IgG Sialylation Research Articles

Sialylation Determines the Nephritogenicity of IgG3 Cryoglobulins

Monoclonal 6-19 IgG3 anti-IgG2a rheumatoid factor derived from lupus-prone MRL-Fas(lpr) mice can induce GN and cryoglobulinemia, but the features that confer nephritogenic potential are not completely understood. Asparagine-linked oligosaccharide chains of 6-19 IgG3 mAb are poorly galactosylated and hardly sialylated, possibly contributing to the pathogenic potential of 6-19 IgG3 rheumatoid factors. Here, we used the 6-19 model of cryoglobulin-associated GN to define the relative contributions of galactosylation and sialylation, in relation to cryoglobulin activity, to the nephritogenic potential of IgG3 antibodies. We generated one highly sialylated and two distinct more galactosylated 6-19 IgG3 rheumatoid factor variants. Although the mere extent of galactosylation had no effect on either the cryogenic and nephritogenic activities of 6-19 IgG3 rheumatoid factor, terminal sialylation attenuated the nephritogenic potential of 6-19 IgG3 by limiting its cryoglobulin activity. These data suggest a protective role of IgG sialylation against the development of cryoglobulin-mediated GN, highlighting the anti-inflammatory activity of sialylated IgG antibodies.

Anti-inflammatory IgG Production Requires Functional P1 Promoter in β-Galactoside α2,6-Sialyltransferase 1 (ST6Gal-1) Gene

The anti-inflammatory properties associated with intravenous immunoglobulin therapy require the sialic acid modification of the N-glycan of the Fc domain of IgG. Sialylation of the Fc fragment is mediated by β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1), acting on the Gal(β4)GlcNAc terminal structure of the biantennary N-glycans on the Fc domain. However, little is known regarding the in vivo regulation of Fc sialylation and its role in the progression of inflammatory processes. Here, we report that decreased Fc sialylation of circulatory IgG accompanies the acute phase response elicited by turpentine exposure or upon acute exposure to either nontypeable Haemophilus influenzae or ovalbumin. However, Fc sialylation was increased 3-fold from the base line upon transition to chronic inflammation by repeated exposure to challenge. The P1 promoter of the ST6Gal-1 gene is critical for Fc sialylation, but P1 does not drive ST6Gal-1 expression in B cells. The Siat1ΔP1 mouse, with a dysfunctional P1 promoter, was unable to produce sialylated Fc in the systemic circulation, despite the presence of Gal(β4)GlcNAc termini on the Fc glycans. The major contribution of P1 action is to synthesize ST6Gal-1 enzymes that are deposited into the systemic circulation. The data strongly indicate that this pool of extracellular ST6Gal-1 in the blood impacts the sialylation of IgG Fc and that defective Fc sialylation is likely a major contributing mechanism for the proinflammatory tendencies previously noted in Siat1ΔP1 animals.

Рівень сіалування антигістонових антитіл сироватки крові хворих на системний червоний вовчак

Anti-histone autoantibodies belong to main marker antibodies used in the diagnostics of systemic lupus erythematosus (SLE) patients. During autoimmune diseases, especially in SLE, prevalent process of inflammation is caused by high titers of auto-antibodies. It is known that the level of IgG sialylation affect their anti-inflammatory properties. The aim of the experiments was to investigate the level of sialylation of anti-histone IgG-antibodies from blood serum of patients with SLE. Investigation was carried out by two methodological approaches. In the first case, the antibodies were purified from blood serum by affine chromatography on histone-Sepharose and protein G-Sepharose followed by Western blot detection of sialic acid residues in the carbohydrate chaines of IgG molecules using sialospecific previously biotinylated Sambucus nigra lectin (SNA). In the second case, IgG-antibodies were isolated from blood serum by affine chromato­graphy on protein G-Sepharose, this fraction was separated on sialylated and not sialy­lated IgG by SNA-Sepharose chromatography followed by checking their affinity to histones by ELISA. In this manuscript, it is shown that the level of sialylation of anti-histone IgG is lower than in control preparation of antibodies. Our data showed that anti-histone autoantibodies are devoid of sialic acid residues at the ends of the carbohydrate chains of Fc-fragments promote their inflammatory properties in patients with SLE. Keywords: blood serum, anti-histone auto-antibodies, systemic lupus erythematosus, carbohydrate chains, sialic acid residues. Note: ELISA – enzyme linked immunosorbent assay, Ab – antibodies, auto-Ab – auto-antibodies.

Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface

Biological activities of immunoglobulin G such as effector functions via Fc receptor interactions are influenced by Fc-linked N-glycans. Here we describe a fast, robust and sensitive nano-LC-ESI-MS method for detailed subclass specific analysis of IgG Fc N-glycosylation. A sheath-flow ESI sprayer was used as a sensitive zero dead volume plug-and-play interface for online MS coupling, generating a very constant spray and ionization over the entire LC gradient. The propionic acid containing sheath-liquid effectively suppressed TFA gas-phase ion-pairing, enabling the use of TFA containing mobile phases. The fixed position of the sheath-flow ESI sprayer, far away from the glass capillary inlet, reduced MS contamination as compared to conventional nano-ESI. The method was found to be suitable for fast and detailed subclass specific IgG Fc N-glycosylation profiling in human plasma. The obtained subclass specific IgG Fc N-glycosylation profiles were processed automatically using in house developed software tools. For each of the IgG subclasses the 8 major glycoforms showed an interday analytical variation below 5%. The method was used to profile the IgG Fc N-glycosylation of 26 women at several time points during pregnancy and after delivery, revealing pregnancy-associated changes in IgG galactosylation, sialylation and incidence of bisecting N-acetylglucosamine.

IgG Fc N-Glycosylation Changes in Lambert-Eaton Myasthenic Syndrome and Myasthenia Gravis

N-glycosylation of the immunoglobulin Fc moiety influences its biological activity by, for example, modulating the interaction with Fc receptors. Changes in IgG glycosylation have been found to be associated with various inflammatory diseases. Here we evaluated for the first time IgG Fc N-glycosylation changes in well-defined antibody-mediated autoimmune diseases, that is, the neurological disorders Lambert-Eaton myasthenic syndrome and myasthenia gravis, with antibodies to muscle nicotinic acetylcholine receptors or muscle-specific kinase. IgGs were purified from serum or plasma by protein A affinity chromatography and digested with trypsin. Glycopeptides were purified and analyzed by MALDI-FTICR-MS. Glycoform distributions of both IgG1 and IgG2 were determined for 229 patients and 56 controls. We observed an overall age and sex dependency of IgG Fc N-glycosylation, which was in accordance with literature. All three disease groups showed lower levels of IgG2 galactosylation compared to controls. In addition, LEMS patients showed lower IgG1 galactosylation. Notably, the galactosylation differences were not paralleled by a difference in IgG sialylation. Moreover, the level of IgG core-fucosylation and bisecting N-acetylglucosamine were evaluated. The control and disease groups revealed similar levels of IgG Fc core-fucosylation. Interestingly, LEMS patients below 50 years showed elevated levels of bisecting N-acetylglucosamine on IgG1 and IgG2, demonstrating for the first time the link of changes in the level of bisecting N-acetylglucosamine with disease.

In Planta Protein Sialylation through Overexpression of the Respective Mammalian Pathway

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

A close look at human IgG sialylation and subclass distribution after lectin fractionation

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti-inflammatory properties depend on sialylation of the N-glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti-inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.

Identification of serum proteins that inhibit lupus immune complex stimulation of plasmacytoid dendritic cells (99.34)

Abstract Regulation of IFN-α produced by plasmacytoid dendritic cells (pDC) is critically important in Systemic Lupus Erythematosus (SLE) as patients have an 'IFN-signature' which correlates with disease severity. We observed that, normalized for IgG, SLE patient cerebrospinal fluid (CSF) induced 800-fold more IFN-α by pDCs compared to paired serum due to potent inhibitory factors in serum. Depletion of IgG from normal serum and addition of purified IgG to SLE immune complex (IC) stimulation assays revealed that normal IgG was one inhibitor in serum. Although the inhibitory capacity of IVIG has recently been attributed to a small subfraction of sialylated IgG in mouse models, sialylation of IgG did not significantly affect IVIG inhibition of IC stimulation of IFN-α. This result indicates either species differences or differences in pDC inflammatory pathways. Deficiencies of early complement components predispose to SLE. Heat inactivation of normal serum and utilization of sera selectively deficient in early complement components revealed that a second serum inhibitor of IC mediated IFN-α stimulation was complement. Suppression of IFN-α produced by pDC was restored by adding back the missing complement component. In summary, we have identified two serum factors that potently attenuate IFN-α induced by SLE ICs. Modulation of these components in vivo could be used therapeutically.

