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Linkage Specific Fucosylation of Alpha-1-Antitrypsin in LiverCirrhosis and Cancer Patients: Implications for a iomarker of

!epatocellular Carcinoma: e1"#1$Comunale% &ary Ann ' (odemich-etesh% Lucy' !afner% )ulie' *ang%

&eng+un' ,orton% Pamela ' et al PLoS One ./ 0Aug "123urn on hit highlighting for speaking 4ro5sers!ide highlighting

Abstract (summary)3ranslate A4stract

ackground

*e previously reported increased levels ofprotein-linked fucosylation 5ith the development of liver

cancerand identified many ofthe proteins containing the altered glycan structures 6ne such

protein is alpha-1-antitrypsin 0A1A32 3o advance these studies% 5e performed ,-linked glycan

analysis on the five ma+or isoformsofA1A3 and completed a comprehensive study ofthe

glycosylation ofA1A3 found in healthy controls% patients 5ith hepatitis C- 0!C72

induced livercirrhosis% and in patients infected 5ith !C7 5ith a diagnosis ofhepatocellular

carcinoma 0!CC2

&ethodology8Principal Findings

Patients 5ith livercirrhosis and liver cancerhad increased levels oftriantennary glycan-containing

outer arm 09-1%2 fucosylation Increases in core 09-1%;2 fucosylation 5ere o4served only on A1A3

from patients 5ithcancer *e performed a lectin fluorophore-linked immunosor4ent assay using

Aleuria Aurantia lectin 0AAL2% specific for core and outer arm fucosylation in over # patients

5ith liverdisease AAL-reactive A1A3 5as a4le to detect !CC 5ith a sensitivity oflycosylation analysis ofthe false positives 5as performed' results indicated that

these patients had increases in outer arm fucosylation 4ut not in core fucosylation% suggesting that

core fucosylation is cancerspecific

Conclusions8Significance

3his report details the step5ise change in the glycosylation ofA1A3 5ith the progression

from livercirrhosis tocancerand identifies core fucosylation on A1A3 as an !CC specific

modification

Full Text 3ranslate Full te?t

3urn on search term navigation

A4stract

ackground
http://search.proquest.com/indexinglinkhandler/sng/au/Comunale,+Mary+Ann/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Rodemich-Betesh,+Lucy/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Hafner,+Julie/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Wang,+Mengjun/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Wang,+Mengjun/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Norton,+Pamela/$N?accountid=62690http://search.proquest.com/pubidlinkhandler/sng/pubtitle/PLoS+One/$N/1436336/DocView/1318937316/fulltext/14102A2BC27328BE03D/2?accountid=62690http://search.proquest.com/indexingvolumeissuelinkhandler/1436336/PLoS+One/02010Y08Y01$23Aug+2010$3b++Vol.+5+$288$29/5/8?accountid=62690http://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690http://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690http://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690#centerhttp://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690#centerhttp://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Rodemich-Betesh,+Lucy/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Hafner,+Julie/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Wang,+Mengjun/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Wang,+Mengjun/$N?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Norton,+Pamela/$N?accountid=62690http://search.proquest.com/pubidlinkhandler/sng/pubtitle/PLoS+One/$N/1436336/DocView/1318937316/fulltext/14102A2BC27328BE03D/2?accountid=62690http://search.proquest.com/indexingvolumeissuelinkhandler/1436336/PLoS+One/02010Y08Y01$23Aug+2010$3b++Vol.+5+$288$29/5/8?accountid=62690http://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690http://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690http://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690#centerhttp://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690#centerhttp://search.proquest.com/docview/1318937316/14102A2BC27328BE03D/2?accountid=62690http://search.proquest.com/indexinglinkhandler/sng/au/Comunale,+Mary+Ann/$N?accountid=62690


