N‑Linked Glycan Structures of the Human Fcγ Receptors Produced inNS0 CellsEoin F. J. Cosgrave,†,‡ Weston B. Struwe,†,§ Jerrard M. Hayes,† David J. Harvey,∥ Mark R. Wormald,∥

and Pauline M. Rudd*,†

†NIBRT Glycobiology Group, National Institute for Bioprocessing Research and Training, Foster’s Avenue, Mount Merrion,Blackrock, County Dublin, Ireland‡Pharmaceutical Life Sciences Group, Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States§Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom∥Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

ABSTRACT: Immune recognition of nonself is coordinatedthrough complex mechanisms involving both innate andadaptive responses. Circulating antibodies communicate witheffector cells of the innate immune system through surfacereceptors known as Fcγ receptors (FcγRs). The FcγRs aresingle-pass transmembrane glycoproteins responsible forregulating innate effector responses toward antigenic material.Although immunoglobulin G (IgG) antibodies bind to a rangeof receptors, including complement receptors and C-typelectins, we have focused on the Fcγ receptors. A total of fivefunctional FcγRs are broadly classified into three families(FcγRI, FcγRII, and FcγRIII) and together aid in controllingboth inflammatory and anti-inflammatory responses of the innate immune system. Due to the continued success of monoclonalantibodies in treating cancer and autoimmune disorders, research is typically directed toward improving the interaction ofantibodies with the FcγRs through manipulation of IgG properties such as N-linked glycosylation. Biochemical studies usingrecombinant forms of the FcγRs are often used to quantitate changes in binding affinity, a key indicator of a likely biologicaloutcome. However, analysis of the FcγRs themselves is imperative as recombinant FcγRs differ greatly from those observed inhumans. In particular, the N-linked glycan composition is significantly important due to its function in the IgG−FcγR interaction.Here, we present data for the N-linked glycans present on FcγRs produced in NS0 cells, namely, FcγRIa, FcγRIIa, FcγRIIB,FcγRIIIa, and FcγRIIIb. Importantly, these FcγRs demonstrate typical murine glycosylation in the form of α-galactose epitopes,N-glycolylneuraminic acid, and other key glycan properties that are generally expressed in murine cell lines and therefore are nottypically observed in humans.

KEYWORDS: glycosylation, antibody, IgG, Fcγ receptors, N-linked glycan, α-galactosylation, N-glycolylneuraminic acid, HPLC,mass spectrometry, N-acetyllactosamine, CD16, CD32, CD64

■ INTRODUCTION

Clearance of foreign material from the host system is achievedin part by the activity of immunoglobulins (Igs), an importantclass of glycoproteins responsible for the recognition of nonselfepitopes and the subsequent broadcasting of this identificationto the innate and adaptive arms of the immune system.However, recognition of potentially harmful foreign antigensfollowed by opsonization is insufficient for their removal, andimmunoglobulins rely on activation of innate effector cells thatcarry out the elimination process. Coupling of solubleimmunoglobulins to innate host cell membranes is facilitatedthrough binding to membrane-bound receptors (Fc receptors)specific for the Fc region of Igs, which then trigger immuneactivation. This leads to clustering of receptors, immunecomplex formation, and triggering of an intracellular signalingcascade involving phosphorylation/dephosphorylation of

ITAMs (immuno-tyrosine-like activatory motifs) and ITIMs(immuno-tyrosine-like inhibitory motifs).Key to the ability of immunoglobulin G (IgG) in

coordinating immune responses is glycosylation, a non-template-driven post-translational modification that whenabsent adversely affects the structural integrity of the antibodymolecule, therein eliminating or significantly reducing bindingto its Fc receptors C1q and MBL.1−5 It is now widelyappreciated that this binding and activation event is mediatedby glycosylation. Carbohydrates attached to IgG, thepredominant class of Ig found in human serum, play a criticalrole in its function of linking antigen recognition to immuneeffector functions.6 Antibody-mediated immune responses

Received: April 14, 2013

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coordinated by IgG are defined by interaction with receptorsspecific for the Fc region of the IgG molecule (Fcγ receptors,FcγRs). The Fcγ receptors are widely distributed across thehematopoietic system and are instrumental in the activation ofinnate effector cells following interaction with immunecomplexes. They are categorized into three main classes: (i)the high-affinity (kD ≈ 10−9 M) FcγRI (CD64) class consistingof FcγRIa, (ii) the low-affinity (kD ≈ 10−7 M) FcγRII (CD32)class consisting of the activatory FcγRIIa and inhibitoryFcγRIIb, and (iii) the low-affinity (kD ≈ 10−6 M) FcγRIII(CD16) class consisting of FcγRIIIa and the GPI-anchoredFcγRIIIb. It has been shown that the glycan composition of IgGsignificantly affects the ability of FcγRIIIa to bind IgG. Mostnotably, the loss of core α(1−6) fucose on the Fc glycans atAsn-297 on each heavy chain significantly increases not onlythe binding affinity to FcγRIIIa7−9 but also the associatedcellular cytotoxicity through NK cell activation.10−13 Theprecise mechanism for this phenomenon is well-defined andinvolves glycosylation of the Fcγ receptor itself. Thecarbohydrate at Asn-162 on FcγRIIIa is instrumental to theactivity of afucosylated IgG, and elimination of the glycan atthis position significantly reduced the binding affinity of IgG byup to an order of magnitude.14 Further studies of FcγRIIIaglycosylation have demonstrated that N-linked glycosylation atposition Asn-45 reduces the binding affinity of IgG while apoint mutation of Asn-45 positively influences affinity.15 Inlight of such findings, it is becoming increasingly evident thatglycosylation of the Fcγ receptors as well as the IgG Fc domainplays a significant role in the ultimate binding event betweenthese two molecules and subsequent downstream activation.This has critical implications for the glycoengineering oftherapeutic monoclonal antibodies (mAbs) and the potentialapproach the biopharmaceutical industry takes toward develop-ment of new candidate mAb therapies.There are multiple levels of complexity in FcγR biology,

including cellular distribution, regulation, activation, andfunction; however, these topics have been discussed else-where,16−21 and in the present study we will focus on cellularFcγR epitopes. Fc−FcγR interactions represent an area ofintense focus in the generation of monoclonal antibodytherapeutics destined for use in cancer immunotherapy.Targeted elimination of tumorigenic cells relies on theidentification of tumor-associated antigens (TAAs) and therecruitment of immune cells capable of destroying aberrantcancer cells. Antibody-mediated tumor eradication depends ontwo principal mechanisms targeted for activation, complement-dependent cytotoxicity (CDC) and antibody-dependentcellular cytotoxicity (ADCC). ADCC is primarily faciliated bythe activity of NK cells through interaction of IgG immunecomplexes with FcγRIIIa, although other innate effector cellssuch as neutrophils22 and eosinophils23 are also capable ofperforming this function. Enhancing the cytotoxic activity ofNK cells through engineering of monoclonal antibodies hasbeen of paramount interest to the pharmaceutical industry as itpresents opportunites to generate therapies with enhancedbiological activity and therefore increased potency and efficacyin tumor eradication.Guidelines governing the biological activity of candidate

mAbs specify that precise information on the binding affinityand specificity of the proposed mAb with FcγRs is required.24,25

