Modifications of human total serum N-glycome during liver fibrosis–cirrhosis, is it all about immunoglobulins?

REVIEW

Modifications of human total serum N-glycome during

liver fibrosis–cirrhosis, is it all about immunoglobulins?

Andre Klein1,2, Jean-Claude Michalski2 and Willy Morelle2

1 Laboratoire de Biochimie et de Biologie Moleculaire, UAM de glycopathologies, Centre de Biologie et Pathologie,CHRU Lille, Bld du Professeur Jules Leclercq, Lille, France

2 Unite Mixte de Recherche CNRS/USTL 8576, ‘Glycobiologie Structurale et Fonctionnelle’, IFR 147, Batiment C9,Universite des Sciences et Technologies de Lille 1, Villeneuve d’Ascq, France

Received: July 10, 2009

Revised: October 21, 2009

Accepted: December 3, 2009

The study of the total serum N-glycome during liver cirrhosis has demonstrated numerous

alterations. The identification of the glycoproteins carrying these modifications and their

relative contribution to the modification of the total serum N-glycome has shown the

important role of IgA and IgG. The possible mechanisms of glycosylation alteration of the Igs

and of liver secreted glycoproteins, the consequences in the pathophysiology of cirrhosis and

their relation to the biomarkers of liver diseases are also discussed in the present review.

Keywords:

Glycosylation / Liver diseases / N-glycome

1 Introduction

Recent technological advances have permitted the develop-

ment of protocols allowing the rapid high-throughput

analysis of the human total serum N-glycome (TSNG). The

TSNG represents the serum glycoprotein N-glycosylation,

the most common post translational modification of plasma

proteins [1]. Alterations of the TSNG have been described in

various pathological conditions such as congenital disorders

of glycosylation, cancers, inflammatory disorders and liver

fibrosis–cirrhosis.

An initial demonstration of TSNG modifications during

cirrhosis and fibrosis was made by Callewaert et al. using an

oligosaccharide electrophoresis technique based on a DNA

sequencer [2]. Elucidation of the N-glycan structures

constituting the TSNG, using different mass spectrometric

approaches, completed the description of the glycosylation

alterations [3].

The search for glycosylation-based biomarkers of liver

fibrosis and hepatocellular carcinoma (HCC) has been

stepped up in the past 5 years and the development of non-

invasive assays for the follow-up and screening of liver

diseases is critical as an alternative to liver biopsy. Unfor-

tunately, glycobiomarkers are not sufficiently sensitive and

specific to be a ‘‘gold standard’’ for the hepatologist.

For these reasons, we wanted to understand on which

glycoproteins the modifications of glycosylation encoun-

tered in liver cirrhosis were located. Our studies demon-

strated the causes for the distribution of secreted

glycoproteins between liver and B lymphocyte and their

relative importance in the modifications of the TSNG [4, 5].

In this review, we present a summary of the TSNG

modifications, the possible mechanisms of glycosylation

alterations of the Igs and liver-secreted glycoproteins, the

consequences in the pathophysiology of cirrhosis and their

relation to the biomarkers of liver diseases.

2 Modifications of the TSNG in liverfibrosis and cirrhosis

The first demonstration of TSNG modifications was made

by the electrophoretic study of desialylated oligosaccharides

using a DNA sequencer; these were characterized by an

increase in glycans with a bisected N-acetylglucosamineAbbreviations: AFP, a-fetoprotein; HCC, hepatocellular carci-

noma; TSNG, total serum N-glycome

Correspondence: Dr. Andre Klein, Laboratoire de Biochimie et de

Biologie Moleculaire, UAM de glycopathologies, Centre de

Biologie et Pathologie, CHRU Lille, Bld du Professeur Jules

Leclercq, Lille 59037 Cedex, France

E-mail: [email protected]

Fax: 133-3-20-44-49-57

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com

372 Proteomics Clin. Appl. 2010, 4, 372–378DOI 10.1002/prca.200900151

residue and an increase in agalactosylated structures. A new

biomarker of cirrhosis was defined – the GlycoCirrhoTest –

and its efficiency was evaluated (AUC 5 0.87, specificity

100% and sensitivity 5 75%); nevertheless, it was not rele-

vant for less advanced fibrosis stages [2, 6].

