 10 paired lymph nodes, respectively. This shows thatthe elevated levels of free KDN in human cancers is not unique toOVCA; (2) In contrast to free KDN, the level of free Neu5Ac inthese tumors and nodes accounted for only 6.4% and 5.12% of thetotal amount of Neu5Ac, respectively. Thus, �95% of the totalamount of Neu5Ac in both tumor and lymph nodes was conju-gated; (3) While the total amount of Neu5Gc in throat cancers wasonly 0.31% of the total Neu5Ac (26 ng/g) vs. 85.18 ug/g), �2/3was bound and 1/3 was free. The central importance of these newfindings are two-fold. First, the elevated expression levels of freeKDN in throat cancers that showed no lymphatic metastasis andwere poorly/moderately differentiated suggests that free KDN maybe an Òearly warningÓ biomarker for detecting early-stagecancers at biopsy, and be of possible prognostic value in determin-ing the potential degree of malignancy. Second, these findingshighlight the importance of further studies to elucidate the molecu-lar mechanisms underlying why elevated levels of free KDN existsin human cancers, and the biological significance of this surprisingfinding.

(241) Expression of TriSia & PolySia in Human Cancers:Potential Role for Diagnostic & Prognostic Biomarkers

for Cancer ScreeningFrederic A. Troy1,3, Shan Yi3, Huili Yu3, Bing Wang2,3

1Univ of California Sch of Med, Davis, CA; 2School of MoleculeBioscience, University of Sydney, Australia; 3Xiamen University

Medical College, Xiamen City, China

Introduction: The a2,8-linked polySia chains that post-translationally modify NCAM is an anti-adhesive glycotope that


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plays a critical role in modulating cell-cell adhesive and cellmigration interactions during embryonic development, neural plas-ticity and tumor metastasis. When re-expressed on the surface ofseveral human tumors it can facilitate their detachment and metas-tasis to different sites. The a2,8 triSia structure was recently shownby C. Sato and colleagues for the first time to reside on four gly-coproteins expressed in a developmental stage-dependent mannerin mouse brain (Glycobiology, 2010). The exact function of thetriSia glycoproteins is unknown, but has been implicated in oligo-dendrocyte differentiation, similar to the function of the triSiastructure on gangliosides. Objective: To determine the level ofa2,8-tri-Sia and polySia expression in human carcinomas.Experimental Method: Western blot analyses with antibodiesspecific for recognizing either triSia or polySia. Results: (1)Expression of the triSia and polySia glycotopes on human carci-nomas are independent of each other; (2) Of the total of 192breast, lung, stomach and colon cancers analyzed, 72% werepolySia positive and 64% were triSia positive; (3) PolySia was dif-ferentially expressed at higher rates in lung tumors/lymph nodescompared with triSia (93% vs 63%), while triSia was differentiallyexpressed in 81% of the colon cancers vs. 66% for polySia; (4)Both glycotopes were expressed at high rates in stomach CA(87.5% for polySia) and 93.7% for triSia); (5) Expression of bothtriSia and polySia were found principally on glycoprotein withMr �160, 140 & 110. Conclusions: (1) An unexpectedly high per-centages of human cancers were found to express both the triSiaand polySia glycotopes, suggesting that these glycoproteins maybe potentially novel diagnostic and prognostic biomarkers forcancer screening; (2) It will be important to determine the functionof these tumor-associated trisialylated glycoproteins.

(242) HNK-1 Glycan Functions as a Tumor Suppressorfor Astrocytic Tumor

Misa Suzuki-Anekoji1, Masami Suzuki1,4, Tatsuya Kobayashi2,Yoshiko Sato3, Jun Nakayama3, Atsushi Suzuki1,4,Kiyohiko Angata1, Xingfeng Bao1, Minoru Fukuda1

1Sanford-Burnham Medical Research Institute, La Jolla, CA;2Neurosurgery, Shinshu University Sch Medicine, Matsumoto,Japan; 3Molecular Pathology, Shinshu University Sch Med,Matsumoto, Japan; 4Keio University School of Medicine,

Tokyo, Japan

Astrocytic tumor is the most prevalent primary brain tumor.However, the role of cell surface carbohydrates in astrocytic tumorinvasion is not known. In a previous study, we showed that polysialicacid facilitates astrocytic tumor invasion, thereby tumor progression(1). Here, we examined the role of HNK-1 glycan in astrocytic tumorinvasion. Kaplan-Meier analysis of 45 patients revealed that higherHNK-1 expression levels were positively associated with increasedsurvival of patients. To determine the role of HNK-1 glycan we trans-fected C6 glioma cells, which lack HNK-1 glycan expression, withβ1,3-glucuronyltransferase-P cDNA, generating HNK-1 positivecells. When these cells were injected into the mouse brain, the resul-tant tumors were 60% smaller than tumors emerging from injectionof the mock-transfected HNK-1 negative parent C6 cells.HNK-1-positive C6 cells also grew more slowly than mock-