Determination of binding kinetics is largely achieved throughmeasurement of protein−protein interactions using techniquessuch as surface plasmon resonance (SPR) or isothermal

titration calorimetry (ITC), each of which typically usesrecombinant forms of the individual Fcγ receptors. Productionof recombinant receptors is performed in similar cell expressionsystems used for antibody generation such as NS0, CHO, BHK,or HEK cells largely due to the fact that the Fcγ receptors areheavily glycosylated proteins and glycosylation is required fortheir biological activity. Typically these cell lines perform all thenecessary post-translational modifications required for produc-tion of complex glycoproteins. While glycosylation is performedby these various cell lines, it is done so in a manner divergent tonormal human glycosylation. This raises obvious concern inlight of compounding evidence for the role of FcγRglycosylation in IgG biological activity.Nonhuman glycosylation of Fcγ receptors used in these

assessments could potentially generate data which are notbiologically relevant. It is therefore necessary to characterize theglycosylation of recombinant Fcγ receptors to determine wherepossible how the heterologous forms compare to their naturalcounterparts. Several groups have employed recombinant Fcγreceptors from NS0 cells for use in affinity studies withoutdetailed knowledge of the carbohydrate content of thereceptors or how glycosylation affects antibody binding andkinetics.26−31 In this study we therefore set out tocomprehensively characterize the N-glycans present oncommercially available and industrially relevant Fcγ receptorsproduced in the murine cell line NS0. Specifically, N-glycansfrom FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb wereinvestigated. The information provided here is of vitalimportance to the biopharmaceutical industry where NS0-expressed receptors are used to assess the activity of candidatemonoclonal antibodies. Using both HPLC- and MS-basedapproaches, we have demonstrated that NS0-derived recombi-nant soluble human FcγRs are substantially heterogeneous withmultiantennary structures containing extensive immunogenicα(1,3)-galactosylation, outer arm fucosylation, poly(N-acetyl-lactosamine), and N-glycolylneuraminic acid. These findingsshow that significant differences are likely to exist in theglycosylation of Fcγ receptors which are recombinantlyproduced and those which are found in humans. Thesereceptors could potentially produce misleading IgG bindinginteraction data, and the information provided here could leadto a more physiologically relevant assessment of monoclonalantibody function when using murine-derived receptors whichcould potentially have differences in protein structure as well ascarbohydrate epitopes. This also raises the question of whetherhuman-derived receptors should be exclusively used for theanalysis of therapeutic monoclonal antibodies.

■ EXPERIMENTAL SECTION

N-Linked Glycan Release and 2-Aminobenzamide (2AB)Labeling

Three separate lots of Fcγ receptors were purchased from R&DSystems (Minneapolis, MN) and delivered as lyophilizedproducts. Each receptor was reconstituted in phosphate-buffered saline (PBS) or PBS containing 0.1% bovine serumalbumin (BSA) to 1 mg mL−1, on the basis of themanufacturer’s recommendation. N-Linked glycans of eachreceptor were released using a 0.1 U mL−1 concentration of thepeptide N-glycanase PNGase F (ProZyme, Hayward, CA).Glycans were subsequently isolated, labeled with 2AB (Ludger,United Kingdom), and reconstituted in deionized water aspreviously described.32

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Hydrophilic Interaction High-Performance LiquidChromatography (HI-HPLC)

2AB-labeled glycans were prepared as 80% (v/v) acetonitrilesolutions for injection onto a TSKgel amide-80, 5 μm (4.6 mm× 250 mm) column as previously described.32 All separationswere performed over a 180 min period at 30 °C using 50 mMammonium formate, pH 4.4, as solvent A and 100% (v/v)acetonitrile as solvent B. All separations were performed usingWaters Alliance 2695 instruments coupled with a Waters 2475fluorescence detector with glycans detected through fluores-cence excitation at 420 nm and emission at 330 nm. Each 180min separation was achieved by running a 20−58% gradient ofsolvent A over a 152 min time frame and a flow rate of 0.4 mLmin−1. HPLC instrumentation was calibrated using 2AB-labeleddextran as an internal calibration standard. Retention times forpeaks arising in the dextran hydrolysate separation were used tofit a fifth-order polynomial distribution curve using WatersEmpower software (Milford, MA), which provided the meansfor converting chromatographic retention times into stand-ardized glucose unit (GU) values as previously described.32

Weak Anion Exchange (WAX) HPLC

The extent of terminal sialylation and charged peakfractionation was determined using WAX-HPLC. A GlykoGlycosep C (7.5 mm × 75 mm) column (ProZyme) was usedfor separation of charged carbohydrate structures. Solvent Awas prepared as 100 mM ammonium acetate, pH 7, in 20% (v/v) methanol, while solvent B was 20% (v/v) acetonitrile. AllWAX-HPLC separations were performed on a Waters Alliance2695 instrument coupled with a Waters 474 fluorescencedetector. Carbohydrate samples were separated over a 30 mingradient method. The relative number of terminal chargedglycan structures was determined by comparison to released N-linked glycans from fetuin. For WAX fractionation procedures,glycan pools were first separated by WAX-HPLC and peaksarising from this separation were manually collected. Excesssolvent in each glycan collection was then evaporated, and theresulting isolated glycans were reconstituted in a predeterminedvolume of doubly distilled water (ddH2O). Each fraction wasprepared for injection onto TSKgel amide-80 columns usingthe aforementioned 180 min gradient method.

