IgA1 Hinge Research Articles

Abnormal IgA1 O-Glycosylation in a Multi-ethnic population of IgA Nephropathy Patients in KwaZulu Natal, South Africa.

Abnormal IgA1 O-Glycosylation in a Multi-ethnic population of IgA Nephropathy Patients in KwaZulu Natal, South Africa.

Quantitative change of IgA hinge O-glycan composition is a novel marker of therapeutic responses of IgA nephropathy

Aberrant O-glycosylation in the hinge region of serum IgA is suggested to be involved in the pathogenesis of IgA nephropathy (IgAN), because the hypoglycosylation including N-acetylneuraminic acid or galactose has been reported in the mucin-type O-glycan of the hinge portion (HP) of IgA deposited in the IgAN patients’ kidney. These aberrant glycosylation has been assessed in most of the previous reports by qualitative but not quantitative methods. In the present study, the molar ratios of GalNAc or Gal to HP were analyzed for serum IgA from IgAN patients. The GalNAc/HP ratio was increased in the patients who achieved remission after a combination therapy of tonsillectomy and intravenous corticosteroid, suggesting any non-innate factors to affect the IgA O-glycosylation in IgAN that is thought to be inherently determined. Furthermore, the O-glycosylation status was different among three groups: IgAN patients in the pretreatment stage, IgAN patients in the remission stage after treatment and healthy controls. These results indicated that aberrant O-glycosylation of serum IgA in the IgAN patients would be inherently present and, to some extent, affected by therapeutic intervention. Finally, the quantitative change of O-glycan composition is a novel marker of therapeutic response of IgAN.

Development of a Polyclonal Antibody Against Synthetic Human Immunoglobulin A1 Hinge Glycopeptide

Background: Undergalactosylated IgA1 has been found to be increased in IgA nephropathy (IgAN) by an ELISA assay using Helix aspersa agglutinin (HAA) that recognizes N-acetylgalactosamine (GalNAc). In this study, we developed a polyclonal antibody (anti-sHGP antibody) against a synthetic IgA1 hinge peptide with five GalNAc residues. Methods: The specificity of the anti-sHGP antibody was evaluated through the incremental treatment of IgA with corresponding glycosidases. Then, the susceptibility of the IgA to anti-sHGP antibody was compared among IgAN patients (n=39), patients with other forms of kidney diseases (OKD, n= 36) and healthy controls (n=37), using ELISA assay. The association of the binding abilities between anti-sHGP antibody and HAA were evaluated blindly using same 85 sera. Results: The binding ability of the anti-sHGP antibody was increased relative to the incremental treatments of neuraminidase (desialo-IgA), galactosidase (desialo/degalacto IgA). The binding levels of anti-sHGP antibody against serum IgA were significantly higher in IgAN patients compared to both healthy controls (P=0.008) and those with OKD (P=0.049). The binding levels of anti-sHGP antibody were closely related to those of HAA ELISA in the same patient sera (RR2=0.5964). Conclusions: It was certified that the anti-sHGP antibody recognized GalNAc residues in the hinge peptide of human IgA1 as well as HAA. The increased antigenicity of IgA against the antibody in IgAN suggested that a serum IgA1 exposing GalNAc residue was increased in IgAN. It would be necessary to identify the precise structure of O-glycans specific to IgAN for developing a more specific antibody.

O-Glycosylated IgA Rheumatoid Factor Induces IgA Deposits and Glomerulonephritis

Structural aberrations of O-linked glycans present in the IgA1 hinge region are associated with IgA nephropathy, but their contribution to its pathogenesis remains incompletely understood. In this study, mice implanted with hybridoma secreting 6-19 IgA anti-IgG2a rheumatoid factor, but not 46-42 IgA rheumatoid factor bearing the same IgA allotype, developed mesangial deposits consisting of IgA, IgG2a, and C3. Studies in immunoglobulin- and C3-deficient mice revealed that the development of these glomerular lesions required the formation of IgA-IgG2a immune complexes and subsequent activation of complement. The proportion of polymeric and monomeric forms, the IgG2a-binding affinity, and the serum levels of IgA-IgG2a immune complexes were similar between 6-19 IgA- and 46-42 IgA-injected mice. In contrast, the analysis of oligosaccharide structures revealed highly galactosylated O-linked glycans in the hinge region of 6-19 IgA and poorly O-glycosylated in the hinge region of 46-42 IgA. Furthermore, the structure of N-linked glycans in the CH1 domain was the complex type in 6-19 IgA and the hybrid type in 46-42 IgA. In summary, this study demonstrates the presence of O-linked glycans in the hinge region of mouse IgA and suggests that 6-19 IgA rheumatoid factor-induced GN could serve as an experimental model for IgA nephropathy.