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*e previously reported increased levels ofprotein-linked fucosylation 5ith the development of liver

cancerand identified many ofthe proteins containing the altered glycan structures 6ne such

protein is alpha-1-antitrypsin 0A1A32 3o advance these studies% 5e performed ,-linked glycan

analysis on the five ma+or isoformsofA1A3 and completed a comprehensive study ofthe

glycosylation ofA1A3 found in healthy controls% patients 5ith hepatitis C- 0!C72

induced livercirrhosis% and in patients infected 5ith !C7 5ith a diagnosis ofhepatocellular

carcinoma 0!CC2

&ethodology8Principal Findings

Patients 5ith livercirrhosis and liver cancerhad increased levels oftriantennary glycan-containing

outer arm 09-1%2 fucosylation Increases in core 09-1%;2 fucosylation 5ere o4served only on A1A3

from patients 5ithcancer *e performed a lectin fluorophore-linked immunosor4ent assay using

Aleuria Aurantia lectin 0AAL2% specific for core and outer arm fucosylation in over # patients

5ith liverdisease AAL-reactive A1A3 5as a4le to detect !CC 5ith a sensitivity oflycosylation analysis ofthe false positives 5as performed' results indicated that

these patients had increases in outer arm fucosylation 4ut not in core fucosylation% suggesting that

core fucosylation is cancerspecific

Conclusions8Significance

3his report details the step5ise change in the glycosylation ofA1A3 5ith the progression

from livercirrhosis tocancerand identifies core fucosylation on A1A3 as an !CC specific

modification

Citation: Comunale &A% (odemich-etesh L% !afner )% *ang &% ,orton P% et al 0"12 Linkage

Specific Fucosylation ofAlpha-1-Antitrypsin in LiverCirrhosis and CancerPatients: Implications for

a iomarker of!epatocellular Carcinoma PLoS 6,@ .0/2: e1"#1$

doi:11


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!epatitis Foundation% and an appropriation from 3he Common5ealth ofPennsylvania 3he

funders had no role in study design% data collection and analysis% decision to pu4lish% or

preparation ofthe manuscript

Competing interests: 3he authors have declared that no competing interests e?ist

Introduction

Infection 5ith hepatitis virus 0!72 or hepatitis C virus 0!C72 is the ma+or

etiology ofhepatocellular cancer0!CC2 1G-#G oth !7 and !C7 cause acute and

chronic liverinfections% and most chronically infected individuals remain asymptomatic for many

years .G A4out 1= to #= ofall chronic !7 carriers eventually develop liver cancer% and it is

estimated that over one million people 5orld5ide die 4ecause of!7- and !C7-associated liver

cancer"G% ;G%


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false positives identified outer-arm fucosylation as 4eing the cause offalse positivity In contrast%

increases in core fucosylation 5ere found only in patients 5ith cancer 3he reasons for this change

and the clinical usefulness ofthis change are discussed

&aterials and &ethods

@thics Statement

oth the Ere?el Bniversity College of&edicine and the Saint Louis Bniversity Institutional (evie5

oards approved the study protocol% 5hich 5as consistent 5ith the standards esta4lished 4y the

!elsinki Eeclarationof1$el @lectrophoresis

A total of< Ol ofserum 5as diluted in Ol ofsolu4iliJation 4uffer containing < & urea% " &

thiourea% #= C!APS% ;. m& E33% . m& tri4utylphosphine% and a #= mi?ture ofcarrier


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ampholytes 0Servalyt p! "-#% p! -1% p! $-1% 1:":12 Samples 5ere vorte?ed periodically for 1 h

and applied to an 1/-cm p! -< ,L immo4iliJed p! gradient 0IP>2 strip 0Amersham% Piscata5ay%

,)% BSA2 >el rehydration 5as carried out for 1# h at . 7 and focused using the IP>Phor

0Amersham2 isoelectric focusing apparatus After focusing% gel strips 5ere first reduced then

alkylated in ;& urea% "= SES% = glycerol% . m& 3ris% p! ;/% and either m& E33 or els 5ere fi?ed and stained 5ith colloidal Coomassie >els 5ere imaged using the 6dyssey

