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Glycosylation of Serum Proteins in Inflammatory Diseases

Glycosylation of Serum Proteins in Inflammatory Diseases Disease Markers 25 (2008) 267–278 267 IOS Press Glycosylation of serum proteins in inflammatory diseases Olga Gornik and Gordan Lauc* Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia Abstract. Inflammatory diseases are accompanied by numerous changes at the site of inflammation as well as many systemic physiological and biochemical changes. In the past two decades more and more attention is being paid to changes in glycosylation and in this review we describe some of the changes found on main serum proteins (α1-acid glycoprotein, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, α2-macroglobulin, C-reactive protein, and others). Molecular background and physiological importance of most of these changes are yet to be discovered, but it is evident that glycosylation plays an important role in the inflammatory response. Maybe the greatest value of these changes currently lays in their potential diagnostic and prognostic usage, either in combination with current diagnostic markers or on their own. However, determining glycan structures is still technically too complex for most clinical laboratories and further efforts have to be made to develop simple analytical tools to study changes in glycosylation. Keywords: Glycosylation, inflammatory diseases, serum proteins, rheumatoid arthritis, systemic lupus erythematosus, pancreati- tis, sepsis 1. Introduction one of the crucial steps for the initiation of inflamma- tion [5], but this has been extensively reviewed else- where [6] and is not subject of this review. Inflammation is a complex biological response of Glycosylation is the most diverse post-translational an organism to harmful stimuli, such as pathogens, protein modification that provides numerous elaborate damaged cells, or irritants. This protective attempt ways to modulate protein function [7–9]. Oligosaccha- to remove injurious stimuli and to initiate the process ride structures are covalently bound to proteins through of healing is a part of almost all pathological condi- nitrogen atom of asparagine or oxygen atoms of serin tions [1]. Both acute and chronic inflammation are or threonin side chains, forming N-and O-linked glyco- highly complex and diverse processes and despite sig- proteins, respectively [10]. N-linked oligosaccharides nificant efforts invested in studying it, our knowledge start with N-acetylglucosamine linked to asparagine how to diagnose, understand and control inflammato- and have a glycan core made of five mannose units. ry response, is still very limited. Every inflammatory They can further differ in branching, to form oligoman- process is accompanied by numerous changes at the nose, complex or hybrid types of glycans. Oligoman- site of inflammation as well as many systemic physi- nose type has two to six extra mannoses, while com- ological and biochemical changes [2], but in the past plex type glycans have two or more branches with at two decades more and more attention is being paid to least one N-acetylglucosamine and galactose and pos- changes in glycosylation [3,4]. Actually, the interac- sibly one sialic acid on each branch. Hybrid type of tion between selectins and their glycoprotein ligands is glycans is a mixture of two types and has one branch of complex structure and one, or more, oligomannose branches (Fig. 1). Corresponding author. Faculty of Pharmacy and Biochemistry, Glycans can be present in various structural forms Ante Kovaci ˇ ca ´ 1, Zagreb, HR 10 000, Croatia. Tel.: +385 1 4818 757; Fax: +385 1 4856 201; E-mail: glauc@pharma.hr. on the same protein, at the same glycosylation site, re- ISSN 0278-0240/08/$17.00  2008 – IOS Press and the authors. All rights reserved 268 O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases Fig. 1. Examples of oligomannose (top), complex (middle) and hybrid (bottom) glycans. All branches of an oligomannose type glycan end in mannoses. Complex type glycans have two or more branches with at least one N-acetylglucosamine and galactose and possibly one sialic acid on each branch. Hybrid type of glycans is a mixture of two types and has one branch of complex structure and one, or more, oligomannose branches. sulting in different glycoforms of the same molecule. tein glycosylation offer a good basis for diagnosis and It is considered that these changes reflect the origin of prognosis of many diseases. the molecule, telling about physiological and biochem- Numerous changes in glycosylation of serum pro- ical condition of an organism at the moment of the re- teins have been reported for inflammatory diseases. lease of the exact molecule. The most important feature Most of these changes are studied in chronic inflam- of glycoproteins is their heterogeneity. It can be ex- matory conditions, while information on glycosylation pressed from minor to considerable differences through in acute inflammation is a bit modest, probably due to higher branching, loss of monosaccharides from one the fact that acute inflammation is difficult to catch in of glycan branches, through absence or presence of its starting point, since this highly dynamic and diverse certain monosaccharide such as sialic acid, fucose, N- process includes a cascade of biochemical events and acetylglucosamine or through type of linkage between involves many diverse systems. In this review we will sugars. present some of the numerous changes in glycosyla- Many diseases are associated with changes in glycan tion of selected serum proteins that were reported to be structures [11]. Since there is no genetic blueprint for associated with different inflammatory diseases. glycans, individual glycan structures vary depending on the current level of expression and intracellular local- ization of biosynthetic enzymes (glycosyltransferases 2. α1-acid glycoprotein and glycosidases). Consequently, altered glycan struc- tures are often attached to the same protein backbone α1-acid glycoprotein (AGP) is a serum acute phase as a consequence of a patophysiological processes oc- protein. Its concentration raises several fold during curring in a cell that produces the protein. These alter- an acute phase reaction like severe burns or trauma, ations can be very specific, and studies of serum pro- as well as under chronic pathological conditions like O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases 269 rheumatoid arthritis [12]. It possesses five N-linked (biantennary) glycoforms of AGP was observed in pa- complex type glycans [13,14], which may be present tients with ulcerative colitis, a chronic inflammatory as bi-, tri- and tetraantenary structures. Some of these disease of unknown etiology, even after its remission. structures may be α1,3-fucosylated to form a structure In acute inflammation the increase in biantennary struc- known as sialyl Lewis X antigen (Neu5Ac α2,3Gal tures was found to reach the maximum value in the ear- β1,4(Fuc α1,3) GlcNAc-R). ly phase of inflammation (2nd day after surgical trau- One of the most interesting features of AGP is that its ma), after which it decreased to control levels [21,27, glycosylation microheterogeneity was found to be al- 28]. Kinetics of this change in acute inflammation dif- tered in many diseases. AGP is a heavily glycosylated fers from the variation in the content of fucosylated gly- serum protein and its both binding and immunomod- cans [21]. Maximum values of fucosylation, reached ulatory functions seem to depend on its glycosylation. within the first few days, persisted even 10–30 days It represents a good model for glycosylation analysis after patients were released from the hospital. Since and is thus, together with IgG, maybe the most stud- these structures can suppress inflammation, it has been ied glycoprotein in different diseases. Among other postulated that this increase might have beneficial ef- physiological functions, AGP binds basic and neutral fects by protecting the organism from overreaction that drugs and its binding activity is influenced by ionic can occur during inflammation [29]. Since the enzyme interactions involving sialic acid and antennary struc- responsible for the addition of such fucose is α1–3 fu- tures [15]. cosyltransferase, levels of this enzyme are crucial. A Changes in biantennary structures of AGP, as well as study on AGP suggested that inflammatory cytokines α1,3 fucosylated N-glycan structures, have been report- regulate the expression of α1–3 fucosyltransferase VI ed in patients with acute inflammation [16,17] as well responsible for α1–3 fucosylation in liver tissue [17,21] as with chronic conditions, such as reumathoid arthritis as well as the expression of α2–3 sialyltransferase re- and diabetes mellitus [18,19]. First findings have main- quired for sialyl-Lewis X formation (since α2–3-linked ly been made by using lectin affinity methods through sialylation is a prerequisite for α1–3 