transfected C6 cells in anchorage-dependent and anchorage-indepen-dent assays. C6-HNK-1 cells migrated well after treatment of anti-β1integrin antibody while the same treatment inhibited cell migration ofmock-transfected C6 cells. Similarly, HNK-1 glycans are attached toα-dystroglycan different from those containing the laminin-bindingglycans, supporting the above conclusion that C6-HNK-1 cellsmigrate independently from β1-integrin mediated signaling.Moreover, HNK-1 positive cells exhibited attenuated activation ofERK 1/2 compared to mock-transfected C6 cells, while FAK acti-vation was equivalent in both cell types. Overall, these results indi-cate that HNK-1 expression leads to attenuated cell growth andaltered cell migration, indicating that HNK-1 glycan functions as atumor suppressor (2).

References1. Suzuki M, Suzuki M, Nakayama J, Suzuki A, Angata K, Chen S,Kasai K, Hagihara K, Yamaguchi Y, Fukuda M. Glycobiology(2005) 15: 887–894.2. Suzuki-Anekoji M, Suzuki M, Kobayashi T, Sato Y, Nakayama J,Suzuki A, Bao X, Angata K, Fukuda M. J Biol Chem (2011) inpress

(243) A Simple and Fast Protocol to Obtain Urine N-glycansHui Zhou, Andrew Briscoe, Richard Lee

Harvard Medical School / Childrens Hospital Boston, Boston, MA

Changes of glycosylation have long been recognized as the hallmarkof many diseases, including cancer and infection. The directmeasurement of these changes is possible because of the advances inmass spectrometry techniques. Numerous candidate N-glycan bio-markers have been proposed by analyzing plasma, serum and tissues.Urine is an attractive source for biomarker discovery because of itsease of acquisition and abundance. However, to date, there are noestablished protocols for the analysis of urine N-glycans. The diffi-culty arises because of the complex metabolic nature of urine, whichcreates significant analytical challenges for glycan purification.

We report a simple and fast protocol to obtain the N-glycomefrom human urine, by utilizing a two-step spin filter purification,and conventional (house-hold) microwave irradiation. Briefly, rawurine was initially concentrated, and albumin depleted by ourpreviously published “One-Step” method (Vaezzadeh, A. et. al.J. Proteome. Res. 2010, 9, 6082). The depleted urine proteins werereduced and alkylated in 8M urea, and followed by high-speed cen-trifuge in second spin filter. The spin filters effectively remove allnon-protein components in the urine, such as metabolites, salts, etc.The purified urine proteins were then recovered in 50 mM ABCbuffer, and subjected to a 20 min microwave-assisted enzymaticde-N-glycosylation. Purified N-glycans were obtained through aC18 SPE, and then undergo per-methylated modifications.Compared to special microwave-reactors, we found that fullde-N-glycosylation can be achieved in 20 min with a house-holdmicrowave oven. Furthermore, this protocol permits multiplexing,owning the great potential for high-throughput. Using this protocolwe obtained MALDI-MS profiles of urine N-glycans from severalnormal adult urine samples. They depict similar heterogeneouscomposition profiles including many large and complex


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sialo-glycans. Analyses with a high mass accuracy LTQ-Orbitrapinstrument, more than 70 compositions were identified from asingle urine sample.

(244) The O-MannomeStephanie H. Stalnaker1, Michael Pierce1, Huaiyu Hu2,David Live1, Margreet A. Wolfert1, Geert-Jan Boons1,

Lance Wells11University of Georgia-CCRC, Athens, GA; 2SUNY Upstate

Medical University, Syracuse, NY

In mammals, it has been estimated that 30% of all the reportedO-linked glycan structures detected in mouse brain are O-mannoseinitiated. The importance of O-mannosylation is illustrated by thefact that O-mannose glycan structures play pivotal roles in a varietyof cellular processes including viral entry, metastasis, adhesion, andneuronal development. However, to date O-mannosylation has onlybeen well documented on a single mammalian protein, alpha-dystroglycan (α-DG). Recently we have shown that the relativeabundance of O-mannose initiated glycols remains unchanged inthe brain of an α-DG knockout mouse model compared to its litter-mate control. Also, unlike most forms of muscular dystrophy, thereare often major neurological complications associated with CMD.In order to account for all of the O-mannose glycan structurespresent in the mammalian brain, it has been proposed that theremust be a significant number of other proteins that bear this modifi-cation. The most predominantly observed O-mannose glycan has acore structure of Siaα3Galβ4GlcNAcβ2Manα-Ser/Thr, which issynthesized by a series of glycosyltransferases. We have recentlyfocused on the activity of POMGnT1 and shown that POMGnT1 isessential for the extension of O-mannose initiated glycan residueswith a GlcNAc, yielding both the classical and branchedO-mannose glycan structures. We are currently using chemoenzy-matic approaches with proteins extracts prepared from thePOMGnT1 -/- brains to label, isolate, and identify these novelO-mannosylated proteins. We are also working to develop andapply O-man-specific monoclonal antibodies to enrich and identifythe O-mannome.