Exoglycosidase Arrays

Full carbohydrate characterization in terms of sequence,composition, and linkage specificities was facilitated by use ofexoglycosidase digestion arrays. All digestion reactions wereperformed with enzymes purchased from ProZyme. Fluo-rescently labeled glycans were digested in 50 mM sodiumacetate, pH 5.5, at 37 °C overnight using a preselected panel ofenzymes, with each digestion reaction brought to a final volumeof 10 μL using ddH2O. Digested glycans were then separatedfrom the enzyme mixtures using Nanosep 10 kDa MWCOcentrifugal filters (Pall Corp., Port Washington, NY). Digested2AB-labeled glycans were then prepared for separation onhydrophilic interaction liquid chromatography (HILIC)−HPLC using the 180 min gradient method as previouslydescribed.32 Specific monosaccharides were removed as follows:terminal sialic acid in all linkages was removed with 1 mU μL−1

Arthrobacter ureafaciens sialidase (ABS), terminal galactosemonosaccharides were removed using 0.5 mU μL−1 bovinetestes β-galactosidase (BTG), which releases both β(1,3)- andβ(1,4)-linked galactose, terminal galactose-α(1,3)-galactose wasreleased using 40 mU μL−1 coffee bean α-galactosidase (CBG),terminal N-acetylglucosamine (GlcNAc) monosaccharides werereleased with 40 mU μL−1 Streptococcus pneumoniae hexosami-nidase (GUH), capable of cleaving β-linked GlcNAc moieties,core α(1,6)-fucose was selectively removed using 1 mU μL−1

bovine kidney α-fucosidase (BKF), terminal outer arm α(1,2)-linked fucose was investigated using 0.8 mU μL−1 Xanthomonasmanihotis α-fucosidase, and α-linked mannose was removedusing 150 mU μL−1 jack bean α-mannosidase (JBM).

MALDI MS

Released N-glycans from 50 μg of Fcγ receptors were releasedin a volume of 50 μL and subsequently diluted with 200 μL ofH2O following reaction. N-Glycans were purified by retentivesolid-phase extraction using porous graphitized carbon (PGC)as previously described33 with minor alterations. In brief, N-glycans were loaded to the top of the PGC bed, washed withHPLC-grade H2O, and eluted with 25% acetonitrile/0.05%trifluoroacetic acid (TFA). N-Glycans were dried, reconstitutedin 100 μL of H2O, and aliquoted into 5 × 20 μL fractions forexoglycosidase digestions. N-Glycans were digested with ABS,

Table 1. Summary of Properties for Commercially Acquired Fc Receptorsa

aFc receptors were purchased from R&D Systems. For each Fc receptor, a structure is presented with putative N-linked glycan sites illustrated in red.The mass recorded is based on the amino acid sequence.

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ABS + CBG, ABS + CBG + BTG, and ABS + CBG + BTG +BKF as described above. Exoglycosidases were removed viaPGC solid-phase extraction (SPE), and N-glycans werereconstituted in 20 μL of H2O and 5 μL of MeOH. Glycansamples (3 μL) were spotted on 1 μL of 2,5-dihydroxylbenzoicacid matrix (10 mg mL−1 in 50% (v/v) acetonitrile aqueoussolution) on a stainless steel MALDI target plate. Sample spotswere recrystallized with 0.7 μL of ethanol. MALDI spectra wereacquired using a Micromass MALDI-Micro MX massspectrometer (Waters, Manchester, U.K.) operated in positivereflectron mode. Instrument control, spectral acquisition, andprocessing were performed with MassLynx software version 4.1(Waters, Milford, MA).ESI−MS/MS

Samples (3 μL) of Fcγ receptor N-glycans following PGCcleanup were placed on the surface of a Nafion 117 membrane(Aldrich, Poole, Dorset, U.K.) which was atop water for furtherdesalting. Spots were removed, and 1 μL of a 1:1 (v/v) mixtureof water/methanol containing 0.2 M ammonium phosphatewas added. The samples were transferred to a Proxeonborosilicate capillary (Proxeon Biosystems, Odense, Denmark)and injected into a Micromass QToF Ultima Global massspectrometer (Waters, Manchester, U.K.). MS/MS spectra(collision-induced dissociation) were acquired in negative ionmode. Data acquisition and processing were conducted withWaters MassLynx version 4.1.

■ RESULTS

The Carbohydrates of Fcγ Receptors Consist ofHigh-Mannose and Complex Multiantennary Structures

Antennarity Is Varied between FcγRs. The principal aimof this study focused on the N-glycan composition ofcommercial Fcγ receptors produced in NS0 cells. Specifically,five Fcγ receptors (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, andFcγRIIIb) were investigated. Table 1 summarizes the keyfeatures of each Fcγ receptor. To ascertain the complexity ofglycosylation present on each FcγR, N-linked glycans werereleased with PNGase F, fluorescently labeled with 2AB, andseparated using HILIC with fluorescence detection (FLD).Assessment of the resulting chromatograms indicated extensivevariability and complexity within and across each Fcγ receptorsample (Figure 1). Using GU values together with structuralassignments via exoglycosidase arrays and subsequent con-firmation by mass spectrometry, several structures were foundto be common to all receptor subtypes. Numerous structureswere also found to be exclusive to individual receptors. A totalof 12 glycan structures were common to all Fcγ receptors,although the relative distribution of these common structuresvaried significantly between receptors (Table 2). Mono- andbiantennary branched structures were found predominantly onFcγRIa and FcγRIIa, while tri- and tetraantennary structuresconstituted the majority of branched structures on theremaining FcγRs (Figure 2).FcγRs Demonstrate Lot-to-Lot Variance. To determine

lot-to-lot variability, three separate lots of each FcγR werepurchased from R&D Systems and analyzed for N-linked glycancontent. The glycan pool composition was found to beconsistent for each Fcγ receptor with the same peaks observedacross three separately released batches (data not shown).However, significant differences were found in the distributionof the common structures within the glycan pool. As a result,the relative standard deviation observed for individual peaks in

each Fcγ receptor glycan pool was found to be significantlyhigh, suggesting variability in the relative abundance ofcommon structures across different batches of Fcγ receptors(Table 3).

Sialylation of FcγRs Demonstrates Variability inAbundance and Heterogeneity. Analysis of sialylation wasperformed using either WAX-HPLC with fluorescencedetection or HILIC-FLD. HILIC-based analysis of sialic acidcontent was determined through comparative assessment of theundigested Fcγ receptor glycan samples with equivalent ABS-treated samples. Monitoring the effect of ABS on each glycanpool provided a means of identifying the peaks containing sialicacid as well as their relative percentages. For each sample, thetotal peak area change due to ABS treatment was measured and

Figure 1. N-Linked glycans released from Fcγ receptors demonstratesignificant complexity and heterogeneity. Fcγ receptors containingmurine-derived N-linked glycosylation were assessed for glycancontent by enzymatically releasing the carbohydrate structures andanalyzing using hydrophilic interaction HPLC. Each glycan pool wasfluorescently labeled with 2-aminobenzamide and separated usingTSKgel amide-80 columns. Peaks common to all FcγR glycan poolsare identified by vertical dashed lines and numerical annotation. Key:(A) FcγRIa, (B) FcγRIIa, (C) FcγRIIb, (D) FcγRIIIa, and (E)FcγRIIIb.