Using mass spectrometric techniques, Morelle et al.biochemically characterized the primary structure of the

different glycans constituting the TSNG and described three

groups of glycosylation modifications: (i) the significant

presence of bisected GlcNAc N-glycans; (ii) the increase in

a1,6 fucosylated structures; and (iii) the presence of neutral

agalactosylated oligosaccharides (Figs. 1A and 2A) [3]. The

TSNG is constituted by the addition of the various individual

glycoprotein N-glycomes and is influenced by their

concentration variations and their specific glycosylation. For

example, the serum Ig level increase is a characteristic

feature of chronic liver disease and in the case of alcoholic

liver diseases, this is particularly important for IgA [7]. So,

the Ig glycosylation, among others, in the TSNG, is more

represented during fibrosis–cirrhosis. Furthermore, altera-

tions of the liver functions are extremely notable during

fibrosis–cirrhosis and affect the secretion, concentration of

glycoproteins and their catabolism. To evaluate the influ-

ence of the various major glycoproteins in the modifications

of TSNG, we have recently purified by affinity chromato-

graphy, transferrin, IgA, and IgG, and studied their glyco-

sylation; we completed the description of modifications of

glycosylation found in patients with liver cirrhosis using bi-

dimensional electrophoresis and ‘‘in-gel PNGase F’’ diges-

tion. The glycosylation alterations observed were either

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Figure 1. MALDI-TOF-MS of N-glycans

released from a healthy donor serum before

(A) and after (B) depletion of albumin/IgG/

IgA, IgGs (C) and IgAs of this healthy donor

serum (D). Depletions of albumin and IgG

were performed on a HiTrapTM Blue HP

column, and on a HiTrapTM rProtein AFF

column, respectively. IgGs were eluted from

the HiTrapTM rProtein AFF column and IgAs

were purified from the albumin/IgG-depleted

serum by affinity chromatography on Jacalin

agarose beads; methods are described in [4,

5]. N-glycans were released from the differ-

ent glycoprotein fractions by peptide

N-glycanase F and were permethylated

before MALDI-TOF analysis. A minor portion

of the monofucosylated glycans carries

fucose on an antenna rather than the core.

Galactose (open circles); mannose (closed

circles); GlcNAc (closed squares); fucose

(open triangles); NeuAc (closed diamonds).

Proteomics Clin. Appl. 2010, 4, 372–378 373


glycoprotein-specific or common to different glycoproteins

[4, 5].

3 Glycosylation modifications of IgG inliver cirrhosis

IgGs are the most abundant glycoproteins in the serum;

carbohydrate moieties account for 2–3% of the molecular

weight. Glycans are mainly located on the CH2 domain of

the heavy chain. Approximately, 30 different oligosaccha-

rides have been described on IgG; they mainly are of the

biantennary complex type, predominantly a1,6 core fucosy-

lated; finally, some of the glycans present a bisecting

GlcNAc residue (Fig. 1C).

Glycans are distributed among four groups – the group of

sialylated oligosaccharides (14%) and three groups of

neutral oligosaccharides, according to the number of term-

inal galactose residues respectively: the agalactosylated (G0,

35%), the monogalactosylated (G1, 35%) and the bigalacto-

sylated structures (G2, 16%) [8, 9].

In cirrhotic patients, the glycosylation modification

observed on IgG mainly is an increase in agalactosylated

structures (Fig. 2C). The agalactosylation of IgG has been

observed in numerous inflammatory and autoimmune

diseases [1]. The agalactosylation of a natural antibody,

directed against the heterophilic Galili antigen Gala1,3Gal

[10], has been described in the serum of patients infected

with hepatitis C virus, occurring with the development of

fibrosis [11]. Serum Ig increase is a well-known feature of

chronic liver diseases; numerous physiopathological

mechanisms might be involved: (i) the ‘‘leaky gut’’ observed

in alcoholic liver disease with the alteration of the gut

permeability by alcohol, causing bacteria from the intestinal

tract to escape into the blood stream, these antigens

stimulate the antibody response; (ii) in alcoholic liver

disease, it is postulated that acetaldehyde and malondialde-

hyde generate modified self proteins after reaction on lysine

residues; these adducts could induce specific immune

responses and explain the presence of circulating auto-

antibodies [12]; (iii) an impaired Ig clearance by the liver.