Infrared Imaging System 0Li-Cor iosystems% Lincoln% ,e4raska2 and analyJed using ,onLinear

Eynamics Progenesis *orkstation gel imaging soft5are 0,onLinear Eurham% ,C% BSA2

&atri?-Assisted Laser Eesorption8IoniJation-3ime ofFlight 0&ALEI-36F2 &ass Spectrometry

Protein spots 5ere e?cised from colloidal Coomassie 4lue stained gels% destained% and digested

5ith trypsin (ecovered peptides 5ere concentrated and desalted using ip3ip C1/ 0&illipore%

edford% &A% BSA2 according to the manufacturerQs directions and prepared for &ALEI-36F mass

spectrometry 4y mi?ing . mL ofpeptide mi?ture 5ith . mL of1 mg8mL 9-cyano-#-

hydro?ycinnamic acid and 1= formic acid in .= acetonitrile and allo5ing the droplet to dry on

the &ALEI plate Peptide mass maps 5ere o4tained using a 7oyager-E@ Pro &ass Spectrometer

0Applied iosystems% Life iotechnologies% Carls4ad% CA2 operated in positive ion reflectron mode

Proteins 5ere identified from the peptide mass maps using the &ASC63 online data4ase

0555matri?sciencecom2 to search the nonredundant protein data4ase

>lycan Analysis

3he "E@ gel spots 5ere e?cised and destained using #= methanol ase F2 5as

diluted 5ith " m& ammonium 4icar4onate p! < and allo5ed to adsor4 into the gel plug 3he gel

plug 5as then covered 5ith the same solution and allo5ed to incu4ate overnight at


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column 03S amide / columns2 complemented 5ith a *aters fluorescence detector and Huantified

using the &illennium Chromatography &anager 0*aters Corporation% &ilford% &A2 3he mo4ile

phase consisted ofsolvent A 0. m& ammonium formate% p! ##2 and solvent 0acetonitrile2 3he

gradient used 5as as follo5s: linear gradient from "= to ./= solvent A at # mL8minute for

1." min follo5ed 4y a linear gradient from ./= to 1= solvent A for the ne?t min 3he flo5

rate 5as increased to 1 mL8minute' the column 5as 5ashed in 1= solvent A for . min

Follo5ing the 5ash step% the column 5as eHuili4rated in "= solvent A for "" min in preparation

for the ne?t sample >lycan structures 5ere identified 4y calculating the glucose uptake value and

e?oglycosidase digestion% as descri4ed previously "1G

Lectin-Fluorophore-linked Immunosor4ent Assay 0FLISA2

3he capture anti4ody 0mouse antihuman A1A3% A4E Serotec% (aleigh ,C% BSA2% 5as incu4ated 5ith

1 m& sodium periodate for 1 h at #KC 3his treatment ensures the lectin is una4le to react 5ith

the glycosylation ofthe anti4ody and does not affect anti4ody su4strate 4inding An eHual

volume ofethylene glycol 5as added% and the o?idiJed anti4ody 5as 4rought to a

concentration of1 Og8mL 5ith sodium car4onate 4uffer% p! $. Anti4ody 0. Og85ell2 5as added

to the plate and% follo5ing incu4ation% 5as 5ashed 5ith 1= 35een "m& phosphate 4uffered

saline p!