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used to approximate the amount of sialylation in each sample.WAX-HPLC was also used to study the sialic acid content ofthe receptors. This technique was used to analyze the

composition of charged glycan structures present in each Fcγreceptor glycan pool and assisted in categorizing the sialic acidcontent into mono-, di-, tri-, and tetrasialylated species (datanot shown). A representative result of sialic acid contentdetermination is presented in Figure 3.FcγRIa contained approximately 24% sialylation, present

primarily as monosialylated structures and distributed acrossfive separate structures (see Figure 3 and Table 2). FcγRIIacontained an equivalent amount of approximately 24%,although sialylation was observed as mono-, di-, andtriantennary structures distributed over 11 peaks. In contrast,FcγRIIb contained less overall sialylation of only approximately18% predominantly as two monosialylated structures. FcγRIIIacontained four monosialylated structures totaling approximately27% of the relative peak area, while FcγRIIIb contained nearly35% sialylation with a total of seven structures ranging frommonosialylated to trisialylated. Generally speaking, the FcγRsdisplayed heterogeneity in both the amount of sialylation andthe extent of terminal sialic acids present. It is particularlyinteresting to compare the different FcγR classes, which containextensive homology in their extracellular domains. FcγRIIacontained more sialylation and greater diversity of sialylatedstructures in general than FcγRIIb. Similarly, FcγRIIIbcontained a greater extent and diversity of sialylation comparedto FcγRIIIa. Reasons for this are unclear, but it is interesting toobserve the different molecular functions of each receptor andwhat impact glycosylation may have on their cellular role.

High-Mannose Structures Vary in Abundance andDistribution across FcγR. Early processing of N-linkedglycans generates a number of high-mannose structures in theendoplasmic reticulum, typically producing structures from M9down to M4, which are usually processed to complex glycans.To measure the oligomannose content, N-linked glycans weresequentially digested with exoglycosidase arrays that exposedterminal mannose residues. Exoglycosidase treatment andsubsequent separation of the digested glycans by HILIC-FLDallowed for the quantitation of the various high-mannosestructures. All tentative high-mannose structures were con-firmed by further digestion of the glycan pool with JBM, whichexhibits enzymatic activity only on structures containingterminal mannose.In the present study high-mannose structures refer only to

structures bearing four terminal mannose (M4) to nine

Table 2. Summary of Common N-Glycan Structures Identified in All Fcγ Receptorsa

FcR relative peak area (%)

peak GU m/z composition structure IA IIA IIB IIIA IIIB x ̅ σ

1 6.21 1257.42 Hex5HexNAc2 M5 14.41 0.27 0.16 0.38 2.65 3.57 6.152 6.84 Fuc1Hex3HexNAc6 F(6)A4 0.59 0.29 0.13 1.14 0.49 0.53 0.393 7.88 1581.53 Hex5HexNAc2 F(6)A3G1Gal1 0.79 1.68 1.66 0.54 1.48 1.23 0.534 8.07 2012.72 Fuc1Hex5HexNAc5 F(6)A3G2 5.02 1.89 2.87 3.64 4.97 3.68 1.355 8.48 Fuc1Hex5HexNAc6 F(6)A4G1Gal1 6.83 2.71 1.54 4.66 3.11 3.77 2.046 8.90 Fuc1Hex6HexNAc5 F(6)A3G3 5.67 4.54 4.37 4.84 6.17 5.12 0.777 9.61 2336.83 Fuc1Hex7HexNAc5 F(6)A3G3Gal1 2.80 7.36 4.58 4.66 4.87 4.85 1.638 9.99 Fuc1Hex7HexNAc5 F(6)A3G2Gal2 3.16 4.68 6.82 4.01 4.46 4.63 1.369 10.45 2498.88 Fuc1Hex8HexNAc5 F(6)A3G3Gal2 5.23 11.70 7.53 5.08 13.89 8.69 3.9510 10.96 Fuc1Hex7HexNAc5NeuAc1 F(6)A3G3Gal1S1 3.24 17.69 3.36 4.47 16.02 8.96 7.2511 11.24 2660.93 Fuc1Hex9HexNAc5 F(6)A3G3Gal3 15.93 16.05 20.12 24.08 13.41 17.92 4.2012 12.38 Fuc1Hex11HexNAc6 F(6)A4G4Gal4 1.22 1.88 4.27 2.21 2.15 2.35 1.14

aFollowing characterization of each Fcγ receptor, common structures were identified and tabulated. GU values together with m/z values andexoglycosidase digestions confirmed the presence of each structure. The relative peak area for each common structure is presented with the averagepeak area (x)̅ reported with the standard deviation (σ). Glycan naming is performed as outlined in the Oxford notation.

Figure 2. Fcγ receptor N-glycans contain varying amounts of branchedstructures. The abundance of antennary structures within glycan poolsobtained from each Fcγ receptor was determined by enzymaticallyreducing each glycan population to branched, hybrid, and high-mannose structures. Symbols denoted on the right of eachchromatogram indicate the individual monosaccharides removedthrough exoglycosidase digestion. Peaks are identified with structuresreported in Oxford notation. Key: (A) FcγRIa, (B) FcγRIIa, (C)FcγRIIb, (D) FcγRIIIa, and (E) FcγRIIIb. Peaks denoted by an asteriskrefer to a structure with an unconfirmed structural composition.

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Table 3. Total N-Glycan Structural Composition Determined across All Fcγ Receptorsa

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Table 3. continued

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terminal mannose (M9) residues. FcγRs expressed in NS0 cellswere selectively found to contain M4 to M9. The totalproportions of oligomannose structures ranged from less than2% to as high as 16% of the total glycan population forindividual Fcγ receptors (Figure 4). FcγRIa demonstrated thelargest content of high-mannose structures (16%), with over13% of the total glycan pool dominated by M5 alone,suggesting that it is at one site protected from furtherprocessing by subsequent glycosyltransferases. The remaining3% was comprised of M4, M6, and M7. The increased level ofhigh mannosylation did not appear to affect the ability for fullglycan processing of the remaining glycan pool as the level ofsialylation was still comparable to that of the other FcγRs.FcγRIIa contained the least amount of high-mannosestructures, with just under 2% of the total glycan populationrepresented by M5 and M6. FcγRIIb contained nearly 4% highmannose structure in the form of M6, M7, and M8, while bothFcγRIIIa and FcγRIIIb contained nearly 6% of each high-mannose structure. Despite similar peak area percentages,FcγRIIIa and FcγRIIIb were markedly different in the diversity

of high-mannose structures. FcγRIIIa contained M4, M5, andM6, while FcγRIIIb contained all structures from M4 to M9.Reasons for the differences in high-mannose diversity are notclear at this point, but represent a unique and defining featurebetween the various FcγRs.