fucosylatation). increased binding of Concavalin A (Con A) [20] and Increase in biantennary structures over tri- and tetraan- Aleuria aurantia lectin (AAL) [21] to AGP in patients tennary ones suggests the increased expression of β1,4 with inflammation. It is interesting to mention that galactosyltransferase, and the decreased expression of studies on different inflammatory diseases showed dif- N-acetylglucosaminyltransferases IV and/or V in in- ferent AGP microheterogeneity variants [12]. For in- flamed hepatocytes. stance, while a decrease in Con A reactivity was re- Work on the prognostic value of α1-acid glycopro- ported in ankylosing spondiliytis [22], Con A reactiv- tein glycosylation in septic shock [30], indicated that ity was normal in systemic lupus erythematosus [23] a modest elevation in biantennary glycans in combina- and inflammatory lung disease [24], and increased in tion with a strong increase in sialyl-Lewis X was as- acute pancreatitis [12]. However, all these findings can sociated with higher mortality than a high transient in- vary depending on the type of patients disease activity crease in biantennary glycans with gradually increasing and presence of concurrent infection. In reumathoid sialyl-Lewis X expression. This clearly demonstrates arthritis patients it was shown that the reactivity of AGP that the manner of changes in glycan structures can be with Con A correlates with disease activity [25], while associated with disease severity. a study on systemic lupus erythematosus showed an increased Con A reactivity in patients with concurrent infection [23]. Studies on chronic and acute infection 3. Immunoglobulin G showed a transition from initially elevated to decreased reactivity to Con A as disease became chronic [26]. Immunoglobulin G (IgG) is a glycoprotein with a Complete glycosylation patterns of AGP can be elu- conserved N-glycosylation site in the Fc region [31, cidated only by using more complex methods, such 32], and variable glycosylation (either O- or N-linked) as HPLC (high performance liquid chromatography), in the Fab region [32]. IgG molecules are glycosylated HPAEC (high performance anion exchange chromatog- by biantennary complex glycan structures at Asn 297 in raphy), CE (capillary electrophoresis) and MS (mass the Fc domain that can vary in the presence or absence spectrometry) techniques [17–19], and many findings of sialic acid, galactose, bisecting N-acetylglucosamine made by lectin studies have also been confirmed by or fucose (Fig. 2). Glycosylation of IgG molecules is these methods. Relative increase of Con A reactive essential for its binding to all Fcγ receptors (FcγR) 270 O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases Sia Sia been reported for this chronic inflammatory disease with autoimmune aspects in many studies using differ- α(2g 6) α(2g 6) ent analytic methods [43–45]. It was suggested that dif- ferent glycoforms of IgG may interact differently with Gal Gal rheumatoid factor auto antibody (RF) in the manner β(1g 4) β(1g 4) that IgG molecules containing less terminal galactose are preferentially recognized by IgG RF [46]. Since GlcNAc GlcNAc IgG is less galactosylated in rheumatoid arthritis, and β(1g 2) β(1g 2) glycoforms having 0, 1 or 2 galactose residues (G0, G1 GlcNAc and G2) are usually scanned for rheumatoid arthritis pa- Man Man tients, it has been suggested to include quantification of β(1g 4) G0 values in routine investigation of rheumatoid arthri- α(1g 3) α(1g 6) Man tis patients and to use G0 levels as prognostic marker for these patients. Very good agreement between dif- β(1g 4) ferent analytical methods (5 different chromatographic GlcNAc protocols and a lectin assay) for measuring G0 values β(1g 4) in rheumatoid arthritis patients [41] and the fact that a decrease in galactosylation happens in the very ear- Fuc GlcNAc α(1g 6) ly stage of rheumatoid arthritis provide further support for this suggestions. It is interesting to mention that an increase in G0 glycoforms for more than two stan- Asn (297) dard deviations was shown to have positive predictive Fig. 2. The largest biantennary complex oligosaccharide found at value of 80% for rheumatoid arthritis in an individual the Fc region of human serum IgG. There is a common pentasac- patient [47]. In the case when patient had both positive charide core containing two mannose (Man) residues attached to a rheumatoid factor and increased G0 value this predic- β-mannosyl-di-N-acetylchitobiose unit. Individual glycans can lack one or more of the monosaccharides in striped boxes (sialic acid, tive value rose up to 94%. The ratio of G0 was shown galactose, bisecting N-acetylglucosamine, or core fucose). Structure to be the best predictive marker for disease course [48] lacking both terminal galactose units is called G0 glycoform and is as well as the best marker of joint destruction [49, increased in rheumatoid arthritis and some other diseases. 50]. Similar changes have been observed in juvenile rheumatoid arthritis [51,52], although lectin study [53] through maintenance of an open conformation of the reported no difference between galactosylation of juve- two heavy chains [33], and deglycosylated IgG anti- nile rheumatoid arthritis patients and the control group bodies are unable to mediate in vivo triggered inflam- until patients were divided in a group of those having matory response [34]. One class of Fc-FcγR inter- acute phase of disease and those in remission. Patients actions generates pro-inflammatory effects of immune with currently active juvenile rheumatoid arthritis had complexes and cytotoxic antibodies [35]. On the oth- significantly lower levels of galactose than those in re- er hand, therapeutic intravenous gamma globulins and mission, in whom galactose levels were comparable to their Fc fragments are anti-inflammatory [35–38]. In- the control group. Interestingly, fucose levels in both travenous gamma globulin is a purified IgG fraction groups were significantly higher than in controls, sug- obtained from the pooled serum of healthy donors and gesting that fucosylation, and not only galactosylation is used to treat inflammatory diseases through admin- of IgG might be an interesting diagnostic marker. istration at high doses [39]. When this kind of anti- There is a great need to understand whether this al- bodies was deglycosylated, they were no longer able tered glycosylation pattern in autoimmune disorders in- to mediate anti-inflammatory activity in vivo [35]. The fluences antibody-mediated effector functions. Some same effect can be accomplished by only desialylat- ed intravenous gamma globulin, suggesting that sialic in vitro studies suggested that G0 antibodies gain acid could be the sugar that is essential for its anti- the capacity to activate the complement pathway via inflammatory activity [35]. mannose-binding lectin (MBL), which could contribute Among all inflammatory conditions, rheumatoid to antibody-mediated inflammation. Recent study in arthritis is the one where glycosylation of IgG has been mice with a genetic deletion of MBL showed that the studied the most [40–42]. Decreased sialylation and activity G0 antibodies is unimpaired in these mice [45] galactosylation of Fc fragments of IgG molecules have and is fully dependent on the presence of activating Fc O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases 271 receptors. Although this argues against the functional with decrease in galactosylation of IgG in animal mod- role of interactions between MBL and G0, it should el [61,62], as well as during pregnancy [63]. However, not be neglected that roles of individual glycans (and interleukin-6 is probably just one of the numerous fac- glycoforms) can be significantly different in mouse and tors contributing to regulation of galactosylation and, human. in general, glycosylation of IgG as well as other serum In studies on small vessel vasculitides: Wegener’s proteins. granulomatosis, microscopic polyangiitis and Churg- Strauss syndrome, that are characterized by circulating IgG of antineutrophil cytoplasmic antibodies, changes 4. Immunoglobulin A (IgA) in IgG glycosylation have been reported as well [54]. Similar to rheumatoid arthritis and juvenile rheuma- Glycosylation also plays an important role in IgA toid arthritis, these conditions are also accompanied by nephrophaty, a form of glomerulonephritis, an inflam- an increased amount of agalactosylated IgG molecules. mation of the glomeruli of the kidney characterized Since the main pathophysiological model of these dis- by deposition of the IgA antibody, especially IgA1 eases is activation of neutrophiles by cytokines within subclass, in glomeruli. IgA1 is rich in carbohy- the microvasculature, changes in IgG structure could drates, carrying N-linked moieties in common with contribute to increased activation of cytokines [54]. IgG, but also O-linked sugars, which are rare in oth- In Sjogren ¨ syndrome the same changes were ob- er serum proteins. Aberrant IgA1 O-linked glycosy- served