premature IgA1 sialylation hold the key to the pathogenic changes in

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1 Is sialylation of IgA the agent provocateur of IgA nephropathy?does Formatted: Font: Italic premature IgA1 sialylation hold the key to the pathogenic changes in IgA1 O-glycosylation in IgAN? Alice Smith, Karen Molyneux, Comment [mc1]: This title may not appeal to many nephrologists, as it sound too biochemical. Can you make it more clinical? John Feehally and Jonathan Barratt Department of Infection, Immunity and Inflammation, University of Leicester, and John Walls Renal Unit, University Hospitals of Leicester NHS Trust, Leicester. Corresponding author Dr Jonathan Barratt Senior Lecturer / Honorary Consultant Nephrologist Department of Infection, Immunity & Inflammation John Walls Renal Unit Leicester General Hospital Gwendolen Road Leicester, LE5 4PW Tel: Fax: e mail: jb81@le.ac.uk 1

2 Suzuki H, Moldoveanu Z, Hall S, Brown R, Vu HL, Novak L, Julian BA, Tomana M, Wyatt RJ, Edberg JC, Alarcón GS, Kimberly RP, Tomino Y, Mestecky J, Novak J. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Invest 2008 Feb;118(2): Summary In IgA nephropathy (IgAN), the mechanisms responsible for deposition of IgA1 in the glomerular mesangium, and subsequent initiation of glomerular injury in some patients, remain unclear. Much research evidence now demonstrates that the O- glycosylation of IgA1 is abnormal in IgAN, and that this is potentially pathogenic. However, the exact nature of the O-glycoyslation abnormality, and the metabolic processes resulting in its production, are not well understood. gress in this field has partly been limited by the lack of a suitable animal model, because the IgA of laboratory animals is not O-glycosylated. In this paper, Suzuki and colleagues have established B cell lines from patients with IgAN that produce IgA1 with abnormal O- glycosylation. This has allowed them to show that abnormal IgA1 carries an increased proportion of O-glycans terminating in sialic acid rather than the normal galactose units (Figure 1), and that this is associated with increased mrna expression and activity of the sialylating enzyme ST6GalNAcII, and decreased expression and Comment [mc2]: Suggest to illustrate this with a figure (including the enzymes involved). activity of the galactosylating enzyme C1GalT1. These findings contribute new insights to the study of defective IgA1 O-galactosylation and mark a significant step forward in the field of IgAN research. 2

3 Review of field Altered O-glycosylation of IgA1 has been recognised as a potentially pathogenic abnormality in IgAN for nearly 20 years [1]. The 18-amino acid hinge region of IgA1 can carry from 0-6 O-glycan moieties, each of which is a relatively short and simple sugar chain, but there are up to 6 different potential forms [2]. The variability in the number and location of occupied O-glycosylation sites, and the different possible forms each of these chains (Figure 2) result in a vast array of potential IgA1 O- glycoforms, and this immense diversity has hindered the precise structural definition of the abnormality in IgAN. Comment [mc3]: Again, can you make that part of the illustration, as requested above? O-glycosylation is a post-translational modification effected by a series of O- glycosyltransferases which are highly specific in respect to the acceptor and donor sugars and the linkage between them (Figure 1). Alterations in the expression or activity of one or more of these O-glycosyltransferases may underlie abnormal IgA1 O-glycosylation in IgAN, but the available evidence to date has been inconclusive or conflicting [3-6]. The confusion partly arises from studies being carried out on mixed cell populations: even isolated B cells contain variable proportions of cells differing in maturity, activation status and Ig isotype production, all of which are likely to influence expression of O-glycosyltransferases. In this paper, Suzuki and colleagues have established IgA1-secreting EBVtransformed B cell lines derived from the peripheral blood of patients with IgAN and controls. As each of these cell lines is a clone, this has provided them with sufficient cells and IgA1 of a single type for detailed characterisation of O-glycosyltransferases and IgA1 O-glycosylation. Previous studies have shown that patients with IgAN are able to produce normally O-glycosylated proteins [7-9], and do not have a global 3

