Exposed peptide core of IgA1 hinge region in IgA nephropathy.

BACKGROUND: The human IgA1 hinge region is a very unique O-linked glycopeptide, and its sialylation and galactosylation recently were reported to be defective in the serum IgA1 derived from patients with IgA nephropathy (IgAN). This study was performed to examine the underglycosylation of the IgA1 hinge region and consequent exposure of the peptide core in IgAN. METHODS: A polyclonal antibody against a synthetic human IgA1 hinge peptide, PVPSTPPTP SPSTPPTPSPS, (anti-sHP ab) was raised in rabbits and shown specifically to recognize the IgA1 which was treated with neuraminidase, beta-galactosidase and alpha-N-acetylgalactosaminidase. The reactivity of the anti-sHP ab against the purified serum IgA1 was compared among the following three groups: 39 patients with IgAN, 30 patients with other renal diseases (ORD) and 21 healthy controls (HC) using an enzyme-linked immunosorbent assay. RESULTS: The reactivity was significantly higher in the IgAN group (mean +/- SD of OD 490 nm: 0.327 +/- 0.059) than in the ORD group (0.274 +/- 0.043, P=0.0002) and in the HC group (0.265 +/- 0.037, P<0.0001). No significant difference was observed between the latter two groups. The frequency of positive cases (> mean +/- 2SD of HC) was 46.2% (18/39) in the IgAN group, 6.7% (2/30) in the ORD group and 0% (0/21) in the HC group. CONCLUSIONS: It was suggested that the peptide core of the IgA1 hinge region is exposed aberrantly by a defective N-acetylgalactosaminylation and plays a possible role in the pathogenesis of IgAN.     

Mass spectrometry analysis of IgA1 hinge region in patients with IgA nephropathy

BACKGROUND: Physicochemical alterations of the IgA molecule are supposed to play a pathogenetic role in IgA nephropathy (IgAN). The present study was carried out to analyze the structural variety of O-glycans on the IgA1 hinge region in IgAN. Sera from 9 IgAN patients and 9 healthy controls were individually examined to evaluate the IgA1 content and binding lectins (jacalin and Helix aspersa), using enzyme-linked immunosorbent assay (ELISA) techniques. The IgA1 from pooled sera were separated by affinity chromatography (jacalin), and the fragment containing the hinge region was prepared by pyridylethylation and trypsin treatment. The IgA fragments containing the hinge glycopeptide (33-mer hinge peptide core (HP) + O-glycans) were separated by jacalin affinity chromatography. Because we used jacalin, we only analyzed the Gal-3GalNAc residue containing IgA. The molecular weight (MW) of the IgA1 fragments was estimated using an ion trap mass spectrometer equipped with an electrospray ion source (ESI/MS). RESULTS: IgA1 concentration in pathological sera was higher than in the control serum (p<0.01). Compared with controls, serum IgA1 from IgAN patients showed significantly greater binding to the 2 lectins, jacalin (p<0.01) and Helix aspersa (HA, p<0.001), which are specific for O-linked Gal-beta1,3-GalNAc and GalNAc, respectively. Analyses of pooled sera showed that the number of O-glycosidic chains was comparable in IgAN and normal sera. With regards to the individual residues, we found that IgAN sera contained less sugar and galactose and sialic acid moieties than sera from control subjects, was reduced in IgAN sera, while terminal N-acetylgalactosamine levels were higher when compared with normal serum.CONCLUSIONS: Abnormalities of hinge region O-linked glycans were confirmed using advanced spectrometry technology. The pathogenetic implications for aggregation and defective removal of IgA1 are discussed.     

Humoral immunity against the proline-rich peptide epitope of the IgA1 hinge region in IgA nephropathy.