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curves 5ere constructed using all possi4le cutoffs for each assay 3he area under the receiver

operating characteristic curve 5as constructed and compared as descri4ed previously A t5o-tailed

P value of. 5as used to determine statistical significance All analyses 5ere performed using

>raphPad Prism 0San Eiego% CA% BSA2

(esults

Levels ofA1A3 and Eegree ofSialyation in Patients 5ith LiverCirrhosis and !CC

Pooled sera from healthy patients 0n "2% patients 5ith livercirrhosis 0n "2 and patients 5ith

!CC 5ith a 4ackground ofcirrhosis 0n "2 5ere resolved via "-E gel electrophoresis 0"E@2

03a4le 12 Figure 1A sho5s a representative "-E@ ofnormal human serum and Figures 1% 1C and

1E sho5 a focus on the . isoforms 0&1% &"% % &;% and &2

and fell 5ithin normal serum concentrations 3his result 5as confirmed 4y analyJing A1A3 an

@nJyme-linked immunosor4ent assay 0@LISA2% 5here4y the A1A3 levels 5ere " mg8mL in the

composite from the healthy patients% mg8mL in the composite from the cirrhotic patients% and

mg8mL in the composite from the !CC patients

Figure omitted% see PEFG

Figure 1 35o-dimensional gel purification ofalpha-1-antitrypsin isoforms

Sera from pools ofhealthy controls 0A% % @2 and cirrhosis 0C% F2 and cancer0E% >2 patients 5ere

focused using IP>Phor -< ,L first dimension strips follo5ed 4y SES-PA>@ separation on /= to

1/= acrylamide gels 3he pIofselected gel spots are &1 #$1% &" #$.% .% &;

..% and &< .1 Panels @% F% and > sho5 the relative a4undance ofeach gel spot

3a4le 1 Patients BtiliJed in Study

*e performed glycan analysis on the &1% &"% % &;% and &< isotypes from the pools ofsera from

normal participants and cirrhotic and !CC patients 0Figures 1-1E2 and e?amined the sialylation

patterns ofeach isotype Figure S1 sho5s a representative glycan profile for the isotype from

the controls Consistent 5ith results ofprevious studies "#G% the 4iantennary glycan is the most

a4undant species Figure S1- sho5s the level ofthe sialyated 4iantennary and triantennary

glycan associated 5ith each isoforms from each patient group As S1- sho5s% the

level ofsialyation on the 4i-anntennery glycan 0A">"S1 or A">"S"2 5as similar in the different

patient groups Similarly% the level ofsialyation 5as not altered on the triantennary glycan

!o5ever% there 5as an increase in the level ofthe tri-sialyated 9-1% linked fucosylated outer arm


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fucosylated glycan 0AF021>S2 on A1A3 from the cancerpatients% as compared to the healthy

and cirrhotic patients 3hese peak are indicated in figure S1A T 5ith an asterisk

Increased Levels ofCore and 6uter Arm Fucosylation Are 64served on A1A3 from Patients 5ith

!CC

3o test if the increased level oftri-sialyated 9-1% linked fucosylated outer arm fucosylated glycan

0AF021>S2 5as the result ofan increase in sialyation or an increase in the total

amount ofparent glycan% sialic acid 5as removed enJymatically and the sample re-analyJed

Figure " sho5s the simplified desialylated glycoprofile ofthe five ma+or isoforms for each ofpatient

group follo5ing treatment 5ith neuraminidase 0Arthro4acter ureafaciens2 3hree peaks ofinterest%

indicated 5ith a star in Figure "% are reproduci4ly altered in the &1% &"% and isoforms as the

diseased liverprogresses from cirrhosis to cancer SeHuential e?oglycosidase digestion 0data not

sho5n2 sho5ed these peaks to 4e a core fucosylated 4iantennary glycan 0F0;2A">"2% a

triantennary glycan 0A>2% and a triantennary glycan 5ith a single 9 1% linked outer arm fucose

residue 0AF021>2 Figure A sho5s a representative desialylated profile 5ith the corresponding

glycan structure identified for each peak 3a4le " is a Huantitation ofeach glycan structure present

on the five ma+or isoforms for each patient group Specific changes in glycosylation are o4served

in the A1A3 isoforms 5ith the progression to cirrhosis and !CC 6n &;% the isoform 5ith very little

tri and tetra-antennary structures% the 4iantennary core fucosylated glycan 0F0;2A">"2% represents

