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      Glycosylation Profile of Differently Charged IgA1 and Their Binding Characteristics to Cultured Mesangial Cells in IgA Nephropathy

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          Abstract

          Background: IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA1 (pIgA1), yet the pathogeneic mechanism remains unresolved. In the present study, we examined the glycosylation profile of differently charged IgA1 from IgAN patients. The binding characteristics of these IgA1 fractions to cultured human mesangial cells (HMC) and hepatoma cell lines (HepG2) were studied. Methods: Differently charged IgA1 were isolated by ion exchange chromatography. The glycosylation profile in the carbohydrate moieties of these differently charged IgA1 was analyzed by galactose (Gal)-, galactose-acetylgalactosamine (Gal-GalNAc)-, or sialic acid-specific enzyme-linked lectin binding assays (ELLA). The binding characteristic of these IgA1 to HMC was examined by flow cytometry and competitive binding assay. Results: Anionic pIgA from IgAN patients showed less reactivity in (Gal)- and (Gal-GalNAc)-specific ELLA (p < 0.01). There was higher reactivity for anionic pIgA1 in α(2,6)-linked sialic acid-specific ELLA (p < 0.01). Anionic pIgA1 from IgAN patients exhibited increased binding to cultured HMC and the binding was significantly reduced after neuraminidase treatment (p < 0.05). In contrast, anionic pIgA1 from IgAN patients bound less to cultured HepG2 cells and the binding was enhanced following neuraminidase treatment (p < 0.05). Conclusions: We demonstrated an unusual glycosylation and sialylation pattern of anionic pIgA1 in IgAN which may have an important effect on its pathogenesis.

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          Most cited references 16

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          Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies.

          Circulating immune complexes (CICs) isolated from sera of patients with IgA nephropathy (IgAN) consist of undergalactosylated, mostly polymeric, and J chain-containing IgA1 and IgG antibodies specific for N-acetylgalactosamine (GalNAc) residues in O-linked glycans of the hinge region of IgA1 heavy chains. Antibodies with such specificity occur in sera of IgAN patients, and in smaller quantities in patients with non-IgA proliferative glomerulonephritis and in healthy controls; they are present mainly in the IgG (predominantly IgG2 subclass), and less frequently in the IgA1 isotype. Their specificity for GalNAc was determined by reactivity with IgA1 myeloma proteins with enzymatically removed N-acetylneuraminic acid (NeuNAc) and galactose (Gal); removal of the O-linked glycans of IgA1 resulted in significantly decreased reactivity. Furthermore, IgA2 proteins that lack the hinge region with O-linked glycans but are otherwise structurally similar to IgA1 did not react with IgG or IgA1 antibodies. The re-formation of isolated and acid-dissociated CICs was inhibited more effectively by IgA1 lacking NeuNAc and Gal than by intact IgA1. Immobilized GalNAc and asialo-ovine submaxillary mucin (rich in O-linked glycans) were also effective inhibitors. Our results suggest that the deficiency of Gal in the hinge region of IgA1 molecules results in the generation of antigenic determinants containing GalNAc residues that are recognized by naturally occurring IgG and IgA1 antibodies.
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            The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fcα receptor interactions.

            The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction with various Fc receptors (Fc Rs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1 Fab and Fc as well as three recombinant IgA1 molecules, including two N-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated that O-glycans are located at Thr228, Ser230, and Ser232, while O-glycan sites at Thr225 and Thr236 are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where < 10% contain sialic acid. This paper contains the first report of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα 2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary structures. Further analysis of these data suggested that processing at one Fc N-glycosylation site affects the other. Neutrophil Fcα R binding studies, using recombinant IgA1, indicated that neither the tailpiece region nor the N-glycans in the C alpha 2 domain contribute to IgA1-neutrophil Fcα R binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the Fcγ R. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence of both the primary sequence and S-S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα 2 N-glycans provides an explanation for the increased sialylation and galactosylation of IgA1 Fc over that of IgG Fc N-glycans, which are confined in the space between the two Cγ 2 domains. This also suggests why in contrast to IgG Fc, the IgA1 N-glycans are not undergalactosylated in rheumatoid arthritis.
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              Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.

              IgA1 proteins from sera of patients with IgA nephropathy (IgAN) are galactosylated to a lesser degree than those from healthy controls. The increased reactivity of intact or de-sialylated serum IgA1 with N-acetylgalactosamine (GalNAc)-specific lectins, Helix aspersa (HAA) and Caragana arborescens (CAA) and de-sialylated IgA1 with Helix pomatia (HPA) and Bauhinia purpurea (BPA) indicated that the Gal deficiency is in glycans located in the hinge region of IgA1 molecules. De-sialylated IgA from sera of 81 IgAN patients bound biotin-labeled lectin HAA more effectively than did de-sialylated IgA from 56 healthy controls (P < 0.0001). Similar results were observed for 67 IgAN patients and 52 controls with second lectin, CAA (P < 0.001). The binding patterns for 9 patients with mesangial proliferative glomerulonephritis of non-IgA origin were similar to those for controls. Incompletely galactosylated IgA1 capable of binding GalNAc-specific lectins was detected in complexes with IgG as demonstrated by ELISA, size-exclusion chromatography and sucrose gradient ultracentrifugation. The formation of IgA1-IgG complexes may affect the serum level of IgA1 by reducing the rate of its elimination and catabolic degradation by the liver.
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                Author and article information

                Journal
                NEE
                Nephron Exp Nephrol
                10.1159/issn.1660-2129
                Cardiorenal Medicine
                S. Karger AG
                1660-2129
                2007
                November 2007
                23 October 2007
                : 107
                : 3
                : e107-e118
                Affiliations
                Departments of aMedicine and bSurgery, Queen Mary Hospital, University of Hong Kong, and cDepartment of Surgery, Queen Elizabeth Hospital, Hong Kong, SAR, China
                Article
                109980 Nephron Exp Nephrol 2007;107:e107–e118
                10.1159/000109980
                17957128
                © 2007 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 7, Tables: 1, References: 36, Pages: 1
                Categories
                Original Paper

                Cardiovascular Medicine, Nephrology

                Polymeric IgA, IgA nephropathy, Mesangial cell, IgA1

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