in a certain subgroup of patients. In this sub- lation of the IgA1 hinge region is the most consistent group decreased galactosylation, as well as increased finding of all abnormalities of the IgA immune sys- level of bisecting N-acetylglucosamine were found. tem reported in IgA nephrophaty [64,65]. The de- Patients with normal IgG glycosylation profile had fect comprises reduced galactosylation of O-linked N- serologically negative RF factor and small risk for de- acetylgalactosamine residues with or without changes veloping rheumatoid arthritis, while patients with G0 in the terminal sialylation of the O-linked sugars. These IgGs and high RF titer had increased risk for rheuma- changes have great implications for the pathogenesis toid arthritis [55]. of IgA nephropathy, since O-linked sugars lie in an im- Glycosylation of IgG was also studied in many other portant functional location, close to the ligand recog- non-rheumatic diseases, mainly malignant states, but nition site of Fc receptors. Changes in the carbohy- since this is not the topic of this paper, changes found drates of IgA1 can therefore affect interactions with re- in these conditions will not be discussed here. An in- ceptors and extracellular proteins [66], and lead to IgA crease in the proportion of serum IgG molecules pos- immune complex formation and mesangial deposition, sessing an altered Fab glycosylation pattern (designat- which can cause proliferation of mesangial cells and ed asymmetric antibodies) was also reported for chron- start inflammatory response. ic parasitic diseases [56]. The study on sera of rats in which an acute inflammatory response was produced by subcutaneous inoculation of turpentine oil [56] al- 5. Transferrin so showed an alteration in the synthesis and glycosy- lation of IgG. During acute inflammation there was a Transferrin is a serum glycoprotein whose main role decrease in the synthesis of IgG which was not affected is transport of iron in blood [67]. It is a negative acute by prior oral administration of dexamethasone, howev- phase protein, meaning that its concentration decreas- er, the turpentine-induced increase in IgG binding to es during acute phase response. Decreased transfer- concanavalin A was found to be inhibited upon prior rin values can also be found in the cases of liver dis- administration of the anti-inflammatory agent. eases, chronic infections, malnutrition, protein losing It is important to stress that changes in galactosyla- enterophaty, trauma or any other severe disease. tion of IgG are also present in normal, or better to say, Transferrin is made of one polypeptide chain with physiological conditions. It is very well known that 679 amino acids and has two N-linked glycan structures IgG galactosylation is age [57,58] and sex [59] related. on asparagines 413 and 611. Its glycan structures can It also changes during pregnancy, with reversible in- be biantennary or triantennary and terminate in sialic crease in galactosylation [60]. Iterleukin-6 (IL-6) is of- acids. Changes in branching, fucosylation or sialyla- ten mentioned as one of the possible modulation factors tion of transferrin have been observed in many malig- in IgG glycosylation, since activation of IL-6 correlates nant [68,69], hereditary (galactosemia [70], CDG [71]) 272 O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases and other severe conditions [72], but in inflammatory sepsis indicated that transferrin sialylation decreases rapidly in septic patients (within first 24 hours), but diseases as well. Study on transferrin microheterogene- then normalises to the initial values in the next few ity patterns in sera of nonanemic rheumatoid arthritis days [84]. Sialylation of transferrin apparently follows patients, iron deficient rheumatoid arthritis patients and the intensity of inflammatory response and can predict patients with the anemia of chronic disease showed in- its outcome. It seems that decrease in transferrin sia- creased branching of transferrin glycans in all rheuma- lylation is a part of acute phase response in septic pa- toid arthritis groups which correlated to the disease tients and that either more expressed decrease in trans- activity (most pronounced in anemia of chronic dis- ferrin sialylation, or no decrease at all, is associated ease) [73]. Increased branching of transferrin glycans, with more severe ways of disease, such as severe sepsis together with increased sialylation, was also reported and septic shock. for ulcerative colitis patient [74] where changes in gly- Different mechanisms of transferrin desialylation cosylation of α1-antichymotripsin and α1-acid glyco- were proposed. Regarding the long transferrin half life protein were not observed. In this disease glycosyla- of 16 days [83], rapid desialylation of transferrin can tion patterns of transferrin did not differ according to be explained either by higher activity of neuraminidas- the activity index of ulcerative colitis [74]. es, or by higher clearens of highly sialylated transfer- The majority of transferrin molecules (85%) in the rin from serum [77]. Same authors speculated that circulation of healthy individuals are glycosylated with this phenomenon is specific for bacterial infection [77]. two simple biantennary glycans terminating in α2,6 However, we observed the same pattern of sialylation linked sialic acids and proper sialylation was found to changes in the early course of acute pancreatitis [84]. be important for transferrin function [75–77]. Remain- The fact that bacterial infection is not present in early ing 15% of transferrin molecules are penta- or trisialy- acute pancreatitis argues against this theory, but con- lated, while the proportion of less sialylated molecules cerning the role of transferrin as iron transporter [67], is negligible [78]. The transferrin of lowered sialylation the possible physiological role of transferrin desialyla- is called carbohydrate-deficient transferrin, and since tion in infection should not be neglected. Bacteria need its appearance reflect disturbances in glycosylation ma- iron for their metabolism and transferrin is its main chinery it is already being routinely used to diagnose source in the host. Since desialylated transferrin has chronic alcoholism [79,80] and congenital disorders of faster clearance from serum and infection is evolution- glycosylation [71]. ary the most usual trigger for inflammation, this mech- Increased serum levels of carbohydrate-deficient anism probably developed as a part of the inflammatory transferrin have also been reported in patients with response, regardless of actual presence or absence of chronic obstructive pulmonary disease, using HP- infection. LC [81]. Levels of carbohydrate-deficient transferrin found did not depend on smoking status, although the 6. Haptoglobin cigarette smoking is the main risk for chronic obstruc- tive pulmonary disease, and were in inverse correlation Haptoglobin is another serum glycoprotein whose with lung function test values, suggesting that defects glycosylation was reported to be altered in rheumatoid of glycosylation might be involved in the pathogenic arthritis [83]. Among these alterations are high levels mechanisms behind the development of this chronic of abnormally fucosylated forms found in the blood pulmonary disease [81]. of individuals with active rheumatoid arthritis. These It was reported that elevated carbohydrate-deficient molecules can be detected by using fucose-specific transferrin predicted prolonged intensive care unit lectin Lotus tetragonolobus and discriminate between stay in traumatized man [82] and major intercur- active and inactive form of disease. However, elevation rent complications were significantly increased in the in fucosylation was not found to be disease specific, high carbohydrate-deficient transferrin group (alcohol- since it was also found in sero-negative patients. Ele- withdrawal syndrome, tracheobronchitis, pneumonia, vation in haptoglobin fucosylation, together with high- pancreatitis, sepsis, congestive heart failure... ). er branching, was also observed in patients with liv- The work on transferrin sialylation in sepsis, using er diseases initiated by alcohol abuse [86,87]. Alco- capillary zone electrophoresis, reported decreased sia- hol abusers, patients with alcoholic cirrhosis and pa- lylation in septic shock patients compared to healthy tients with primary biliary cirrhosis had changed hap- individuals as well as rapid decrease in sheep model toglobin glycosylation pattern, in contrast to patients of septic shock [77]. Our recent study in patients with with chronic active hepatitis [88]. O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases 273 Table 1 Common methods for studying glycosylation changes Method Information Obtained Advantages Limitations Conclusion Lectin affinity methods presence of specific easy to perform, uses na- provide limited informa- applicable for screening structure(s) tive proteins, inexpensive tion, detects