Fcγ Receptors Contain Extensive Gal-α(1,3)-Gal Epitopes

Murine-derived recombinant proteins incorporate monosac-charide linkages not typically found in humans, such asgalactose linked to galactose in an α(1−3) orientation.34 Thisterminal disaccharide is known to be immunogenic inhumans,35,36 and therefore, it is important to identify anyglycans bearing this epitope. To determine if such moietiesexisted in the NS0-expressed Fcγ receptors, two approacheswere utilized, including HILIC-FLD with exoglycosidase arraysand negative mode ESI−MS/MS. To identify structurescontaining galactose-α(1−3)-galactose, CBG causes a signatureGU shift on N-glycans containing the Gal-α-Gal epitope, whereglycan peaks shift by 0.8−0.9 GU per α-galactose monomerwhen assessed by HILIC-FLD. Glycans released from eachreceptor were used in targeted digestions using enzyme

Table 3. continued

aStructures identified within each Fcγ receptor sample are tabulated. All N-glycans are reported in GU values with structure and structure nameusing the Oxford notation. For each GU value, the relative peak area (RPA) is reported with the standard deviation (±σ). Standard deviation valuesare based on the analysis of three separate lots of each Fcγ receptor. The presence of values within a cell position indicates the identification of thegiven structure within the NS0 FcγR sample. The relative standard deviation (RSD, %) is reported to give a reflection of the variability observedbetween different lots of the same NS0 Fcγ receptor. All peaks with a relative peak area greater than 0.5% are reported.

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compositions with and without CBG. All digestion reactionswere separated by HILIC, and shifts corresponding to losses ofα-linked galactose monomers were observed following

reactions containing CBG. A representative chromatographiccomparison is illustrated in Figure 5. Many of the dominantpeaks in each chromatogram demonstrated a shift correspond-ing to a loss of at least one and at most four galactosemonomers linked in an α(1−3) position.The overall change in peak area due to CBG activity was

subsequently used to determine the total amount of the glycanpool containing α-galactose. Of the five Fcγ receptors analyzed,FcγRIIb contained the highest amount of α-galactosylation withan approximate amount of 71% of the total glycan pool. FcγRIacontained the lowest amount of α-galactose with anapproximate amount of 35%. The lower amount of α-galactosepresent on FcγRIa is possibly due to the elevated levels of high-mannose structures observed with FcγRIa, although the exactrelationship between mannose content and α-galactosylation asit pertains to Fcγ receptor biology remains to be determined.FcγRIIa was found to contain approximately 56% α-galactosylation, with FcγRIIIa and FcγRIIIb determined tohave 61% and 44%, respectively. It is evident from thesenumbers that α-galactose is a prominent monosaccharide onNS0-derived FcγRs.

FcγRs Contain N-Glycolylneuraminic Acid

With evidence supporting the presence of sialic acids on eachFcγ receptor and reports indicating the prevalence of N-glycolylneuraminic acid (Neu5Gc) in NS0 cells,37 wedetermined if any of the receptor-associated sialic acids werepresent as Neu5Gc. ESI−MS/MS confirmed the presence ofNeu5Gc within the NS0 Fcγ receptor N-glycan pool. Neu5Gcstructures were identified by tandem ESI−MS of [M − H +H2PO4] precursor ions (Figure 6). The fragmentation spectraof sialic acid containing negative ions results predominantly inthe detection of the sialic acid monosaccharide peaks. The B-type fragment of Neu5Gc is detected at 307 m/z (N-acetylneuraminic acid (Neu5Ac) B-type ions are detected at290 m/z). This and the isolated precursor mass distinguishedNeu5Gc-containing structures.

Glycans Contain poly(N-acetyllactosamine)

Core branched structures are identified and quantified using aseries of exoglycosidases that truncate the glycan pool tostructures bearing terminal GlcNAc residues. In most cases, thelargest branched structure identified is the tetraantennarystructure A4 with a GU value of approximately 6.4. In situationswhere structures with GU values greater than 6.4 persist, otherglycan modifications are possible that can be identified withselected exoglycosidase digestions. Glycans that are likely toappear with a larger mass than the core antennary structuresafter an ABS/BTG/CBG/BKF digestion typically includeeither high-mannose structures or branched structures contain-ing poly(N-acetyllactosamine) (LacNAc) repeats. Glycan peakscontaining repeating units of 1.3 GU values in combinationwith the core antennary structures indicate the presence ofLacNAcs. This is based on the composition of a LacNAcconsisting of a GlcNAc (0.5 GU) and a galactose (0.8 GU).The presence of higher mass structures suggested eithermultiple high-mannose structures (M5 to M9) or terminalglycan structures that prevented complete digestion of theglycan pool with the exoglycosidases ABS, BTG, BKF, andCBG used in each reaction (Figure 7).Reducing end glycan termination by motifs such as outer arm

fucosylation or LacNAc repeats could potentially explain thepersistence of higher order glycan structures. To confirm thepresence of LacNAcs, glycans were digested using ABS, CBG,

Figure 3. FcγR sialylation is identified by enzymatic digestion.Sialidase digestion of each Fcγ receptor glycan pool was performed todetermine the abundance of sialylation. FcγRIIIb N-glycans were usedas a representative for each Fcγ receptor glycan pool. (A) WAX-HPLC-FLD was used to separate the FcγRIIIb glycan pool on thebasis of charge, where 2AB-labeled N-glycans from fetuin were used toidentify retention positions of neutral glycans and glycans containingone (mono), two (di), three (tri), and four (tetra) terminal sialic acids.Each sialylated peak was confirmed by separating ABS-digested glycansfrom the FcRIIIb glycan pool and identifying missing peaks. Additionalcharges not arising from sialylation were also observed and aredenoted by an asterisk. (B) HILIC-FLD chromatograms for both theundigested and ABS-digested N-glycans from FcγRIIIb were used toidentify individual peaks containing sialic acid. Peak migrations fromsialylated to neutral structures are denoted by dashed arrows betweeneach chromatogram. Sialylated peaks are annotated using the Oxfordlinear notation.

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BTG, BKF, and GUH, and the resulting chromatogram wasanalyzed and compared to the same digest without GUH.Following digestion with GUH, all higher mass structuresdigested to the M3 core, indicating that all structures above GUvalues of 6.4 most likely correlated to structures containingLacNAc repeats. Using the known incremental shifts in GUvalues due to exoglycosidase activity, the composition of thevarious LacNAc-containing structures was determined. ThisHILIC and exoglycosidase approach identified structurescontaining both one and two terminal LacNAcs. The relativeproportions of LacNAc structures was low in all cases,contributing from as low as 1% to no greater than 6% of thetotal glycan pool in all the Fcγ receptors studied.