Solid-phase synthesis of a pentavalent GalNAc-containing glycopeptide (Tn antigen) representing the nephropathy-associated IgA hinge region

Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcα1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host–pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyze anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen-expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for β1-3-galactosylation using recombinant T-synthase (β1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan–antigen-associated diseases.

Recognition of Galactose-Deficient O-Glycans in the Hinge Region of IgA1 by N-Acetylgalactosamine-Specific Snail Lectins: A Comparative Binding Study

Aberrancies in IgA1 glycosylation have been linked to the pathogenesis of IgA nephropathy (IgAN), a kidney disease characterized by deposits of IgA1-containing immune complexes in the glomerular mesangium. IgA1 from IgAN patients is characterized by the presence of galactose (Gal)-deficient O-glycans in the hinge region that can act as epitopes for anti-glycan IgG or IgA1 antibodies. The resulting circulating immune complexes are trapped in the glomerular mesangium of the kidney where they trigger localized inflammatory responses by activating mesangial cells. Certain lectins recognize the terminal N-acetylgalactosamine (GalNAc)-containing O-glycans on Gal-deficient IgA1 and can be potentially used as diagnostic tools. To improve our understanding of GalNAc recognition by these lectins, we have conducted binding studies to assess the interaction of Helix aspersa agglutinin (HAA) and Helix pomatia agglutinin (HPA) with Gal-deficient IgA1. Surface plasmon resonance spectroscopy revealed that both HAA and HPA bind to a Gal-deficient synthetic hinge region glycopeptide (HR-GalNAc) as well as various aberrantly glycosylated IgA1 myeloma proteins. Despite having six binding sites, both HAA and HPA bind IgA1 in a functionally bivalent manner, with the apparent affinity for IgA1 related to the number of exposed GalNAc groups in the IgA1 hinge. Finally, HAA and HPA were shown to discriminate very effectively between the IgA1 secreted by cell lines derived from peripheral blood cells of patients with IgAN and that from cells of healthy controls. These studies provide insight into lectin recognition of the Gal-deficient IgA1 hinge region and lay the groundwork for the development of reliable diagnostic tools for IgAN.

Down-regulation of core 1 1,3-galactosyltransferase and Cosmc by Th2 cytokine alters O-glycosylation of IgA1

Background. Patients with IgA nephropathy (IgAN) have an increased amount of abnormally O-glycosylated IgA1 in circulation, in glomerular deposits and produced by tissue cells in vitro. Although increased production of Th2 cytokines by peripheral blood lymphocytes and a functional abnormality of core 1 β1,3-galactosyltransferase (C1β3Gal-T) have been proposed as mechanisms underlying pathogenesis of IgAN, they are still obscure and are not connected.Methods. To clarify the effect of T-cell cytokines, we analysed the mRNA levels of C1β3Gal-T and its molecular chaperone Cosmc, C1β3Gal-T activity and subsequent O-glycosylation of IgA1 in a human B-cell line stimulated with these cytokines. The surface IgA1-positive human B-cell line was cultured with recombinant human IFN-γ, IL-2, IL-4 or IL-5. The production and glycosylation of IgA1 were determined by sandwich ELISA and enzyme-linked lectin binding assay, respectively. The mRNA levels of C1β3Gal-T and Cosmc were quantitatively measured by real-time PCR. C1β3Gal-T activity was analysed using high-performance liquid chromatography.Results. IgA1 production by IL-4-stimulated cells was significantly higher than controls or after IFN-γ or IL-5. The terminal glycosylation of secreted IgA1 was altered in response to IL-4. IL-4 stimulation significantly decreased the mRNA levels of both C1β3Gal-T and Cosmc and of C1β3Gal-T activity. IL-4 stimulation was clearly blocked by recombinant human IL-4 soluble receptor.Conclusions. It appears that Th2 cytokine IL-4 may play a key role in controlling glycosylation of the IgA1 hinge region.

Sugars and immune complex formation in IgA nephropathy

In vitro evidence suggests that immune complex formation in IgA nephropathy is determined by the sugar content of the IgA1 hinge region. Absence of galactose residues in this region renders the IgA1 molecule immunogenic.