only some and preliminary tests structures Lectin affinity unspecific binding of all suitable for separating chromatography proteins with certain gly- purified proteins accord- can structure ing to glycosylation Crossed affino- great flexibility (could in- cannot handle larger immunoelectrophoresis vestigate wide range of number of samples, proteins by using different semi-quantitative prob- lectins and antibodies) lems with reproducibility Electrophoresis + protein separation and insufficient protein Western blotting + lectin detection separation lectin detection 2D electrophoresis + higher resolution of multiple steps included Western blotting + protein separation lectin detection Enzyme linked lectin fast, high throughput, limited reliability suitable for high- assay (ELLA) throughput screening Chromatographic type of oligosaccharides, expensive, require good methods presence of specific analytical skills and data monosaccharides, type interpretation and number of monosac- charides, sequence and linkages, positions of an- tennae, anomeric configurations HPLC Quantitative detailed in- requires purified gly- provides high amount of formation obtained, pos- can structures, demand- information, especially sible coupling with other ing sample preparation, in combination with exo- analytical methods glycan labelling glycosidase sequencing HPAEC-PAD no glycan labelling requires purified glycan good method for either required, quantitative structures glycan or monosaccha- ride content analysis Mass spectrometry type of oligosaccharides, no glycan labelling expensive equipment, re- presence of specific required, high sensitivity, quire good analytical monosaccharide, type high amount of data, skills and knowledge for and number of reliable data interpretation, limit- monosaccharides, ed quantification sequence and linkages, antennae positions, anomeric configurations 7. α2-Macroglobulin ed that the concentration of total monosaccharides on alpha 2-macroglobulin purified from serum samples Using lectin blots in conjunction with peptide map- of systemic lupus erythematosus patients was signif- ping, alpha 2-macroglobulin purified from systemic lu- icantly higher than in normal donors [89]. In the pus erythematosus patients was shown to be abnor- same study was also examined if there are any cor- mally glycosylated, suggesting the occurrence of com- relations between the levels of mannose and glucose plex glycosylation in this pathological condition [89]. on alpha 2-macroglobulin and systemic lupus erythe- When serum samples of normal donors, systemic lu- matosus. The concentrations of mannose and galac- pus erythematosus patients, rheumatoid arthritis pa- tose on alpha 2-macroglobulin from systemic lupus tients, a mixed connective tissue disease patient and a erythematosus patients were significantly higher than Sjogren’s syndrome patient were analysed for carbo- from normal donors, in contrast to concentration of hydrate content of alpha 2-macroglobulin it was not- glucose where no difference was found between exam- 274 O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases ined groups. These results suggested that quantifica- mal model of septic shock [77]. The same study also tion of carbohydrates in selected glycoproteins, such as showed the increase in sialylation of total serum pro- alpha 2-macroglobulin, may be a novel and alternative teins. While following up sialylation of transferrin and clinical marker for systemic lupus erythematosus. De- total serum proteins during sepsis and acute pancreati- velopment of an enzyme-linked immunosorbent assay tis we also observed an increase in sialylation of total (ELISA) using a monoclonal antibody directed against serum proteins that occurred at the early course of dis- abnormally glycosylated serum alpha2-macroglobulin ease, followed by normalization afterwards. The most which was capable of recognizing changes of glyco- probable explanation for this initial increase is the in- sylation in systemic lupus erythematosus patients con- creased production of highly sialylated acute phase pro- tributed further to this findings and may be useful in teins such as α1-acid glycoprotein [2,93]. Further in- differential diagnosis [90]. vestigation on sialylation of plasma proteins in inflamed mice showed that the increase in sialylation involves all types of sialic acids (α2,3, α2,6 and α2,8) [94]. 8. C-reactive protein Beside the increase in total sialylation, when acute phase response was stimulated in mice by turpentine Although C-reactive protein (CRP) is the “most pop- oil injection, a decrease in total fucosylation was also ular" and the oldest molecular marker of acute phase reported to occur as an early event in the acute phase response in both acute and chronic inflammation, work response [95]. α1-acid glycoprotein (a positive acute on glycosylation of this serum protein is very scarce. phase protein), α1-macroglobulin (a non acute phase CRP is a pentraxin, calcium binding protein, whose val- protein) and α1-inhibitor3 (a negative acute phase pro- ue rises up to 100 times in the first 24 hours as response tein) showed similar alterations in sialylation and fuco- to initial stimulus, especially bacterial infection. It has sylation in this case, in contrast to α2-macroglobulin been reported that CRPs show variation in both their that contained no significant amount of fucose during amino acid sequences and glycosylation patterns in dif- acute phase response [95]. These studies demonstrated ferent pathological conditions [91]. These changes in that changes mentioned happen on pre-existing plasma tryptophan contents, together with glycosylation and proteins (since they occur too rapidly for new protein specific sialylation changes play a contributory role synthesis to have visible effects) and that they also in- in their binding characteristics (e.g. to antibodies, and volve non-acute phase proteins. Secretion of sialyl- other plasma proteins) [92]. transferases and fucosidases in plasma, reversible en- Beside the difference in sialic acid content, it’s dif- docytosis of proteins or the action of membrane bound ferent linkage was found in different diseases, by us- enzymes are only some of the possible mechanisms that ing Sambucus nigra agglutinin (recognizes α2,6 bound could explain the observed effects. sialic acid) and Maackia amurensis (recognizes α2,3 Changes of glycosylation of serum proteins have al- bound sialic acid) lectins. CRPs from Visceral Leish- so been detected in psoriatic arthritis where good cor- manisis, tuberculosis and Systemic Lupus Erythemato- relation was observed between total Con A reactivity sus contain α2,3 linked sialic acids, while proteins of serum and serum levels of CRP and IL-6 [96], which from some other diseases which are not of inflamma- was discussed before as a putative regulator of glycosy- tory character have α2,6 linked sialic acids [92]. Thus lation pattern of proteins upon inflammation. Increased routine use of quantifying CRP may be further supple- reactivity to Con A for two serum proteins, AGP and mented with determination of qualitative alterations of antichymotripsin, was also detected in acute phase re- this protein to obtain a unique marker for diagnosis and sponse after hip arthroplasty, with no correlation with monitoring of an acute phase response in inflammation. protein concentrations [97]. Due to development of modern and sensitive tech- niques for studying glycan structures, the modern ap- 9. Other and total serum proteins proach to study serum protein glycosylation patterns includes first the screening of total serum glycans and Many studies done on protein glycosylation in in- then the identification of exact serum proteins responsi- flammation involved total serum proteins, or combined ble for glycan structures involved in changes observed. the glycosylation patterns of few of them. As men- This approach result in detailed information on gly- tioned before, changes in sialylation of transferrin have can structures present in the serum sample and, al- been observed in septic patients as well as in ani- though it is still time consuming, reveals many differ- O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases 275 ences. High performance liquid chromatography meth- Acknowledgements ods [98–100] and mass spectrometric methods [99,101] Work in author’s laboratory is supported by grants are techniques mostly used for this approach and are of- #219-0061194-2023 and #006-0061194-1218 from the ten further supplemented with different lectin methods Croatian Ministry of Science, Education and Sport, and (Table 1). by EuroPharm grant from the European Commission. 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Glycosylation of Serum Proteins in Inflammatory Diseases

Disease markers , Volume 25 (4-5) – Jan 6, 2009

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Pubmed Central
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Copyright © 2008 Hindawi Publishing Corporation.