(245) Mechanisms for Cancer Metastasis in the Formationof Trimeric Tn Antigen Modified by pp-GalNAc-T13 GeneYasuyuki Matsumoto1, Quig Zhang1, Kazunori Hamamura1,

Takenosuke Yoshida1, Kaoru Akita2, Hiroshi Nakada2,Tetsuya Okajima1, Keiko Furukawa3, Takeshi Urano4,

Koichi Furukawa11Nagoya University Graduate School of Medicine, Nagoya,Japan; 2Kyoto Sangyo University, Kyoto, Japan; 3Chubu

University, Kasugai, Japan; 4Shimane University of Medicine,Shimane, Japan

The development of metastasis is a major cause of death in manyhuman cancers. Mechanisms for the acquisition of metastatic poten-tial are, however, not well understood. In addition, the seriousproblem is that molecular targeted therapies affect not only tumorsbut also normal tissues, which could be resolved by high specificitytoward cancer. So, it is important to identify specific carbohydratestructures that are exclusively involved in cancer metastasis.

In order to analyze the mechanisms for cancer metastasis, highmetastatic sublines, C4-sc and C4-ly were obtained by repeatedinjection of mouse Lewis lung cancer (C4). pp-GalNAc-T13 gene,which acts for the initiation of O-linked glycosylation on mucin-likeproteins, was identified as a highly-expressed gene along withincreased metastasis by the DNA microarray analysis. Stable trans-fection of an expression vector of pp-GalNAc-T13 into C4(T13-TF) resulted in increased invasion and migration activity.Then, immunoblotting and flow cytometry were performed by anti-trimeric Tn antibody (MLS128), showing that the expression levelsof trimeric Tn antigen (tTn) increased in T13-TF clones. Moreover,immunoprecipitation and immunoblotting revealed that Syndecan-1(Sdc1) might be a carrier protein of tTn.

To address the significance of pp-GalNAc-T13 in cancer metasta-sis, transfectant cells of C4-sc with an shRNA vector of the gene(T13-KD) were established. Metastasis experiments revealed lowcoalescence to fascia and significantly reduced lung metastasis inT13-KD cells after subcutaneous injection compared with thecontrol transfectants. These data suggested that it might provide apotential biomarker for cancer metastasis.

(246) Glycomics Analysis of Dried Blood SpotsL. Renee Ruhaak1, Suzanne Miyamoto2, Carlito B. Lebrilla1

1UC Davis, Davis, CA; 2UC Davis Cancer Center, Sacramento,CA

N-glycosylation is now considered as a potential pool for bio-markers. The have been proposed as potential biomarkers forseveral diseases including several forms of cancer, but alsoAlzheimer’s, and diabetes. Blood spots have been of recent inter-ests due to their convenience; the use of dried blood spots (DBS)for –omics types of studies as this sampling method is much lessinvasive than ‘classical’ blood drawing, and also facilitates ship-ping and storage. Most blood-based –omics studies have been con-ducted on serum or plasma, and two differences between DBS andserum/plasma have to be considered in method development: 1)Blood cells are still present in DBS compared to serum/plasma. 2)Sample volumes of DBS are limited (� 15 µl).

Here we present a procedure for N-glycan profiling of DBSsamples comprising reconstitution of DBS in N-glycan release buffer,protein denaturation, enzymatic N-glycan release and PGC Solidphase extraction (SPE) for purification. Samples are then analyzedusing nHPLC-PGC-chip-TOF-MS to generate N-glycan profiles.Using this method, around 150 N-glycan structures can be monitored,originating from around 40 N-glycan compositions that can be ana-lyzed with good repeatability (%CVaround 20%). To assess the stab-ility of the N-glycans during storage, DBS samples were stored at RTand -80°C. No major differences in N = glycan composition could beobserved. Moreover, upon comparison of the N-glycan profile ofDBS with profiles obtained from serum, which is a classical matrixfor N-glycan profiling, similar patterns were observed.

The method presented here will be very useful, as it does notonly allow the use of DBS already collected in biorepositories, butalso facilitates future sample collection for N-glycan profiling,especially for research related to children and the elderly, who havelimited blood supplies, as well as animal studies in small mammals.


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Program and abstracts for the 2011 Meeting of the Society for Glycobiology

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Transcript of Program and abstracts for the 2011 Meeting of the Society for Glycobiology