4 defect in the enzymes involved. Therefore, abnormally glycosylated IgA1 may arise from specific subpopulations of IgA1-producing B cells in IgAN. Suzuki and colleagues have established IgAN B cell lines producing such abnormal IgA1, and carried out a series of elegant experiments comparing these cells and their products to those from healthy subjects. In this way, they have shown that the abnormality in IgAN takes the form of premature sialylation of the core N-acetylgalactosamine moiety, preventing its more usual galactosylation and resulting in truncated O-glycan chains. This is clearly associated with increased gene expression and activity of one of the various forms of sialyltransferases, ST6GalNAcII, and downregulation of the O- galactosylating enzyme C1GalT1 and its chaperone protein Cosmc. The results of this study shed new light on this important pathogenic feature of IgAN, and highlight ST6GalNAcII as a novel target of interest. We now need to understand the factors that govern expression and activity of O-glycosyltransferases as it is apparent from this study that subtle changes in the balance between them have important consequences. Our group have has found that IgAN B cells produce normally O-galactosylated IgD [8], suggesting that the O-glycosyltransferase imbalance does not appear until a later stage of B cell maturation, after classswitching to IgA1. Furthermore, the shift in relative expression of ST6GalNAcII and C1GalT1 and consequent production of IgA1 with truncated O-glycans may be normal in certain immunological situations. We have shown that both IgAN patients and healthy subjects produce such abnormal IgA1 in response to the mucosal antigen Helicobacter pylori, whereas IgA1 antibodies to tetanus toxoid are heavily O- galactosylated in both IgAN and controls. This implies that B cell populations possessing the ability to produce IgA1 with alternative O-glycoforms exist in IgAN and in healthy subjects. Suzuki et al have shown that the O-glycosyltransferase 4

5 expression of blood-derived EBV cell lines correlates with serum IgA1 O- glycosylation of the same subject, providing a new in vitro system in which to study the factors underlying the presence of pathogenic IgA1 in the circulation in IgAN. Clinical implications Suzuki and colleagues have identified and characterized B cell subsets that generate IgA1 O-glycoforms that are widely accepted to have an important pathogenic role in IgA immune complex formation in IgAN [10]. What remains unknown is the origin of these B cells and why in IgAN they appear in the circulation in increased numbers. Undergalactosylation and increased sialylation of IgA1 is a normal feature of IgA1 directed against mucosal antigens and this raises the possibility that the cells identified by Suzuki and colleagues are mucosally-derived trafficking lymphocytes [9,11]. While undergalactosylated and sialylated IgA1 may confer an advantage at mucosal surfaces, when present in the circulation it appears that they promote the formation of immune complexes with a propensity for mesangial deposition. The association between mucosal infections and IgAN has been established for many years but the precise link between the mucosal IgA immune system and mesangial IgA deposition has remained elusive. There is however an increasing body of evidence supporting the displacement of mucosally-primed B cells to systemic sites in IgAN and that. this This might be explained by defects in the expression of cell surface homing receptors by trafficking lymphocytes [12-15]. What is needed now is precise immunophenotyping of the IgA1-committed B cells isolated and studied by Suzuki and colleagues so that we can trace their origins and understand how they arrived in the circulation in IgAN. Pinpointing the origins of these B cells will not only get us closer to understanding the fundamental immune processes operating in 5

6 IgAN but also provide novel therapeutic targets in a disease currently bereft of any form of specific treatment. Take home message B cells programmed to sialylate IgA1 early (and therefore precludevent the later addition of galactose) may be the architects of IgA immune complex formation in IgA Comment [mc4]: English ok here? nephropathy. References 1. Andre PM, Le Pogamp P, Chevet D: Impairment of jacalin binding to serum IgA in IgA nephropathy. J Clin Lab Anal 4: , Tarelli E, Smith AC, Hendry BM, Challacombe SJ, Pouria S: Human serum IgA1 is substituted with up to six O-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. Carbohydr Res 339: , Allen AC, Topham PS, Harper SJ, Feehally J: Leucocyte beta 1,3 galactosyltransferase activity in IgA nephropathy. Nephrol Dial Transplant 12: , Qin W, Zhou Q, Yang LC, Li Z, Su BH, Luo H, Fan JM: Peripheral B lymphocyte beta1,3-galactosyltransferase and chaperone expression in immunoglobulin A nephropathy. J Intern Med 258: , Inoue T, Sugiyama H, Kikumoto Y, Fukuoka N, Maeshima Y, Hattori H, Fukushima K, Nishizaki K, Hiki Y, Makino H: Downregulation of the beta1,3- galactosyltransferase gene in tonsillar B lymphocytes and aberrant lectin bindings to tonsillar IgA as a pathogenesis of IgA nephropathy. Contrib Nephrol 157: ,