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0278-0240
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1875-8630
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10.1155/2008/493289
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Abstract

Disease Markers 25 (2008) 267–278 267 IOS Press Glycosylation of serum proteins in inflammatory diseases Olga Gornik and Gordan Lauc* Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia Abstract. Inflammatory diseases are accompanied by numerous changes at the site of inflammation as well as many systemic physiological and biochemical changes. In the past two decades more and more attention is being paid to changes in glycosylation and in this review we describe some of the changes found on main serum proteins (α1-acid glycoprotein, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, α2-macroglobulin, C-reactive protein, and others). Molecular background and physiological importance of most of these changes are yet to be discovered, but it is evident that glycosylation plays an important role in the inflammatory response. Maybe the greatest value of these changes currently lays in their potential diagnostic and prognostic usage, either in combination with current diagnostic markers or on their own. However, determining glycan structures is still technically too complex for most clinical laboratories and further efforts have to be made to develop simple analytical tools to study changes in glycosylation. Keywords: Glycosylation, inflammatory diseases, serum proteins, rheumatoid arthritis, systemic lupus erythematosus, pancreati- tis, sepsis 1. Introduction one of the crucial steps for the initiation of inflamma- tion [5], but this has been extensively reviewed else- where [6] and is not subject of this review. Inflammation is a complex biological response of Glycosylation is the most diverse post-translational an organism to harmful stimuli, such as pathogens, protein modification that provides numerous elaborate damaged cells, or irritants. This protective attempt ways to modulate protein function [7–9]. Oligosaccha- to remove injurious stimuli and to initiate the process ride structures are covalently bound to proteins through of healing is a part of almost all pathological condi- nitrogen atom of asparagine or oxygen atoms of serin tions [1]. Both acute and chronic inflammation are or threonin side chains, forming N-and O-linked glyco- highly complex and diverse processes and despite sig- proteins, respectively [10]. N-linked oligosaccharides nificant efforts invested in studying it, our knowledge start with N-acetylglucosamine linked to asparagine how to diagnose, understand and control inflammato- and have a glycan core made of five mannose units. ry response, is still very limited. Every inflammatory They can further differ in branching, to form oligoman- process is accompanied by numerous changes at the nose, complex or hybrid types of glycans. Oligoman- site of inflammation as well as many systemic physi- nose type has two to six extra mannoses, while com- ological and biochemical changes [2], but in the past plex type glycans have two or more branches with at two decades more and more attention is being paid to least one N-acetylglucosamine and galactose and pos- changes in glycosylation [3,4]. Actually, the interac- sibly one sialic acid on each branch. Hybrid type of tion between selectins and their glycoprotein ligands is glycans is a mixture of two types and has one branch of complex structure and one, or more, oligomannose branches (Fig. 1). Corresponding author. Faculty of Pharmacy and Biochemistry, Glycans can be present in various structural forms Ante Kovaci ˇ ca ´ 1, Zagreb, HR 10 000, Croatia. Tel.: +385 1 4818 757; Fax: +385 1 4856 201; E-mail: glauc@pharma.hr. on the same protein, at the same glycosylation site, re- ISSN 0278-0240/08/$17.00  2008 – IOS Press and the authors. All rights reserved 268 O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases Fig. 1. Examples of oligomannose (top), complex (middle) and hybrid (bottom) glycans. All branches of an oligomannose type glycan end in mannoses. Complex type glycans have two or more branches with at least one N-acetylglucosamine and galactose and possibly one sialic acid on each branch. Hybrid type of glycans is a mixture of two types and has one branch of complex structure and one, or more, oligomannose branches. sulting in different glycoforms of the same molecule. tein glycosylation offer a good basis for diagnosis and It is considered that these changes reflect the origin of prognosis of many diseases. the molecule, telling about physiological and biochem- Numerous changes in glycosylation of serum pro- ical condition of an organism at the moment of the re- teins have been reported for inflammatory diseases. lease of the exact molecule. The most important feature Most of these changes are studied in chronic inflam- of glycoproteins is their heterogeneity. It can be ex- matory conditions, while information on glycosylation pressed from minor to considerable differences through in acute inflammation is a bit modest, probably due to higher branching, loss of monosaccharides from one the fact that acute inflammation is difficult to catch in of glycan branches, through absence or presence of its starting point, since this highly dynamic and diverse certain monosaccharide such as sialic acid, fucose, N- process includes a cascade of biochemical events and acetylglucosamine or through type of linkage between involves many diverse systems. In this review we will sugars. present some of the numerous changes in glycosyla- Many diseases are associated with changes in glycan tion of selected serum proteins that were reported to be structures [11]. Since there is no genetic blueprint for associated with different inflammatory diseases. glycans, individual glycan structures vary depending on the current level of expression and intracellular local- ization of biosynthetic enzymes (glycosyltransferases 2. α1-acid glycoprotein and glycosidases). Consequently, altered glycan struc- tures are often attached to the same protein backbone α1-acid glycoprotein (AGP) is a serum acute phase as a consequence of a patophysiological processes oc- protein. Its concentration raises several fold during curring in a cell that produces the protein. These alter- an acute phase reaction like severe burns or trauma, ations can be very specific, and studies of serum pro- as well as under chronic pathological conditions like O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases 269 rheumatoid arthritis [12]. It possesses five N-linked (biantennary) glycoforms of AGP was observed in pa- complex type glycans [13,14], which may be present tients with ulcerative colitis, a chronic inflammatory as bi-, tri- and tetraantenary structures. Some of these disease of unknown etiology, even after its remission. structures may be α1,3-fucosylated to form a structure In acute inflammation the increase in biantennary struc- known as sialyl Lewis X antigen (Neu5Ac α2,3Gal tures was found to reach the maximum value in the ear- β1,4(Fuc α1,3) GlcNAc-R). ly phase of inflammation (2nd day after surgical trau- One of the most interesting features of AGP is that its ma), after which it decreased to control levels [21,27, glycosylation microheterogeneity was found to be al- 28]. Kinetics of this change in acute inflammation dif- tered in many diseases. AGP is a heavily glycosylated fers from the variation in the content of fucosylated gly- serum protein and its both binding and immunomod- cans [21]. Maximum values of fucosylation, reached ulatory functions seem to depend on its glycosylation. within the first few days, persisted even 10–30 days It represents a good model for glycosylation analysis after patients were released from the hospital. Since and is thus, together with IgG, maybe the most stud- these structures can suppress inflammation, it has been ied glycoprotein in different diseases. Among other postulated that this increase might have beneficial ef- physiological functions, AGP binds basic and neutral fects by protecting the organism from overreaction that drugs and its binding activity is influenced by ionic can occur during inflammation [29]. Since the enzyme interactions involving sialic acid and antennary struc- responsible for the addition of such fucose is α1–3 fu- tures [15]. cosyltransferase, levels of this enzyme are crucial. A Changes in biantennary structures of AGP, as well as study on AGP suggested that inflammatory cytokines α1,3 fucosylated N-glycan structures, have been report- regulate the expression of α1–3 fucosyltransferase VI ed in patients with acute inflammation [16,17] as well responsible for α1–3 fucosylation in liver tissue [17,21] as with chronic conditions, such as reumathoid arthritis as well as the expression of α2–3 sialyltransferase re- and diabetes mellitus [18,19]. First findings have main- quired for sialyl-Lewis