The hypergammaglobulinemia and agalactosylation

affect the liver disease. The N-glycans of the Fc fragment

modulate the effector functions of IgG, especially the acti-

vation of the complement system and binding to Fc gamma

receptor (FcR) present on phagocytes. The agalactosylated

IgGs are able to activate the complement system via the

lectin pathway. The terminal GlcNAc residues present at the

oligosaccharide terminal end of the agalactosylated IgGs are

recognized by the Mannan Binding Lectin, a member of the

collectin family that has an important role in innate

immunity. The activation of this pathway can trigger rapid-

enhanced phagocytosis [8]. The agalactosylation of IgG

appears as a proinflammatory mechanism common to

numerous inflammatory diseases. A decreased IgG sialyla-

tion was also observed after immunisation with a test anti-

gen and might correspond to a switch from a steady anti-

inflammatory state to a protective inflammatory state upon

immunization [13]. The modified immune response might

be important in the development of liver fibrosis and in the

acceleration of the liver disease [14].

4 Glycosylation modifications of IgA inliver cirrhosis

In human serum, IgA exists as two isotypes: IgA1 (90%) and

IgA2 (10%). All IgAs contain two N-glycans on the CH2 and

CH3 domains and IgA2 differs by the presence of two or

three additional N-glycans. Thirty percent of IgA1 Fab

fragment is N-glycosylated. The hinge region of IgA1

contains O-glycans.

IgA1 N-glycans are principally biantennary, but they

differ from IgGs as there is a higher number of tri-

antennary structures. More than 90% of glycans are sialy-

lated and less than 2% are agalactosylated. A small portion

of N-glycans shows a bisected GlcNAc residue. Finally,

approximately one out of three glycans is a1,6 core fucosy-

lated [1]. A typical N-glycome of normal IgA is described in

Fig. 1D.

The MALDI-TOF MS analysis of purified IgA N-linked

oligosaccharides of a patient with liver cirrhosis demon-

strates the changed distribution of N-glycans (Fig. 2D).

Different modifications can be characterized during liver

fibrosis: (i) the loss of terminal sialic acid, characterized by

an increase in mono- and in a-sialooligosaccharides; (ii) the

increase in glycans containing a bisected GlcNAc residue

and among that population, the predominance of non-

fucosylated bisected oligosaccharides [4].

Non-fucosylated, agalactosylated bisected GlcNAc oligo-

saccharides (ions at m/z 1907 and 2111) are very repre-

sentative of the IgA in the TSNG of cirrhotic patients, (Figs.

1 and 2).

As for IgG, the glycan modulates the Fc fragment bind-

ing to Mannan Binding Lectin [15] and to the asialoglyco-

protein receptor (ASGP-R); ASGP-R binds to galactose and

N-acetylgalactosamine terminated oligosaccharides and

removes IgA2 from the serum, but not the sialylated struc-

tures of IgA1, explaining their predominance in the serum

[16]. During liver fibrosis–cirrhosis, the increase in the

agalactosylated, non-sialylated IgA might be due to the

alteration of the liver clearance and also to an increased IgA

production. Interestingly, after liver transplantation for

alcoholic cirrhosis, the immediate reduction in IgA, IgG and

IgM was observed, suggesting an increased catabolism [17].

5 Glycosylation modifications of hepaticglycoproteins during liver cirrhosis

The study of the modifications of hepatic glycoprotein

glycosylation after bidimensional electrophoresis and

374 A. Klein et al. Proteomics Clin. Appl. 2010, 4, 372–378


‘‘in-gel’’ digestion demonstrated the increase in the fucosy-

lation of transferrin and haptoglobin and the presence of

oligosaccharides with a bisecting GlcNAc residue on trans-

ferrin [4, 5]. Transferrin N-glycome modifications were

confirmed after purification of the glycoprotein by immu-

noaffinity chromatography [5].

Human serum transferrin normally contains two

N-glycosylation sites. N-glycans present in a normal

subject’s transferrin mainly are the biantennary and the

triantennary fully sialylated oligosaccharides at a ratio of

85:15 and a small proportion is core fucosylated. The

presence of a bisected GlcNAc residue on the sugar chains

of transferrin has been described in HCC [18] and the

increased enzymatic activity (N-acetylglucosaminyl trans-

ferase III (GnT-III)) in serum has been observed in

liver cirrhosis and HCC [19]. The expression of GnTIII is

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Figure 2. MALDI-TOF-MS of permethy-

lated N-glycans isolated from one

serum of cirrhotic patient before (A)

and after (B) depletion of albumin/IgG/

IgA, IgGs(C) and IgAs of this patient (D).