O-linked oligosaccharides of the IgA1 hinge region: roles of its aberrant structure in the occurrence and/or progression of IgA nephropathy

Primary IgA nephropathy (IgAN) has been regarded as an immune complex-mediated glomerulonephritis characterized immunohistologically by the predominant deposition of IgA in the glomerular mesangial area with a variety of histopathologic injuries (Clarkson et al. in Ann Rev Med 38:157-168, 1987). In 1992, the characteristic structure of O-linked oligosaccharides (O-glycans) in the IgA1 hinge and its possible aberrancy were simultaneously and independently proposed by Mesteckey et al. (Cont Nephrol 104:172-182, 1993), and our group (Cont Nephrol 104:217, 1993) at the International Congress of Nephrology (IgA Nephropathy 25th year) held in Nancy, France. Since then, the aberrancy has been confirmed by several research groups and is suspected to play a role in the occurrence and/or the progression of IgAN. At the end of the 1980s, I took an interest in the existence of O-glycans in the hinge region of IgA1 and have pursued the structure of the carbohydrate chains. Since an excellent review on the structure and the role of the carbohydrate in IgA molecules was recently published by Narita et al. (Clin Ex Nephrol 12:332-338, 2008), this review focuses on the process by which I developed the idea of aberrant O-glycosylation in IgA1 molecules in IgAN patients and summarizes our recent observations concerning IgA1 molecules.

The Nonplanar Secretory IgA2 and Near Planar Secretory IgA1 Solution Structures Rationalize Their Different Mucosal Immune Responses

Secretory IgA (SIgA) is the most prevalent human antibody and is central to mucosal immunity. It exists as two subclasses, SIgA1 and SIgA2, where SIgA2 has a shorter hinge joining the Fab and Fc regions. Both forms of SIgA are predominantly dimeric and contain an additional protein called the secretory component (SC) that is attached during the secretory process and is believed to protect SIgA in harsh mucosal conditions. Here we locate the five SC domains relative to dimeric IgA2 within SIgA2 using constrained scattering modeling. The x-ray and sedimentation parameters showed that SIgA2 has an extended solution structure. The constrained modeling of SIgA2 was initiated using two IgA2 monomers that were positioned according to our best fit solution structure for dimeric IgA1. SC was best located along the convex edge of the Fc-Fc region. The best fit models showed that SIgA2 is significantly nonplanar in its structure, in distinction to our previous near planar SIgA1 structure. Both the shorter IgA2 hinges and the presence of SC appear to displace the four Fab regions out of the Fc plane in SIgA2. This may explain the noncovalent binding of SC in some SIgA2 molecules. This nonplanar structure is predicted to result in specific immune properties for SIgA2 and SIgA1. It may explain differences observed between the SIgA1 and SIgA2 subclasses in terms of their interactions with antigens, susceptibility to proteases, effects on receptors, and distribution in different tissues. The different structures account for the prevalence of both forms in mucosal secretions.

No evidence for a role of cosmc-chaperone mutations in European IgA nephropathy patients

Altered IgA1 galactosylation is involved in the pathogenesis of IgA nephropathy (IgAN). The galactosyltransferase core-1 beta3-galactosyltransferase-1 (C1GALT1) and its chaperone cosmc are specifically required for O-galactosylation of the IgA1 hinge region. Mutations in the cosmc gene result in a secondary loss of function of C1GALT1 with subsequent undergalactosylation of glycoproteins. Mosaic mutations of cosmc have been shown to result in autoimmune disease. We hypothesized that cosmc mutations might contribute to the altered IgA1 galactosylation in IgAN patients. We studied cosmc gene sequences in genomic DNA obtained from male patients with biopsy-proven sporadic (n = 33) and familial IgAN (n = 6 patients from different families). To account for a potential mosaicism we sequenced cosmc in 10 different peripheral blood mononuclear cell DNA clones of every patient. To specifically assess potential mosaic mutations in IgA-producing cells, cosmc mutations were also analysed in DNA isolated from CD20+ B-lymphocytes from three male IgAN patients. Despite our extensive genomic analysis, the data revealed no functionally relevant cosmc gene variants in sporadic or familial IgAN cases. A cosmc gene polymorphism, rs17261572, was identified in these IgAN patients in a similar frequency as previously reported in healthy adults. A functional consequence of this polymorphism has not yet been determined. Although decreased C1GALT1 activity has been implicated in the IgAN pathogenesis and cosmc chaperone mutations can cause autoimmune disease, our data provide no evidence for a relevant role of cosmc gene mutations in European patients with sporadic or familial IgAN.