X formation (since α2–3-linked ly been made by using lectin affinity methods through sialylation is a prerequisite for α1–3 fucosylatation). increased binding of Concavalin A (Con A) [20] and Increase in biantennary structures over tri- and tetraan- Aleuria aurantia lectin (AAL) [21] to AGP in patients tennary ones suggests the increased expression of β1,4 with inflammation. It is interesting to mention that galactosyltransferase, and the decreased expression of studies on different inflammatory diseases showed dif- N-acetylglucosaminyltransferases IV and/or V in in- ferent AGP microheterogeneity variants [12]. For in- flamed hepatocytes. stance, while a decrease in Con A reactivity was re- Work on the prognostic value of α1-acid glycopro- ported in ankylosing spondiliytis [22], Con A reactiv- tein glycosylation in septic shock [30], indicated that ity was normal in systemic lupus erythematosus [23] a modest elevation in biantennary glycans in combina- and inflammatory lung disease [24], and increased in tion with a strong increase in sialyl-Lewis X was as- acute pancreatitis [12]. However, all these findings can sociated with higher mortality than a high transient in- vary depending on the type of patients disease activity crease in biantennary glycans with gradually increasing and presence of concurrent infection. In reumathoid sialyl-Lewis X expression. This clearly demonstrates arthritis patients it was shown that the reactivity of AGP that the manner of changes in glycan structures can be with Con A correlates with disease activity [25], while associated with disease severity. a study on systemic lupus erythematosus showed an increased Con A reactivity in patients with concurrent infection [23]. Studies on chronic and acute infection 3. Immunoglobulin G showed a transition from initially elevated to decreased reactivity to Con A as disease became chronic [26]. Immunoglobulin G (IgG) is a glycoprotein with a Complete glycosylation patterns of AGP can be elu- conserved N-glycosylation site in the Fc region [31, cidated only by using more complex methods, such 32], and variable glycosylation (either O- or N-linked) as HPLC (high performance liquid chromatography), in the Fab region [32]. IgG molecules are glycosylated HPAEC (high performance anion exchange chromatog- by biantennary complex glycan structures at Asn 297 in raphy), CE (capillary electrophoresis) and MS (mass the Fc domain that can vary in the presence or absence spectrometry) techniques [17–19], and many findings of sialic acid, galactose, bisecting N-acetylglucosamine made by lectin studies have also been confirmed by or fucose (Fig. 2). Glycosylation of IgG molecules is these methods. Relative increase of Con A reactive essential for its binding to all Fcγ receptors (FcγR) 270 O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases Sia Sia been reported for this chronic inflammatory disease with autoimmune aspects in many studies using differ- α(2g 6) α(2g 6) ent analytic methods [43–45]. It was suggested that dif- ferent glycoforms of IgG may interact differently with Gal Gal rheumatoid factor auto antibody (RF) in the manner β(1g 4) β(1g 4) that IgG molecules containing less terminal galactose are preferentially recognized by IgG RF [46]. Since GlcNAc GlcNAc IgG is less galactosylated in rheumatoid arthritis, and β(1g 2) β(1g 2) glycoforms having 0, 1 or 2 galactose residues (G0, G1 GlcNAc and G2) are usually scanned for rheumatoid arthritis pa- Man Man tients, it has been suggested to include quantification of β(1g 4) G0 values in routine investigation of rheumatoid arthri- α(1g 3) α(1g 6) Man tis patients and to use G0 levels as prognostic marker for these patients. Very good agreement between dif- β(1g 4) ferent analytical methods (5 different chromatographic GlcNAc protocols and a lectin assay) for measuring G0 values β(1g 4) in rheumatoid arthritis patients [41] and the fact that a decrease in galactosylation happens in the very ear- Fuc GlcNAc α(1g 6) ly stage of rheumatoid arthritis provide further support for this suggestions. It is interesting to mention that an increase in G0 glycoforms for more than two stan- Asn (297) dard deviations was shown to have positive predictive Fig. 2. The largest biantennary complex oligosaccharide found at value of 80% for rheumatoid arthritis in an individual the Fc region of human serum IgG. There is a common pentasac- patient [47]. In the case when patient had both positive charide core containing two mannose (Man) residues attached to a rheumatoid factor and increased G0 value this predic- β-mannosyl-di-N-acetylchitobiose unit. Individual glycans can lack one or more of the monosaccharides in striped boxes (sialic acid, tive value rose up to 94%. The ratio of G0 was shown galactose, bisecting N-acetylglucosamine, or core fucose). Structure to be the best predictive marker for disease course [48] lacking both terminal galactose units is called G0 glycoform and is as well as the best marker of joint destruction [49, increased in rheumatoid arthritis and some other diseases. 50]. Similar changes have been observed in juvenile rheumatoid arthritis [51,52], although lectin study [53] through maintenance of an open conformation of the reported no difference between galactosylation of juve- two heavy chains [33], and deglycosylated IgG anti- nile rheumatoid arthritis patients and the control group bodies are unable to mediate in vivo triggered inflam- until patients were divided in a group of those having matory response [34]. One class of Fc-FcγR inter- acute phase of disease and those in remission. Patients actions generates pro-inflammatory effects of immune with currently active juvenile rheumatoid arthritis had complexes and cytotoxic antibodies [35]. On the oth- significantly lower levels of galactose than those in re- er hand, therapeutic intravenous gamma globulins and mission, in whom galactose levels were comparable to their Fc fragments are anti-inflammatory [35–38]. In- the control group. Interestingly, fucose levels in both travenous gamma globulin is a purified IgG fraction groups were significantly higher than in controls, sug- obtained from the pooled serum of healthy donors and gesting that fucosylation, and not only galactosylation is used to treat inflammatory diseases through admin- of IgG might be an interesting diagnostic marker. istration at high doses [39]. When this kind of anti- There is a great need to understand whether this al- bodies was deglycosylated, they were no longer able tered glycosylation pattern in autoimmune disorders in- to mediate anti-inflammatory activity in vivo [35]. The fluences antibody-mediated effector functions. Some same effect can be accomplished by only desialylat- ed intravenous gamma globulin, suggesting that sialic in vitro studies suggested that G0 antibodies gain acid could be the sugar that is essential for its anti- the capacity to activate the complement pathway via inflammatory activity [35]. mannose-binding lectin (MBL), which could contribute Among all inflammatory conditions, rheumatoid to antibody-mediated inflammation. Recent study in arthritis is the one where glycosylation of IgG has been mice with a genetic deletion of MBL showed that the studied the most [40–42]. Decreased sialylation and activity G0 antibodies is unimpaired in these mice [45] galactosylation of Fc fragments of IgG molecules have and is fully dependent on the presence of activating Fc O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases 271 receptors. Although this argues against the functional with decrease in galactosylation of IgG in animal mod- role of interactions between MBL and G0, it should el [61,62], as well as during pregnancy [63]. However, not be neglected that roles of individual glycans (and interleukin-6 is probably just one of the numerous fac- glycoforms) can be significantly different in mouse and tors contributing to regulation of galactosylation and, human. in general, glycosylation of IgG as well as other serum In studies on small vessel vasculitides: Wegener’s proteins. granulomatosis, microscopic polyangiitis and Churg- Strauss syndrome, that are characterized by circulating IgG of antineutrophil cytoplasmic antibodies, changes 4. Immunoglobulin A (IgA) in IgG glycosylation have been reported as well [54]. Similar to rheumatoid arthritis and juvenile rheuma- Glycosylation also plays an important role in IgA toid arthritis, these conditions are also accompanied by nephrophaty, a form of glomerulonephritis, an inflam- an increased amount of agalactosylated IgG molecules. mation of the glomeruli of the kidney characterized Since the main pathophysiological model of these dis- by deposition of the IgA antibody, especially IgA1 eases is activation of neutrophiles by cytokines within subclass, in glomeruli. IgA1 is rich in carbohy- the microvasculature, changes in IgG structure could drates, carrying N-linked moieties in common with contribute to increased activation