FcγRs Contain Outer Arm Fucosylation

The possibility of glycan modification by outer arm fucosylationwas investigated due to the appearance of undigested glycanduring routine exoglycosidase array analysis. To avoid issueswith enzyme specificity and/or activity, mass spectrometry wasused to search for structures containing more than one fucose.Outer arm fucosylation was initially confirmed by ESI−MS/MSof negat ive ions. The MS/MS spectrum of theHex8HexNAc5Fuc2 [M + H2PO4]

− ion from FcγRIIIbidentified fucose linked to the reducing end GlcNAc residuefrom the 2,4AR fragment as well as terminally from the 510 and570 m/z ions (Figure 8C). The outer arm fucose was linked toa GlcNAc residue from the absence of 370 and 325 m/z ions,

corresponding to a Fuc-Hex C- and B-type fragment,respectively. Furthermore, the E-type m/z 1139 ion from the3-arm located the fucose on either GlcNAc present on this arm;however, the linkage position of fucose was not identified. Massspectrometric analysis of FcγRIIIb was able to demonstrate thepresence of multiple structures with outer arm fucosylation and,in some instances, multiple fucose monosaccharide residues perN-linked glycan.To determine the linkage of outer arm fucose in each Fcγ

receptor, α-fucosidases specific for terminal fucose in variouslinkage positions were used. AMF, which cleaves fucose linkedto N-acetylglucosamine in either an α(1−3) or α(1−4)position was used with no effect on any of the glycan samples(data not shown). To ensure full access for the enzyme to itssubstrate, terminal monosaccharides that potentially blockedaccess of AMF were first removed before digestion with AMF.This had no impact on the activity of AMF. The secondfucosidase used was XMF, which specifically removes α(1−2)-fucose linked to galactose. Similar to AMF, no obvious effectwas observed (data not shown) despite similar efforts toremove any interfering terminal monosaccharides.Interestingly and somewhat unexpectedly, BKF (1 mU μL−1)

was found to be more capable of removing outer arm fucosewhen compared to AMF, where the latter demonstrated noactivity on any FcγR glycan pool (data not shown). BKFreleases α(1−2)- and α(1−6)-linked nonreducing terminalfucose residues more efficiently than α(1−3)- and α(1−4)-

Figure 4. Extent of high-mannose structures in various NS0 FcγRs. High-mannose structures were identified for each FcγR through exoglycosidasearray analysis using ABS, CBG, BTG, BKF, and GUH. Total high-mannose structure abundance was determined by quantitation of each high-mannose peak present in HILIC-FLD chromatograms. The relative abundance of high-mannose glycans for each FcγR is illustrated as a bar graph,and the distribution (M4 to M9) for each FcγR is depicted with pie charts.

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linked fucose under the reaction conditions used. Outer armfucosylation was identified in the Fcγ receptor samples throughobservation of an unidentifiable peak during routineexoglycosidase digestion. Subsequent analysis revealed abifucosylated triantennary structure, F(6)A3F1 (Figure 8A).

The migration of the F(6)A3F1 peak to the A3 peak followingBKF digestion provided evidence that BKF was able to removeboth the core α(1−6) fucose and the outer arm fucose. Due tothe ability of BKF to remove both α(1−3) and α(1−4) fucose,the exact linkage of the outer arm fucose could not be

Figure 5. NS0 FcγRs contain extensive α-galactosylation. Released and 2AB-labeled glycans from each FcγR were first digested with the sialidaseABS to remove terminal sialylation (A). The asialylated glycan pool was then digested with coffee bean α-galactosidase (B) followed by bovine testesβ-galactosidase (C) to determine which peaks contained the differentially linked galactose structures. The chromatograms presented are based ondata obtained from FcγRIIa and are representative of a common outcome for all FcγRs. Dashed lines represent the migration of various structuresdue to enzyme activity. All peaks are identified and reported using the Oxford linear notation.

Figure 6. N-Glycolylneuraminic acid residues are expressed on Fcγ receptors. The nano-ESI−MS/MS spectrum of the m/z 1430.4 [M − H +H2PO4]

2− ion from FcRγIIIb demonstrated the presence of NGNA residues. The 306.1 m/z peak corresponds to the B-fragment of the NGNAmonosaccharide.

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determined. Despite this, evidence is presented that demon-strates Fcγ receptors expressed in NS0 cells contain both corefucose and outer arm fucose linked to N-acetylglucosamine.

■ DISCUSSION

α-Galactosylation

Analysis of Fcγ receptors expressed in NS0 cells revealedseveral interesting attributes. Perhaps the most significantattribute identified was the presence of extensive galactose-α(1−3)-galactose epitopes throughout all Fcγ receptorsubtypes. Glycan analysis revealed that, in all cases, the mostabundant carbohydrates contained α-galactosylation present onbi-, tri-, and tetraantennary structures (Figure 5). Incomparison to previously published Fcγ receptor glycananalyses from BHK,38,39 HEK293,40 and CHO,40 thecarbohydrate structures presented here differ significantly andfurther reinforce the assertion that Fcγ receptors exhibitdifferent glycoprofiles depending on the expression systemused. FcγRIIIa, for example, when expressed in the human cellline HEK293, will contain mainly biantennary and hybridglycans with terminal GalNAc residues, while in CHO cells itwill predominantly present core fucosylated bi- and trianten-nary glycans.40

Detailed IgG interaction analysis with NS0-expressed FcγRsis required to determine the potential binding effects of α-Galepitopes. To our knowledge this information is not available.

However, because this carbohydrate structure is not found inhumans, any positive or negative effect on IgG binding will notbe representative of a natural IgG interaction. It is also worthnoting that potential therapeutic use of these receptors couldelicit an immune response due to circulating anti-α-Galantibodies, resulting in an undesirable reaction. This has beenfound previously with therapeutic proteins containing α-Galepitopes.35 The presence of terminal α-galactosylation alsocauses a reduction in the extent of sialylation, which may havenegative connotations for FcγR interactions. Sialyltransferasecompetes for the same acceptor substrate as α(1−3)galactosyltransferase, meaning that the presence of an α-Galresidue on a terminal galactose will prevent addition of a sialicacid. This could potentially affect the net charge of thereceptor-associated glycans and could also interfere with IgGbinding.