##/= in normal patients% #% 5as

associated 5ith a decrease in triantennary glycan A> 03a4le "% glycan ;2


Figure " 3he desialylated ,-linked glycan profile for each ofthe five A1A3 isoforms from normal

controls 0top2 and cirrhotic 0middle2 or !CC patients 04ottom2

3he ma+or peaks that are altered are indicated 5ith an asterisk and are 0from left to right2 a core

fucosylated 4ianntennary glycan 0F$;2A">"2% a trianntennary ,-linked glycan% and a trianntennary


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,-linked glycan 5ith a single outer arm fucose residue 0AFG2 3he percent ofeach ofthese

peaks in the different isoforms and in the different patient groups is sho5n in 3a4le "


Figure Specific changes in glycosylation on A1A3 can 4e o4served 5ith the progression

from livercirrhosis toliver cancer

0A2 A representative ,-linked glycan profile from a normal control patient 3he 11 ma+or glycan

structures identified are indicated and given a num4er 3his num4er is used in 3a4le " 5ith

structure names provided 3he relative change in the level ofthe trianntennary ,-linked glycan

5ith a single outer arm fucose residue 0AFG>2 02 and the core fucosylated 4ianntennary

glycan 0F;GA">"2 0C2 in normal to cirrhotic and cirrhotic to !CC is sho5n as percent change As

this figure sho5s% increases in outer arm fucosylation are associated 5ith 4oth cirrhosis and !CC%

5hereas increased core fucosylation is only o4served 5ith !CC

3a4le " 3he ,-linked >lycans Found on A1A3 Isoforms from Controls% Patients 5ith Cirrhosis% or

Patients 5ith !CC

Analysis ofFucosylated A1A3 4y Lectin-FLISA in a Cohort of#./ Patients

3o further e?amine if the changes in 4oth core and outer arm fucosylation could 4e seen in

individual patients and potentially used as a diagnostic marker of cancer% 5e analyJed a patient

cohort consisting of#./ patients for the level offucosylated A1A3 using a lectin-FLISA 4ased

assay In this assay% A1A3 5as captured using a monoclonal anti4ody and the level offucosylation

5as determined using the fucose-4inding AAL AAL recogniJes 4oth outer arm and core fucosylated

glycan 35enty patients 5ith no evidence of liverdisease 5ere used as controls' patients

infected 5ith !7 had an unkno5n level of liverfi4rosis' "1. patients infected 5ith !C7 had an

unkno5n level of liverfi4rosis' ;. patients had livercirrhosis' ;" patients had otherliverdiseases'

and ; patients had liver cancer03a4le 12 Figure A sho5s the relative level offucose lectin-

reactive A1A3 in the si? patient groups 7alues are given as -fold increase in relation to the level in

commercially purchased normal sera 3he mean and $.= confidence interval ofthe mean are

sho5n for each group *e found a clear statistical difference 4et5een the !CC group and all other

groups 0PM12 and 4et5een the cirrhotic group and the control group 0P 1


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single lesion ofless than "cm 11G% had a $1 fold 0U#"2 similar to

those o4served in healthy controls !o5ever% these patients have significant increases in outer arm

fucosylation 0AF021>2% similar to the highest levels seen in patients 5ith !CC 3hat is% the

level ofouter arm fucosylation in these patients ranged from 1= to 1


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It is also interesting to note that lectin reactivity 5as greater than the total change o4served in

fucosylation For e?ample% patient "< had either core or outer arm fucose on ""/"= ofhis or her

,-linked glycans In contrast% A1A3 from a healthy individual had fucose 0core or outer arm2 on

$= ofthe ,-linked glycans !ence% using the lectin FLISA% 5e should have o4served closer to a

".-fold increase and not the 11-fold increase that 5as o4tained 3his difference may 4e an

artifact ofthe lectin-FLISA% the result ofother proteins attached to A1A3% increased or modified 6-

glycosylation% or increased accessi4ility ofthe lectin to the fucose residues Allofthese possi4le

scenarios are under investigation

It is also important to note that increased outer arm fucosylation 5as o4served 4oth in patients