of cytokines [54]. IgG, but also O-linked sugars, which are rare in oth- In Sjogren ¨ syndrome the same changes were ob- er serum proteins. Aberrant IgA1 O-linked glycosy- served in a certain subgroup of patients. In this sub- lation of the IgA1 hinge region is the most consistent group decreased galactosylation, as well as increased finding of all abnormalities of the IgA immune sys- level of bisecting N-acetylglucosamine were found. tem reported in IgA nephrophaty [64,65]. The de- Patients with normal IgG glycosylation profile had fect comprises reduced galactosylation of O-linked N- serologically negative RF factor and small risk for de- acetylgalactosamine residues with or without changes veloping rheumatoid arthritis, while patients with G0 in the terminal sialylation of the O-linked sugars. These IgGs and high RF titer had increased risk for rheuma- changes have great implications for the pathogenesis toid arthritis [55]. of IgA nephropathy, since O-linked sugars lie in an im- Glycosylation of IgG was also studied in many other portant functional location, close to the ligand recog- non-rheumatic diseases, mainly malignant states, but nition site of Fc receptors. Changes in the carbohy- since this is not the topic of this paper, changes found drates of IgA1 can therefore affect interactions with re- in these conditions will not be discussed here. An in- ceptors and extracellular proteins [66], and lead to IgA crease in the proportion of serum IgG molecules pos- immune complex formation and mesangial deposition, sessing an altered Fab glycosylation pattern (designat- which can cause proliferation of mesangial cells and ed asymmetric antibodies) was also reported for chron- start inflammatory response. ic parasitic diseases [56]. The study on sera of rats in which an acute inflammatory response was produced by subcutaneous inoculation of turpentine oil [56] al- 5. Transferrin so showed an alteration in the synthesis and glycosy- lation of IgG. During acute inflammation there was a Transferrin is a serum glycoprotein whose main role decrease in the synthesis of IgG which was not affected is transport of iron in blood [67]. It is a negative acute by prior oral administration of dexamethasone, howev- phase protein, meaning that its concentration decreas- er, the turpentine-induced increase in IgG binding to es during acute phase response. Decreased transfer- concanavalin A was found to be inhibited upon prior rin values can also be found in the cases of liver dis- administration of the anti-inflammatory agent. eases, chronic infections, malnutrition, protein losing It is important to stress that changes in galactosyla- enterophaty, trauma or any other severe disease. tion of IgG are also present in normal, or better to say, Transferrin is made of one polypeptide chain with physiological conditions. It is very well known that 679 amino acids and has two N-linked glycan structures IgG galactosylation is age [57,58] and sex [59] related. on asparagines 413 and 611. Its glycan structures can It also changes during pregnancy, with reversible in- be biantennary or triantennary and terminate in sialic crease in galactosylation [60]. Iterleukin-6 (IL-6) is of- acids. Changes in branching, fucosylation or sialyla- ten mentioned as one of the possible modulation factors tion of transferrin have been observed in many malig- in IgG glycosylation, since activation of IL-6 correlates nant [68,69], hereditary (galactosemia [70], CDG [71]) 272 O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases and other severe conditions [72], but in inflammatory sepsis indicated that transferrin sialylation decreases rapidly in septic patients (within first 24 hours), but diseases as well. Study on transferrin microheterogene- then normalises to the initial values in the next few ity patterns in sera of nonanemic rheumatoid arthritis days [84]. Sialylation of transferrin apparently follows patients, iron deficient rheumatoid arthritis patients and the intensity of inflammatory response and can predict patients with the anemia of chronic disease showed in- its outcome. It seems that decrease in transferrin sia- creased branching of transferrin glycans in all rheuma- lylation is a part of acute phase response in septic pa- toid arthritis groups which correlated to the disease tients and that either more expressed decrease in trans- activity (most pronounced in anemia of chronic dis- ferrin sialylation, or no decrease at all, is associated ease) [73]. Increased branching of transferrin glycans, with more severe ways of disease, such as severe sepsis together with increased sialylation, was also reported and septic shock. for ulcerative colitis patient [74] where changes in gly- Different mechanisms of transferrin desialylation cosylation of α1-antichymotripsin and α1-acid glyco- were proposed. Regarding the long transferrin half life protein were not observed. In this disease glycosyla- of 16 days [83], rapid desialylation of transferrin can tion patterns of transferrin did not differ according to be explained either by higher activity of neuraminidas- the activity index of ulcerative colitis [74]. es, or by higher clearens of highly sialylated transfer- The majority of transferrin molecules (85%) in the rin from serum [77]. Same authors speculated that circulation of healthy individuals are glycosylated with this phenomenon is specific for bacterial infection [77]. two simple biantennary glycans terminating in α2,6 However, we observed the same pattern of sialylation linked sialic acids and proper sialylation was found to changes in the early course of acute pancreatitis [84]. be important for transferrin function [75–77]. Remain- The fact that bacterial infection is not present in early ing 15% of transferrin molecules are penta- or trisialy- acute pancreatitis argues against this theory, but con- lated, while the proportion of less sialylated molecules cerning the role of transferrin as iron transporter [67], is negligible [78]. The transferrin of lowered sialylation the possible physiological role of transferrin desialyla- is called carbohydrate-deficient transferrin, and since tion in infection should not be neglected. Bacteria need its appearance reflect disturbances in glycosylation ma- iron for their metabolism and transferrin is its main chinery it is already being routinely used to diagnose source in the host. Since desialylated transferrin has chronic alcoholism [79,80] and congenital disorders of faster clearance from serum and infection is evolution- glycosylation [71]. ary the most usual trigger for inflammation, this mech- Increased serum levels of carbohydrate-deficient anism probably developed as a part of the inflammatory transferrin have also been reported in patients with response, regardless of actual presence or absence of chronic obstructive pulmonary disease, using HP- infection. LC [81]. Levels of carbohydrate-deficient transferrin found did not depend on smoking status, although the 6. Haptoglobin cigarette smoking is the main risk for chronic obstruc- tive pulmonary disease, and were in inverse correlation Haptoglobin is another serum glycoprotein whose with lung function test values, suggesting that defects glycosylation was reported to be altered in rheumatoid of glycosylation might be involved in the pathogenic arthritis [83]. Among these alterations are high levels mechanisms behind the development of this chronic of abnormally fucosylated forms found in the blood pulmonary disease [81]. of individuals with active rheumatoid arthritis. These It was reported that elevated carbohydrate-deficient molecules can be detected by using fucose-specific transferrin predicted prolonged intensive care unit lectin Lotus tetragonolobus and discriminate between stay in traumatized man [82] and major intercur- active and inactive form of disease. However, elevation rent complications were significantly increased in the in fucosylation was not found to be disease specific, high carbohydrate-deficient transferrin group (alcohol- since it was also found in sero-negative patients. Ele- withdrawal syndrome, tracheobronchitis, pneumonia, vation in haptoglobin fucosylation, together with high- pancreatitis, sepsis, congestive heart failure... ). er branching, was also observed in patients with liv- The work on transferrin sialylation in sepsis, using er diseases initiated by alcohol abuse [86,87]. Alco- capillary zone electrophoresis, reported decreased sia- hol abusers, patients with alcoholic cirrhosis and pa- lylation in septic shock patients compared to healthy tients with primary biliary cirrhosis had changed hap- individuals as well as rapid decrease in sheep model toglobin glycosylation pattern, in contrast to patients of septic shock [77]. Our recent study in patients with with chronic active hepatitis [88]. O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases 273 Table 1 Common methods for studying glycosylation changes Method Information