Serum under-galactosylated IgA1 is increased in Japanese patients with IgA nephropathy

Immunoglobulin A nephropathy (IgAN) is characterized by an aberrant structure of O-glycans in the IgA1 hinge region. Recently, under-galactosylated IgA1 has been found to be increased in Caucasian IgAN patients. Thus, we examined this in Japanese IgAN patients. An enzyme-linked immunosorbent assay of binding between Helix aspersa (HAA) and serum IgA was performed in Japanese IgAN patients and the HAA-IgA binding levels were compared among IgAN patients (n = 41), patients with other forms of kidney disease (OKD, n = 43) and healthy controls (n = 38). The clinicopathological severity of IgAN was then analysed between patients with high and low HAA-IgA binding levels. The levels were also compared in 11 patients before and after the combination of tonsillectomy and steroid pulse therapy. Furthermore, we examined the O-glycan structure of IgA1 hinge glycopeptides by mass spectrometry (MS). The HAA-IgA binding levels were significantly higher in IgAN patients compared with either healthy controls (P = 0.0025) or those with OKD (P = 0.016). To reflect the absolute level of under-galactosylated IgA, we multiplied the HAA-IgA binding level by the serum IgA concentration to produce an indicative value. The specificity and sensitivity of this value were 89% and 66%, respectively. MS showed that peak distribution of IgA1 hinge glycopeptides was shifted to smaller molecular weights in high HAA-IgA-binding IgAN patients. There was no correlation between the HAA-IgA binding level and either disease severity or the use of combination therapy. HAA-IgA binding is significantly increased in Japanese IgAN patients. This potential IgAN marker is not affected by disease severity or therapeutic intervention.

Aberrant IgA1 Glycosylation Is Inherited in Familial and Sporadic IgA Nephropathy

IgA nephropathy (IgAN) is a complex trait determined by genetic and environmental factors. Most IgAN patients exhibit a characteristic undergalactosylation of the O-glycans of the IgA1 hinge region, which promotes formation and glomerular deposition of immune complexes. It is not known whether this aberrant glycosylation is the result of an acquired or inherited defect, or whether the presence of aberrant IgA1 glycoforms alone can produce IgAN. A newly validated lectin enzyme-linked immunosorbent assay (ELISA) was used to determine the serum level of galactose-deficient IgA1 (Gd-IgA1) in a cohort of 89 IgAN patients and 266 of their relatives. High Gd-IgA1 levels (> or =95th percentile for controls) were observed in all 5 available patients with familial IgAN, in 21 of 45 (47%) of their at-risk relatives (assuming autosomal dominant inheritance), and in only 1 of 19 (5%) of unrelated individuals who married into the family. This provides evidence that abnormal IgA1 glycosylation is an inherited rather than acquired trait. Similarly, Gd-IgA1 levels were high in 65 of 84 (78%) patients with sporadic IgAN and in 50 of 202 (25%) blood relatives. Heritability of Gd-IgA1 was estimated at 0.54 (P = 0.0001), and segregation analysis suggested the presence of a major dominant gene on a polygenic background. Because most relatives with abnormal IgA1 glycoforms were asymptomatic, additional cofactors must be required for IgAN to develop. The fact that abnormal IgA1 glycosylation clusters in most but not all families suggests that measuring Gd-IgA1 may help distinguish patients with different pathogenic mechanisms of disease.

Protective role of anti-synthetic hinge peptide antibody for glomerular deposition of hypoglycosylated IgA1

The KM mouse lacks endogenous genes for immunoglobulins and carries the entire human IgH locus and the IgLk transgene. Therefore, human IgA1 does not provoke a hetero-immune response. We had observed mesangial IgA deposits in KM mice given desialo-degalacto (DeS/DeGal) IgA1. In this study, the mice were immunized with synthetic IgA1 hinge (glyco-)peptide before administration of DeS/DeGal IgA1, and the effects of the pre-immunization were evaluated. Mice were divided into sHP, 5GalNAc-sHP and non-immunization groups. In two pre-immunization groups, KLH-conjugated sHP or KLH-5GalNAc-sHP, which has five GalNAc residues, was subcutaneously given three times every 2 weeks. Two weeks after the final pre-immunization, DeS/DeGal IgA1 was administered daily for 5 weeks. Serial serum levels of anti-sHP and anti-IgA1 antibodies were evaluated by ELISA. On the day of the last administration of IgA1, renal biopsy was performed. Mesangial IgA deposits were observed in all non-immunized mice. In pre-immunized mice, IgA deposition was not detected in 6 of 13 sHP mice and 1 of 4 5GalNAc-sHP mice. The intensities of IgA deposits were significantly different between sHP groups and non-immunized (P = 0.003) groups. There was a significant inverse correlation between the intensities of IgA deposits and the anti-sHP antibody titers (P = 0.016). These results suggest that the anti-IgA1 hinge peptide antibody plays a role in the inhibition of glomerular IgA deposition.