Depletions of albumin and IgG were

performed on a HiTrapTM Blue HP

column, and on a HiTrapTM rProtein

AFF column, respectively. IgGs were

eluted from the HiTrapTM rProtein AFF

column and IgAs were purified from

this albumin/IgG-depleted serum by

affinity chromatography on Jacalin

agarose beads, methods are described

in [4, 5]. N-glycans were released from

the different glycoprotein fractions by

peptide N-glycanase F and were

permethylated before MALDI-TOF

analysis. A minor portion of the

monofucosylated glycans carries

fucose on an antenna rather than the

core. Galactose (open circles); mannose

(closed circles); GlcNAc (closed

squares); fucose (open triangles);

NeuAc (closed diamonds).



associated to the regenerative processes of liver and hepato-

carcinogenesis [20]. The remodeling of the hepatic archi-

tecture is constituted of regenerative nodules and fibrosis.

These nodules may evolve into dysplastic nodules or HCCs

[21]. The elevation of transferrin-bisected oligosaccharides

might reflect the intensity of these processes.

The core fucosylation (the addition of an a1,6 linked

fucose residue to the N-acetytlglucosamine residue linked to

the peptide backbone) increase is observed on transferrin

and on the serum N-glycome after depletion of IgG and IgA.

The increased fucosylation has been described on various

other hepatic glycoproteins during liver diseases such as

haptoglobin [22], a-fetoprotein (AFP) [23] and a1 antitrypsin

[24]. In HCC, the levels of the core fucosyltransferase (Fut8)

expression and GDP-fucose synthesis are enhanced [25] and

explain the presence of the specific AFP-L3 HCC marker.

The altered secretion of hepatic glycoproteins into bile

ducts probably is another factor of the increased core fuco-

sylation. It has been shown that a1-antitrypsin, a1 acid

glycoprotein and haptoglobin possess a higher fucosylation

in bile than in serum. Fut8 knock-out mice had a low level of

a1-antitrypsin and a1 acid glycoprotein in bile as compared

to wild-type mice. Core fucosylation would be an addressing

signal to secretion in the bile duct. In liver cirrhosis, the

tissue disorganization with the fibrosis and nodules might

be an important factor in the fucosylation modifications of

the TSNG [14, 26].

Differences of the N-glycome in the serum depleted in

IgA and IgG (Figs. 1B and 2B) in cirrhotic patients as

compared to normal subjects are minor and mainly concern

the relative increase in biantennary and triantennary fuco-

sylated oligosaccharides. Bisected N-acetylglucosamine

residues are found as traces in control subjects (Fig. 1B) and

in very low amounts in cirrhotic patients (Fig. 2B). This also

shows that the modifications of the TSNG are principally

supported by IgG and IgA.

6 Glycosidic biomarkers for the diagnosisand prognosis of liver disease

The search for biomarkers based on glycosylation modifi-

cations in patients with chronic liver diseases is important to

find a substitute for or to complement the liver biopsy for

the diagnosis of liver fibrosis. The use of these surrogate

markers is a great challenge for the monitoring of fibrosis,

grade of activity of the liver disease and for prognostic and

follow-up purposes [27].

Different glycomic biomarkers have been defined by the

group of Callewaert [2, 28]; the GlycoHepatoTest is the

combination of three markers obtained by calculating the

intensity ratios of different oligosaccharides: the Glyco-

CirrhoTest, the GlycoFibroTest and the GlycoHCCTest. The

GlycoCirrhoTest was the first to be described; it is obtained

after desialylation of the TSNG, it corresponds to the loga-

rithmic ratio of two oligosaccharidic peaks – one is a

bisected biantennary core fucosylated oligosaccharide and

the other a triantennary oligosaccharide. This marker can

distinguish compensated cirrhotic from non-cirrhotic

chronic liver disease patients but not less advanced fibrosis

stages (from F0/F1 to F3 according to the Metavir system).