Fcγ Receptor Outer Arm Fucosylation

It has been previously established that BKF is capable ofremoving fucose from GlcNAc in a number of linkagepositions, including α(1−3), α(1−4), and α(1−6). Thepossibility that BKF was removing fucose in a positioninaccessible by either XMF or AMF was considered. Theactivity of BKF on outer arm fucose and its ability to removefucose in either an α(1−3) or α(1−4) linkage to N-acetylglucosamine was an unexpected finding observed in thepresent study. Somewhat surprisingly, both AMF and XMF,

Figure 7. N-Acetyllactosamine is identified in NS0 FcγRs. To identify the presence of N-acetyllactosamine (LacNAc) structures, each 2AB-labeledFcγR glycan pool was first digested with ABS, CBG, BTG, and BKF to reduce the glycan pool to structures containing terminal N-acetylglucosamine(GlcNAc), hybrids, and high-mannose structures (A). A hexosaminidase (GUH) was used to identify peaks containing terminal GlcNAc (B).Comparison of the two resulting chromatograms allowed for identification of LacNAc structures.

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two exoglycosidases capable of removing α(1−3)- or α(1−4)-linked nonreducing terminal fucose, were unable to remove thisresidue.While BKF demonstrated activity where AMF and XMF

could not, terminal fucose structures continued to persist thatwere unable to be modified by any of the available α-fucosidases. N-Glycans digested with ABS, CBG, BTG, andBKF followed by analysis with HILIC-FLD revealed the

persistence of a peak with an approximate GU value of 5.6(Figure 8A). The composition of this peak was determined tobe M3A1F1, indicating an inability of any of our fucosidases forremoving the fucose. Outer arm fucose was found predom-inantly on triantennary structures, but its exact arm specificitycould not be determined. It is possible that one of the terminalGlcNAcs prevented access of BKF to the outer arm fucose,consequently generating a glycan structure in the form of A3F1.

Figure 8. Fcγ receptors contain outer arm fucose. HILIC-FLD and ESI−MS/MS were used to confirm the presence of outer arm fucose on FcRIIIb.For HILIC-FLR analysis, N-linked glycans released from FcγRIIIb were digested with (A) ABS, BTG, and CBG or (B) ABS, BTG, CBG, and BKF toidentify the presence of outer arm fucose. Chromatograms of the two digestion reactions indicated the movement of key peaks due to enzymeactivity. The undigested glycan pool was subjected to static nanoinfusion ESI−MS/MS analysis (C) where diagnostic fragment ions were detectedand confirmed the observations in the HILIC-FLD data.

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If this is the case, it is interesting to observe that BKF is able toaccess outer arm fucose in a manner different from thatobserved for AMF. It is also possible that BKF is able to removeterminal fucose in a new linkage position, namely, a fucose-α(1−2)-GlcNAc, possibly an α(1−6)-linked fucose to anonreducing GlcNAc, or indeed a fucose in an arm-specificnature. However, these suggestions are speculative and requirefurther investigation.

High-Mannose Glycans and Antennarity

The Fcγ receptors differ dramatically in their relative abundanceand diversity of high-mannose structures as well as theirdistribution of antennary structures. The exact function of thisdiversity remains unclear, but future work is aimed at definingthe glycovariants that exist at individual N-linked sites on thesereceptors as site occupancy of the Fcγ receptors is likely to beimportant. An interesting example of this is evident withFcγRIIIa, where one glycosylation site at Asn-162 has beenshown to be important for positive antibody binding and adifferent site at Asn-45 has been shown to have inhibitoryeffects on afucosylated IgG binding.15 Zeck et al.40 haveperformed an extensive site occupancy study of Asn-162 ofFcγRIIIa expressed in both HEK and CHO cells and show thatdifferent glycosylation patterns produced influence binding ofIgG. Unfortunately, no significant structural information isavailable with regard to the glycosylation site occupancy basedon the solved crystal structures of FcγRIIIb41 FcγRIIIa,42

FcγRIIa,43 and FcγRIa.44 In the case of FcγRIa and FcγRIIa,which are expressed in CHO and insect cells, respectively, theonly glycans visible in the electron density map are away fromthe Fc−Fcγ receptor interface. Noting that the presence ofcarbohydrates is usually refractory to crystallization, it is likelythat identified structures were the only crystallizable complexesand therefore not representative of the complete interaction.This is evident in the glycosylated FcγRIIIa structure complexwith or without afucosylated antibody which shows carbohy-drates in the binding interface play a pivotal role in theinteraction and explain the higher affinity for afucosylated IgGto FcγRIIIa.45

Receptor-Associated Neu5Gc

Terminal Neu5Gc is a known feature of murine glycosylationbut is considered a nonhuman sialic acid. It is present in manymammals, including chimpanzees and mice due to activity ofthe enzyme cytidine monophosphate N-acetylneuraminic acidhydroxylase (CMAH), which catalyzes the conversion ofNeu5Ac to Neu5Gc.46 Negative mode ESI−MS/MS of releasedNS0 Fcγ receptor glycans clearly demonstrated the presence ofthis carbohydrate moiety. However, the relative abundance ofthis structure could not be determined in this study. Humansprimarily synthesize the sialic acid Neu5Ac for incorporationinto glycans but due to an inactivating mutation in the genecoding for the enzyme CMAH are unable to hyroxylateNeu5Ac to Neu5Gc.47 Similar to murine α-Gal epitopes,Neu5Gc is capable of acting as an antigen to stimulate theproduction of anti-Neu5Gc antibodies, and circulating anti-Neu5Gc IgG has been found in healthy humans.48 Thismammalian nonhuman sialic acid could therefore induce animmune response or result in neutralization and removal ofbiopharmaceuticals, which reinforces the need for vigilencetoward glycan analysis of glycoprotein therapeutics expressed inmurine cell lines.

Glycosylation of Human Fcγ Receptors Depends on theCellular Expression System

To our knowledge three previous glycan analysis studies havebeen performed for recombinant human Fcγ receptors. Theseinclude both FcγRIIa38 and FcγRIIIb39 expressed in BHK cellsand FcγRIIIa expressed in either HEK293 cells or CHO cells.40