Glycosylation Changes on Serum Glycoproteins in Ovarian Cancer May Contribute to Disease Pathogenesis

Ovarian cancer is the most lethal of all gynaecological cancers among women. Serum CA125 is the only biomarker that is used routinely and there is a need for further complementary biomarkers both in terms of sensitivity and specificity. N-glycosylation changes in ovarian cancer serum glycoproteins include a decrease in galactosylation of IgG and an increase in sialyl Lewis X (SLex) on haptoglobin β-chain, α1-acid glycoprotein and α1-antichymotrypsin. These changes are also present in chronic inflammation but not in malignant melanoma, where there are low levels of inflammatory processes. Acute phase proteins carrying increased amounts of SLex have an increased half-life. Sialylation of acute phase proteins also decreases apoptosis favouring survival of cancer cells. Cancer cells produce inflammatory cytokines which influence glycosylation processing in liver parenchymal cells. Altered glycosylation of the acute phase protein transferrin plays an important role in iron homeostasis. Glycosylated transferrin and its glycans have anti-apoptotic properties and many transferrin receptors in carcinoma could play a role in development of anaemia. Decreased galactosylation and sialylation of IgG increases the cytotoxicity of natural killer cells and complement activation via mannose-binding lectin (MBL). Altered glycosylation of acute phase proteins and IgG suggests that cancer regulates certain pathways favouring cancer cells survival.

Galactosylation and sialylation of terminal glycan residues of human immunoglobulin G using bacterial glycosyltransferases with in situ regeneration of sugar-nucleotides

The N-glycans in human immunoglobulin G were engineered through the bacterial glycosyltransferase-catalyzed galactosylation and sialylation with in situ regeneration of sugar-nucleotides. For the galactosylation and sialylation, β1,4-galactosyltransferase from H. pylori and α2,3-sialyltransferase from N. gonorrhoeae were expressed in Escherichia coli. For the regeneration of UDP-galactose and CMP-NeuAc, the required enzymes were also expressed in E. coli and their extracts were employed as biocatalysts. Using the catalytic system, we carried out the galactosylation of terminal GlcNAc residues with UDP-Gal regeneration and sialylation of terminal Gal residues with CMP-NeuAc regeneration, respectively. When the oligosaccharides in human IgG were analyzed by HPLC (NP and WAX) and MALDI-TOF, the galactosylation reaction showed 86.1% increase in terminal galactosylation of N-linked oligosaccharide in IgG, and the sialylation reaction gave 69.6% increase in terminal sialylation. To achieve a maximum sialylation of IgG, simultaneous galactosylation and sialylation reaction was conducted by incubating all of the biocatalysts required for the galactosylation, sialylation and sugar-nucleotide regenerations in one reactor, resulting in 80.2% increase in terminal sialylation.

Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation.

The occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RF activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.

Effects of ageing on the ultrastructure of rat decidua and endometrium

In patients with active rheumatoid arthritis (RA) decrease of galactosylation is correlated with disease activity. The aim of our study was to evaluate an effect of methotrexate therapy on glycosylation disturbances of IgG in RA patients.IgG glycosylation in 40 patients with active RA treated with methotrexate for 12 months prior to and after treatment were compared. The control group consisted of 20 healthy volunteers. IgG glycosylation was assessed using biotinylated lectins and immunosorbent ELISA assay. For galactose specificity Datura stramonium lectin (DSA), for sialic acid Sambucus nigra (SNA) and Maackia amurensis (MAA) and for fucose residue Areulia auranta (AAA) lectins were used.In RA-cases N-glycan galactosylation and sialylation of IgG before treatment were significantly lower than in healthy subjects (for DSA, MAA lectins p < 0.001 and SNA p < 0.05). Significant increase of IgG galactosylation and sialylation in RA patients after therapy (for DSA, MAA and SNA lectin p < 0.05) was detected. Moreover the glycosylation disturbances of N-glycan IgG were strongly associated with changes of disease activity based on disease activity score. For fucose residues significantly higher absorbency of AAA lectin in RA patients before treatment was observed compared to control subjects (p < 0.05) and slightly, not significantly decreased after MTX therapy.Defect of galactosylation of IgG in RA patients is a useful marker of disease activity that may be used for the assessment of therapy effectiveness. The role of IgG fucosylation and sialylation in RA pathogenesis has still to be determined.