Obtained Advantages Limitations Conclusion Lectin affinity methods presence of specific easy to perform, uses na- provide limited informa- applicable for screening structure(s) tive proteins, inexpensive tion, detects only some and preliminary tests structures Lectin affinity unspecific binding of all suitable for separating chromatography proteins with certain gly- purified proteins accord- can structure ing to glycosylation Crossed affino- great flexibility (could in- cannot handle larger immunoelectrophoresis vestigate wide range of number of samples, proteins by using different semi-quantitative prob- lectins and antibodies) lems with reproducibility Electrophoresis + protein separation and insufficient protein Western blotting + lectin detection separation lectin detection 2D electrophoresis + higher resolution of multiple steps included Western blotting + protein separation lectin detection Enzyme linked lectin fast, high throughput, limited reliability suitable for high- assay (ELLA) throughput screening Chromatographic type of oligosaccharides, expensive, require good methods presence of specific analytical skills and data monosaccharides, type interpretation and number of monosac- charides, sequence and linkages, positions of an- tennae, anomeric configurations HPLC Quantitative detailed in- requires purified gly- provides high amount of formation obtained, pos- can structures, demand- information, especially sible coupling with other ing sample preparation, in combination with exo- analytical methods glycan labelling glycosidase sequencing HPAEC-PAD no glycan labelling requires purified glycan good method for either required, quantitative structures glycan or monosaccha- ride content analysis Mass spectrometry type of oligosaccharides, no glycan labelling expensive equipment, re- presence of specific required, high sensitivity, quire good analytical monosaccharide, type high amount of data, skills and knowledge for and number of reliable data interpretation, limit- monosaccharides, ed quantification sequence and linkages, antennae positions, anomeric configurations 7. α2-Macroglobulin ed that the concentration of total monosaccharides on alpha 2-macroglobulin purified from serum samples Using lectin blots in conjunction with peptide map- of systemic lupus erythematosus patients was signif- ping, alpha 2-macroglobulin purified from systemic lu- icantly higher than in normal donors [89]. In the pus erythematosus patients was shown to be abnor- same study was also examined if there are any cor- mally glycosylated, suggesting the occurrence of com- relations between the levels of mannose and glucose plex glycosylation in this pathological condition [89]. on alpha 2-macroglobulin and systemic lupus erythe- When serum samples of normal donors, systemic lu- matosus. The concentrations of mannose and galac- pus erythematosus patients, rheumatoid arthritis pa- tose on alpha 2-macroglobulin from systemic lupus tients, a mixed connective tissue disease patient and a erythematosus patients were significantly higher than Sjogren’s syndrome patient were analysed for carbo- from normal donors, in contrast to concentration of hydrate content of alpha 2-macroglobulin it was not- glucose where no difference was found between exam- 274 O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases ined groups. These results suggested that quantifica- mal model of septic shock [77]. The same study also tion of carbohydrates in selected glycoproteins, such as showed the increase in sialylation of total serum pro- alpha 2-macroglobulin, may be a novel and alternative teins. While following up sialylation of transferrin and clinical marker for systemic lupus erythematosus. De- total serum proteins during sepsis and acute pancreati- velopment of an enzyme-linked immunosorbent assay tis we also observed an increase in sialylation of total (ELISA) using a monoclonal antibody directed against serum proteins that occurred at the early course of dis- abnormally glycosylated serum alpha2-macroglobulin ease, followed by normalization afterwards. The most which was capable of recognizing changes of glyco- probable explanation for this initial increase is the in- sylation in systemic lupus erythematosus patients con- creased production of highly sialylated acute phase pro- tributed further to this findings and may be useful in teins such as α1-acid glycoprotein [2,93]. Further in- differential diagnosis [90]. vestigation on sialylation of plasma proteins in inflamed mice showed that the increase in sialylation involves all types of sialic acids (α2,3, α2,6 and α2,8) [94]. 8. C-reactive protein Beside the increase in total sialylation, when acute phase response was stimulated in mice by turpentine Although C-reactive protein (CRP) is the “most pop- oil injection, a decrease in total fucosylation was also ular" and the oldest molecular marker of acute phase reported to occur as an early event in the acute phase response in both acute and chronic inflammation, work response [95]. α1-acid glycoprotein (a positive acute on glycosylation of this serum protein is very scarce. phase protein), α1-macroglobulin (a non acute phase CRP is a pentraxin, calcium binding protein, whose val- protein) and α1-inhibitor3 (a negative acute phase pro- ue rises up to 100 times in the first 24 hours as response tein) showed similar alterations in sialylation and fuco- to initial stimulus, especially bacterial infection. It has sylation in this case, in contrast to α2-macroglobulin been reported that CRPs show variation in both their that contained no significant amount of fucose during amino acid sequences and glycosylation patterns in dif- acute phase response [95]. These studies demonstrated ferent pathological conditions [91]. These changes in that changes mentioned happen on pre-existing plasma tryptophan contents, together with glycosylation and proteins (since they occur too rapidly for new protein specific sialylation changes play a contributory role synthesis to have visible effects) and that they also in- in their binding characteristics (e.g. to antibodies, and volve non-acute phase proteins. Secretion of sialyl- other plasma proteins) [92]. transferases and fucosidases in plasma, reversible en- Beside the difference in sialic acid content, it’s dif- docytosis of proteins or the action of membrane bound ferent linkage was found in different diseases, by us- enzymes are only some of the possible mechanisms that ing Sambucus nigra agglutinin (recognizes α2,6 bound could explain the observed effects. sialic acid) and Maackia amurensis (recognizes α2,3 Changes of glycosylation of serum proteins have al- bound sialic acid) lectins. CRPs from Visceral Leish- so been detected in psoriatic arthritis where good cor- manisis, tuberculosis and Systemic Lupus Erythemato- relation was observed between total Con A reactivity sus contain α2,3 linked sialic acids, while proteins of serum and serum levels of CRP and IL-6 [96], which from some other diseases which are not of inflamma- was discussed before as a putative regulator of glycosy- tory character have α2,6 linked sialic acids [92]. Thus lation pattern of proteins upon inflammation. Increased routine use of quantifying CRP may be further supple- reactivity to Con A for two serum proteins, AGP and mented with determination of qualitative alterations of antichymotripsin, was also detected in acute phase re- this protein to obtain a unique marker for diagnosis and sponse after hip arthroplasty, with no correlation with monitoring of an acute phase response in inflammation. protein concentrations [97]. Due to development of modern and sensitive tech- niques for studying glycan structures, the modern ap- 9. Other and total serum proteins proach to study serum protein glycosylation patterns includes first the screening of total serum glycans and Many studies done on protein glycosylation in in- then the identification of exact serum proteins responsi- flammation involved total serum proteins, or combined ble for glycan structures involved in changes observed. the glycosylation patterns of few of them. As men- This approach result in detailed information on gly- tioned before, changes in sialylation of transferrin have can structures present in the serum sample and, al- been observed in septic patients as well as in ani- though it is still time consuming, reveals many differ- O. Gornik and G. Lauc / Glycosylation of serum proteins in inflammatory diseases 275 ences. High performance liquid chromatography meth- Acknowledgements ods [98–100] and mass spectrometric methods [99,101] Work in author’s laboratory is supported by grants are techniques mostly used for this approach and are of- #219-0061194-2023 and #006-0061194-1218 from the ten further supplemented with different lectin methods Croatian Ministry of Science, Education and Sport, and (Table 1). by EuroPharm grant from the European Commission. 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