As expected FcγRIIa and FcγRIIIb produced in BHK cellsexhibit hamster kidney specific glycosylation patterns withα(2−3)-linked sialic acids only, no bisecting GlcNAc, and thepresence of GalNAc residues. Eighteen and thirty-nineindividual structures were identified for FcγRIIa and FcγRIIIb,respectively. Multiantennary structures containing up to fourGlcNAcs with or without sialic acid capping were alsodescribed. While similarities exist between these analyses andthe data presented here for the equivalent receptors from NS0cells, such as the presence of oligomannose and multiantennaryglycans, significant differences also exist, such as the presence ofNeu5Gc and α-Gal and the absence of GalNac epitopes.More recently, a detailed site-specific analysis of the glycans

present at each position of FcγRIIIa expressed in both CHOand HEK293 cells was reported. This illustrated starkdifferences in the glycans observed at individual sites on theFcγ receptor.40 Specifically, an abundance of glycan structurescontaining outer arm fucose and GalNAcs linked to GlcNAcswere found on HEK-expressed FcγRIIIa, while CHO cellsexpressed FcγRIIIa with mainly complex sialylated biantennarystructures. Interestingly, different glycoforms were observed atdifferent sites on FcγRIIIa, providing evidence that glycoformsubsets occur in a site-specific manner. In both HEK and CHO,there were no reports of α-galactosylation; however, outer armfucose was described. Of additional interest was the observationof the effect of outer arm fucosylation on sialylation. Sialylationwas not observed on antennae containing outer arm fucose as itis competitive. It is worth noting that depending on theexpression system used human Fcγ receptors will havesignificantly different glycan contents, with or withoutimmunogenic epitopes, and these glycosylation differences arelikely to affect the interaction with IgG.

Use of Fcγ Receptors in IgG Binding and Therapeutic mAbEvaluation

Recombinant soluble Fcγ receptors are commonly used inindustry to assess the binding of potential therapeuticmonoclonal antibodies and academia to investigate the bindingof IgG and other nonantibody ligands. Through bioanalyticaltechniques such as surface plasmon resonance (SPR) andisothermal titration calorimetry (ITC), the binding affinity ofIgGs to Fcγ receptors and association/dissociation rates can bedetermined and used as a measure of performance andeffectiveness of the antibody. In fact, regulatory agencies suchas the FDA and EMA insist on such measurements beingperformed. Several publications have referenced the use ofNS0-derived Fcγ receptors for use in SPR studies of antibodybinding affinity.26,29,30,49−52 It is worth noting that in manycases these analyses are performed without a detailedknowledge of the glycosylation of the recombinant receptorsused in the assays, and this is one of the main reasons why thework described here was performed. Interestingly, recentstudies evaluating the biological activity of afucosylated anti-CD20 antibody with FcγRIIIa has shown that specific glycansites on the receptor significantly affect the binding ofantibodies. Crystallization of glycosylated FcγRIIIa with anafucosylated antibody revealed a unique interface in the

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antibody−receptor complex consisting of carbohydrate−carbohydrate interactions.45 Significantly, these interactionsare weakened or nonexistent in the complex of receptor andcore-fucosylated antibody. On the basis of these importantobservations, it now seems increasingly evident that a role forglycosylation on FcγRs exists and that binding between IgGsand FcγRs is likely mediated in part by direct carbohydrate−carbohydrate interactions.

Glycosylation of Fcγ Receptors in Their NaturalEnvironment: The Key Challenge

Significant efforts have been made to determine the extent andnature of glycosylation of Fcγ receptors expressed in NS0 cells,principally as it is abundantly clear that the glycosylation stateof Fcγ receptors affects IgG binding.14,15 In situations whereglycosylation of a receptor or a ligand plays a prominent role inthe binding interaction, it is far from ideal to lack detailedknowledge of how each binding partner is glycosylated,particulary in the case of in vitro versus in vivo and in vivoveritas situations. This can lead to spurious results, misleadingdata, and potentially dangerous situations if immunogeniccarbohydrates are used in biotherapeutic applications. However,the key objective for Fcγ receptor glycobiology should not befocused around characterizing the glycosylation of recombinantglycoproteins expressed in cell lines and instead should becentered around understanding the glycosylation status of Fcγreceptors as they appear on innate effector cells such asmacrophages and natural killer cells. Several important papersby Kimberly et al. used traditional glycoprotein analysistechniques to describe the glycosylation status of Fcγ receptorspresent on a variety of immune effector cells.53,54 Many criticalfeatures were described, such as the cell-type-specificglycosylation of Fcγ receptors and differential ligand bindingto these receptors.Edberg and Kimberly demonstrated that FcγRIIIa expressed

by monocytes differed in terms of its glycosylation whencompared to FcγRIIIa expressed by NK cells.55 Whenexpressed by NK cells, this receptor was shown to containhigh-mannose- and complex-type oligosaccharides; however,when found on monocytes, it possessed no high-mannose-typeglycans. This intriguing result suggests that natural glycoformsof Fcγ receptors are present as cell-type-specific glycoformswhich bind IgG differentially. This has implications both foractivation and for modulation of the immune system. Edberg etal. have also proposed that the presence of high-mannosestructures is important for opsonin-independent receptorligation, which is a key feature for the phagocytic uptake ofbacterial pathogens displaying lectin-type adhesins (fimbriatedEscherichia coli, Pseudomonas aeruginosa, and Salmonellatyphimurium). This is fascinating, as it suggests that Fcγreceptors also have a function in nonclassical receptor ligation,although this has yet to be demonstrated experimentally. Thispotentially explains the presence of high-mannose structures inall of the Fcγ receptors described here. It remains a challenge,however, to determine the cell-type-specific glycosylation ofFcγ receptors and how it dictates ligand binding anddownstream effector functions. Until this can be adequatelydescribed, the role of Fcγ receptors in immune function cannotbe fully understood.

Closing Comments

The work presented here highlights the distinct glycanstructural properties observed through recombinant expressionof Fcγ receptors in NS0 cells. Importantly, many of the glycan

structures reported here are not typically found in humans. It iswidely accepted that key glycan epitopes such as α-galactosylation and N-glycolylneuraminic acid are a concernfor regulatory agencies due to their known immunogenicity inhumans, but what impact these particular properties have on invitro binding studies remains to be seen. Neu5Gc and Neu5Acdiffer by one oxygen atom at the C5 position, but is thisunlikely to affect the affinity for IgG or kinetic rates ofassociation and dissociation? Similarly, the replacement ofterminal sialylation with terminal α-galactosylation significantlyaugments the charged glycan composition of Fcγ receptors, butunderstanding the overall impact on FcγR biology is currentlyunclear. Further complicating the matter is the fact that thelocal heterogeneity of glycosylation at individual sitesthroughout each Fcγ receptor is not yet known. Precise glycandistribution at individual sites remains paramount to fullyelucidating the function of glycans in Fcγ receptor biology.Finally, knowledge of glycosylation of recombinant Fcγreceptors will only bear importance when the glycosylationstatus of their naturally occurring counterparts is elucidated, inboth dormant and active states.

■ AUTHOR INFORMATION

Corresponding Author

*E-mail: Pauline.Rudd@nibrt.ie. Phone: +353 (0)1 215 8100.Fax: +353 (0)1 215 8166.

Notes

The authors declare no competing financial interest

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