ヒト血清IｇA１ヒンジ部O-glycosylated peptide のESI イオントラッフ゜質量分析計を用いたglycoproteomics

ヒト血清IｇA１ヒンジ部O-glycosylated peptide のESI イオントラッフ゜質量分析計を用いたglycoproteomics

The pathogenic role of IgA1 O‐linked glycosylation in the pathogenesis of IgA nephropathy (Review Article)

SUMMARY: Numerous abnormalities of the IgA immune system have been reported in IgAN but the most consistent finding remains aberrant IgA1 O‐linked glycosylation of the IgA1 hinge region. The defect comprises reduced galactosylation of O‐linked N‐acetylgalactosamine residues with or without changes in the terminal sialylation of the O‐linked sugars. Aberrant O‐galactosylation has been found in serum IgA1, in IgA1 isolated from tonsillar lymphocytes, and in IgA1 eluted from mesangial deposits. There is evidence that changes in IgA1 O‐galactosylation lead to IgA immune complex formation and mesangial IgA deposition. Mesangial cells exposed to these IgA immune complexes proliferate and adopt a pro‐inflammatory phenotype; they secrete cytokines, chemokines, growth factors and extracellular matrix components promoting glomerular inflammation and glomerulosclerosis. Recent evidence suggests that the control of IgA1 O‐glycosylation is linked to class switching from IgD to IgA1 synthesis and that the pattern of IgA1 O‐glycosylation may be programmed at the time of initial antigen encounter. IgA1 glycosylation varies between systemic and mucosal sites and the association of aberrant IgA1 galactosylation with low affinity, polymeric IgA1 antibodies against mucosal antigens suggests undergalactosylated IgA1 may in fact be a mucosal glycoform of IgA1. Although suited to the mucosal compartment, when these IgA1 glycoforms enter the systemic circulation in appreciable quantities they deposit in the mesangium and trigger glomerular inflammation. This review will discuss the evidence for the role of IgA1 O‐glycosylation in the pathogenesis of IgAN and propose an explanation for the presence of aberrantly O‐glycosylated IgA1 in the circulation of patients with IgAN.

The genetic susceptibility to IgA nephropathy: A novel functional candidate gene for incomplete O-glycosylation of IgA1

Incompleteness of O-glycosylation in the IgA1 hinge has been implicated as a central mechanism in the development of IgA nephropathy. Although underglycosylation was reported to be an acquired abnormality, genes for enzymes of O-glycosylation, such as C1GALT1, may be responsible for susceptibility to IgA nephropathy.

Method for generation of in vivo biotinylated recombinant antibodies by yeast mating

We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and Western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K d = 4.8 × 10 − 9 M and K d = 5.1 × 10 − 9 M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.

Structural analyses of O-glycan sugar chains on IgA1 hinge region using SELDI-TOFMS with various lectins

The aim of the study was to develop a simple and precise method for identifying glycosylation of the IgA hinge region using surface-enhanced laser desorption/ionization (SELDI)-TOFMS with a lectin-coupled ProteinChip array. Serum IgA was isolated using an anti-IgA antibody column. Following reduction, alkylation, and trypsin digestion, the IgA fragments were applied on the ProteinChip coupled with jacalin, peanut agglutinin (PNA), or Vilsa villosa lectin (VVL). The SELDI-TOFMS peaks corresponding to the fragments containing IgA1 hinge glycopeptides trapped by each lectin were compared. The jacalin-, PNA-, and VVL-immobilized ProteinChips detected 13, 4, and 2 peaks, respectively. One major peak was confirmed as a glycopeptide by MS/MS analysis. These results suggest that a lectin-immobilized ProteinChip assay can be used to simplify the procedures for the analyses of the O-glycans in IgA1 hinge. This method potentially makes it possible to identify a disease-specific glycoform by selecting the appropriate ligand-coupled ProteinChip array.

Trial of simple methods for analysis of O‐glycan structure of IgA1 hinge by SELDI‐TOFMS using jacalin‐immobilized protein chip arrays; fundamental study toward application to IgA nephropathy screening

Trial of simple methods for analysis of O‐glycan structure of IgA1 hinge by SELDI‐TOFMS using jacalin‐immobilized protein chip arrays; fundamental study toward application to IgA nephropathy screening