5ith cirrhosis and in those 5ith !CC 3his finding implies that outer arm fucosylation 5as not

specific to cancer4ut rather 5as pro4a4ly associated 5ith inflammation% first from

the livercirrhosis and then from the presence ofthe !CC lesion Indeed% the level ofouter arm

fucosylation may 4e a more specific marker ofinflammation and may 4e predictive of

cancerdevelopment "


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3he sialylated ,-linked glycan profile for each ofthe five A1A3 isoforms from normal% cirrhotic% or

!CC patients 0A2 A representative sialyated profile ofthe A1A3 isoform from healthy

individuals 02 3he relative percentofsialyated 4i-antennary or tri-antennary glycan in each A1A3

isoform

0 & 3IF2

Author Contri4utions

Conceived and designed the e?periments: &AC A& Performed the e?periments: &AC L( )! &*

A& AnalyJed the data: &AC &* P, A&E 3 A& Contri4uted reagents8materials8analysis tools:

A&E A& *rote the paper: &AC A&

References

1 Ei isceglie A& 01$/$2 !epatocellular carcinoma: molecular 4iology ofits gro5th and

relationship to hepatitis virus infection &ed Clin ,orth Am oormastic &% et al 0"#2 (isk factors for

hepatocellular carcinoma in patients 5ith cirrhosis Eig Eis Sci #$: /.-/. Find this article online

/ Alpert &@% Briel )% de ,echaud 01$;/2 alpha fetoglo4lin in the diagnosis ofhuman hepatoma

, @ngl ) &ed "


14/17

1 Ei isceglie A&% !oofnagle )! 01$/$2 @levations in serum alpha-fetoprotein levels in patients

5ith chronic hepatitis Cancer;#: "11


15/17

" Ei isceglie A&% Shiffman &L% @verson >3% Lindsay L% @verhart )@% et al 0"/2 Prolonged

therapy ofadvanced chronic hepatitis C 5ith lo5-dose peginterferon , @ngl ) &ed .$: "#"$-

"##1 Find this article online

"1 >uile >(% (udd P&% *ing E(% Prime S% E5ek (A 01$$;2 A rapid high-resolution high-

performance liHuid chromatographic method for separating glycan mi?tures and analyJing

oligosaccharide profiles Anal iochem "#: "1-""; Find this article online

"" )eppsson )6% @inarsson ( 01$$"2 >enetic variants ofalpha 1-antitrypsin and hemoglo4in typed

4y isoelectric focusing in preselected narro5 p! gradients 5ith PhastSystem Clin Chem /: .ao CF% Laroy *% Ee5aele S% et al 0"lyco4iology 1/: 11.-111/ Find this article online

"/ Arnold ),% Saldova (% !amid B&% (udd P& 0"/2 @valuation ofthe serum ,-linked glycome

for the diagnosis of cancerand chronic inflammation Proteomics /: "/#-"$ Find this article

online

"$ Peracaula (% Sarrats A% Saldova (% Pla @% Fort @% et al 0"12 >lycosylation of liveracute-

phase proteins in pancreatic cancerand chronic pancreatitis P(63@6&ICS - Clinical Applications

In press Find this article online

*ord count: 6061


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D "1 Comunale et al 3his is an open-access article distri4uted under the terms of the Creative

Commons Attri4ution License% 5hich permits unrestricted use% distri4ution% and reproduction in any

medium% provided the original author and source are credited: Comunale &A% (odemich-etesh L%

!afner )% *ang &% ,orton P% et al 0"12 Linkage Specific Fucosylation of Alpha-1-Antitrypsin in

Liver Cirrhosis and Cancer Patients: Implications for a iomarker of !epatocellular Carcinoma

PLoS 6,@ .0/2: e1"#1$ doi:11


17/17

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