7 6. Buck KS, Smith AC, Molyneux K, El-Barbary H, Feehally J, Barratt J: B-cell O-galactosyltransferase activity, and expression of O-glycosylation genes in bone marrow in IgA nephropathy. Kidney Int, Allen AC, Harper SJ, Feehally J: Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy. Clin Exp Immunol 100: , Smith AC, de Wolff JF, Molyneux K, Feehally J, Barratt J: O-glycosylation of serum IgD in IgA nephropathy. J Am Soc Nephrol 17: , Smith AC, Molyneux K, Feehally J, Barratt J: O-glycosylation of serum IgA1 antibodies against mucosal and systemic antigens in IgA nephropathy. J Am Soc Nephrol 17: , Mestecky J, Suzuki H, Yanagihara T, Moldoveanu Z, Tomana M, Matousovic K, Julian BA, Novak J: IgA nephropathy: current views of immune complex formation. Contrib Nephrol 157:56-63, Royle L, Roos A, Harvey DJ, Wormald MR, van Gijlswijk-Janssen D, Redwan el RM, Wilson IA, Daha MR, Dwek RA, Rudd PM: Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems. J Biol Chem 278: , Batra A, Smith AC, Feehally J, Barratt J: T-cell homing receptor expression in IgA nephropathy. Nephrol Dial Transplant, Harper SJ, Allen AC, Layward L, Hattersley J, Veitch PS, Feehally J: Increased immunoglobulin A and immunoglobulin A1 cells in bone marrow trephine biopsy specimens in immunoglobulin A nephropathy [see comments]. Am J Kidney Dis 24: ,

8 14. Harper SJ, Pringle JH, Wicks AC, Hattersley J, Layward L, Allen A, Gillies A, Lauder I, Feehally J: Expression of J chain mrna in duodenal IgA plasma cells in IgA nephropathy. Kidney International 45: , Kennel-de March A, Bene MC, Renoult E, Kessler M, Faure GC, Kolopp- Sarda MN: Enhanced expression of L-selectin on peripheral blood lymphocytes from patients with IgA nephropathy. Clin Exp Immunol 115: , 1999 Formatted: Indent: Left: 0 cm, First line: 0 cm 8

9 Legends to Figures Formatted: Font: Bold Figure 1 O-glycosylation of IgA1. The O-linked sugars of IgA1 are attached to serine or threonine residues in the IgA1 hinge region which lies between the CH1 and CH2 domains of the 1 heavy chain. Formatted: Font: Italic Formatted: Font: Italic Formatted: Font: Symbol The hinge region is made up of 17 amino acids, of which at least six are O-linked glycosylation sites. The O-linked sugar chains are core 1 structures based on N- acetylgalactosamine (GalNAc) in O-linkage with serine (usually) or threonine. This core GalNAc is usually further extended with galactose (Gal) in the 1,3 configuration or sialic acid (N-acetylneuraminic acid, NeuNAc) in an 2,6 configuration. pp-galnac-t: UDP-N-acetyl- -D-galactosamine:polypeptide N- acetylgalactosaminyltransferase ST6GalNAcII: N-acetylgalactosamine-specific 2,6-sialyltransferase Formatted: Font: Symbol C1GalT1: core galactosyltransferase Cosmc: core 1 Figure 2 3 galactosyltransferase -specific molecular chaperone Formatted: Font: Symbol Formatted: Font: Bold The major O-glycan forms of human IgA1. The IgA1 O-glycans are all based on a core 1 structure with N-acetylgalactosamine (GalNAc) units in O-linkage with serine or threonine. This may occur alone or may be extended with sialic acid in 2,6-linkage with GalNAc or 1,3-linked galactose (Gal). Further extension with sialic acid (NeuNAc) in 2,3-linkage with Gal can also occur. 9

10 IgA1 Ser/Thr Step 1 Addition of N-acetylgalactosamine (GalNAc) O-linkage GalNAc pp-galnac-t Increased in IgAN GalNAc GalNAc Decreased in IgAN ST6GalNAcII a2,6 linkage b1,3 linkage C1GalT1 / Cosmc Addition of sialic acid (Nacetylneuraminic acid, NeuNAc) in an a2,6 configuration prevents further addition of galactose to GalNAc NeuNAc Gal There may be further addition of sialic acid to galactose (Gal) in an a2,3 configuration

11 IgA1 Ser/Thr unoccupied Ser/Thr -O- GalNAc Hinge region CH1 224 Ser Thr Thr Ser Ser Thr Thr Ser Ser 240 CH2 Ser/Thr -O- GalNAc NeuNAc Ser/Thr -O- GalNAc Ser/Thr -O- GalNAc Gal Gal NeuNAc