The desialylated bisected fucosylated biantennary oligo-

saccharide corresponds in our work to the sum of the

intensities of two ions, the monosialylated (at m/z 2850) and

the bisialylated oligosaccharide (at m/z 3211), respectively

(Figs. 1 and 2). These bisected glycans are particularly

present on IgG and Ig A, and were found on some hepatic

synthesized glycoproteins (Fig. 2B). The triantennary non-

fucosylated structure corresponds to tri- and bisialylated

oligosaccharides; ions at m/z 3603 and 3241, respectively,

are mainly found in the Igs A- and G-depleted fraction and

in the IgA N-glycome. This biomarker reflects the increase

in Ig concentration and also the increased GnTIII activity in

the regenerative liver nodules.

The GlycoFibroTest is the ratio of the fucosylated

agalactosylated bisected glycan, corresponding to ions at

m/z 2081 in our work, and of the triantennary structure

non-fucosylated, corresponding to sialylated ions at m/z3603 and 3241. Ions at m/z 2081 are mainly found on IgG

(Figs. 1C and 2C) and are not detectable in the IgA- and G-

depleted fractions (Figs. 1B and 2B). The GlycoFibroTest

makes it possible to monitor liver fibrosis and demonstrate a

specific agalatosylation of IgG different from that encoun-

tered in Rheumatoid polyarthritis [28]. The bisected fuco-

sylated agalatosylated glycan involved in the GlycoFibroTest

combines two important effects: the agalactosylation of Ig

glycans and the increased activity of GnTIII.

HCC frequently occurs in combination with liver

cirrhosis. The search for the biomarker of early detection of

HCC is important to improve the prognosis. The

GlycoHCCTest is the third marker of the GlycoHepatoTest

[29]; it is the ratio of an a1,3 fucosylated triantennary

desialylated oligosaccharide to the bisected biantennary

fucosylated oligosaccharide. In the MALDI-TOF MS analysis

of the TSNG, the a1,3 fucosylated triantennary structure

cannot be distinguished from the a1,6 core fucosylated

triantennary structure due to their identical mass (ions at

m/z 3777). The ions at m/z 3777 are mainly found in the Igs

A- and G-depleted fraction, whereas traces are also observed

in the IgA N-glycome (Figs. 1 and 2). Another glycomic

biomarker – the increased ratio of the a1,3 fucosylated tetra-

antennary to the tetra-antennary oligosaccharide used in

combination with the platelet concentration – has been

recently described as a marker of HCC [30]. In a recent

study, the combination of the intensity of three glycans (at

m/z 2472, 3241 and 4052) detected HCC with a sensitivity of

90% and a specificity of 89% [31].

Many glycosylation changes have been described during

HCC, including the increased a1,6 fucosylation of AFP;

AFP-L3 is used for the diagnosis of HCC [14, 32]. Modifi-

cations in the activity of N-acetylglucosaminyltransferases

III and V responsible for the biosynthesis of bisected and



branched oligosaccharides, respectively, have been asso-

ciated to HCC, (reviewed in [33]). Branching and fucosyla-

tion modifications are also found during inflammation [1];

the distinction of the various possible positions of fucose

a1,6 or a1,3 linked to multiantennary glycans is an impor-

tant challenge for the improvement of the sensitivity and

specificity of HCC detection.

7 Concluding remarks

The modifications of the TSNG found during chronic liver

diseases reflect the alteration of the biosynthesis and cata-

bolism of Igs and hepatic glycoproteins. Furthermore, the

quantitative importance of Igs tends to hinder most of the

modifications found on hepatic glycoproteins. Future

studies on the glycosylation modifications encountered

during fibrosis, cirrhosis and HCC will enlighten the roles

of the glycan moiety of the various glycoproteins on the

pathophysiology.

The development of glycosylation biomarkers in liver

disease is rendered difficult by the extreme diversity of the

primary structure of glycans. Technological and analytical

improvements are still required to distinguish the subtleties

of their structures. The TSNG is the direct global approach

to the study of the glycosylation of serum glycoproteins and

provides a lot of information and numerous different

biomarkers characteristic of different clinical effects;

another trail of research is represented by biomarkers based

on a single molecule, AFP-L3, and the agalactosylation of

specific natural antibodies, leading to the development of a

simpler assay. Future explorations of glycosylation modifi-

cations will enhance the specificity and selectivity of the

marker. For comparison sake, carbohydrate deficient trans-

ferrin, the best biomarker of alcoholic consumption, is a

glycosylation biomarker and the pathophysiology is still not

understood more than 20 years after its discovery and 600

studies published.

The authors have declared no conflict of interest.

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