BACKGROUND: The human IgA1 hinge region is a unique mucin-like O-linked proline-rich glycopeptide, and its core peptide was found to be exposed aberrantly by the underglycosylation in IgA nephropathy (IgAN). We describe here the presence of humoral immunity against the IgA1 hinge peptide epitope in IgAN and evaluate the relationship between the underglycosylation of the IgA1 hinge region and humoral immunity. METHOD: The serum anti-IgA1 hinge peptide antibody (anti-alpha1HP ab) titre was measured and compared between the IgAN (n=37) and control groups (n=34) by enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide corresponding to the human IgA1 hinge region, PVPSTPPTPSPSTPPTPSPS, as an antigen. Next, to evaluate the relationship between the underglycosylation of the IgA1 hinge region and the humoral immunity, the reactivity of the serum IgG from the patients with IgAN against monoclonal IgA1 which had been digested enzymatically to remove the carbohydrates from the IgA1 hinge region was measured by ELISA. RESULTS: The anti-alpha1HP ab titre was significantly higher in the IgAN group than in the control group (OD value: IgG class, 0.564+/-0.344 vs 0. 331+/-0.154, P=0.0014; IgM class, 0.272+/-0.148 vs 0.141+/-0.072, P<0.0001) and it was positive in approximately 40% of the patients with IgAN. In addition, the reactivity of the serum IgG from the IgAN patients against the monoclonal IgA1 was found to be increased as the carbohydrates were enzymatically removed from the IgA1 hinge region (when native=100; asialo, 122+/-9.5; agalacto, 167+/-11.5; naked, 188+/-3.9). CONCLUSION: These results suggested that the peptide epitope of the IgA1 hinge region which was aberrantly exposed by underglycosylation could induce the humoral immune response in IgAN.     

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.     

Underglycosylation of IgA1 hinge plays a certain role for its glomerular deposition in IgA nephropathy

This study was performed to isolate and investigate the IgA1 that could accumulate in glomeruli (glomerulophilic IgA1). IgA1 was fractionated by the electric charge and the reactivity to Jacalin. Serum IgA1 of IgA nephropathy patients was separated and fractionated using a Jacalin column and subsequent ion-exchange chromatography. The fractions were divided into three groups of relatively cationic (C), neutral (N), and anionic (A). IgA1 was also divided into Jacalin low (L), intermediate (I), and high (H) affinity fractions by serial elution using 25, 100, and 800 mM galactose. The left kidneys of Wistar rats were perfused with 2, 5, or 10 mg of each group of IgA1. The rats were sacrificed 15 min, 30 min, 3 h, or 24 h after the perfusion. The accumulation of each IgA1 in the glomeruli was then observed by immunofluorescence. The IgA1 of the fractions N and H separated by the two methods was definitely accumulated in the rat glomeruli with a similar pattern. The electrophoresis revealed that the macromolecular IgA1 was increased in fraction H compared with other fractions. Therefore, Jacalin high-affinity IgA1(fraction H) was applied on a diethylaminoethyl column and divided into electrically cationic (HC), neutral (HN), and anionic (HA). Only the asialo-Galbeta1,3GalNAc chain was identified in the fraction HN IgA1 by gas-phase hydrazinolysis. Furthermore, the IgA1 fraction was strongly recognized by peanut agglutinin, Vicia Villosa lectins, and antisynthetic hinge peptide antibody. These results indicated that the IgA1 molecules having the underglycosylated hinge glycopeptide played a certain role in the glomerular accumulation of IgA1 in IgA nephropathy.     

Detection of gender difference and epitope specificity of IgG antibody activity against IgA1 hinge portion in IgA nephropathy patients by using synthetic hinge peptide and glycopeptide probes

Background and Aims: There are many reports on the presence of an incompletely glycosylated O‐linked oligosaccharide(s) on the IgA1 hinge region in some immunoglobulin (IgA) nephropathy patients. Furthermore, the production of an antibody against the naked hinge peptide portion was reported in an IgA nephropathy patient. In this report, characterization of the IgG antibody against the hinge portion was carried out by using synthetic hinge glycopeptide probes. Methods and Results: The following synthetic hinge peptide and glycopeptides were prepared: 19mer peptide, V‐P‐S‐T‐P‐P‐T‐P‐S‐P‐S‐T‐P‐P‐T‐P‐S‐P‐S (designated HP), the peptide having a single α‐linked GalNAc residue at positions 4, 7, 9, 11 and 15 (4 GN ? 15 GN, respectively) and the same peptide having five GalNAc residues at all five positions (GN5). The mean value of the antibody activity against these probes was compared with each other. The highest activity against the naked hinge peptide (HP) and lowest activity against the fully glycosylated hinge peptide (GN5) were obvious. As attachment of GalNAc to position 4 or 11 on the peptide brought about a significant reduction of the activity against the naked hinge peptide, the P‐S‐T‐P sequence included in both positions was thought to be the most probable site recognized by these antibodies. As an additional unexpected observation, a gender difference in this antibody activity against all the probes was found. The antibody activity in a female was significantly higher compared with that in a male. Conclusion: Because the frequency of incidence of IgA nephropathy is known to be slightly higher in males, this gender difference might indicate a protective meaning to remove aberrantly glycosylated molecules from the patient's serum.     

Abnormalities of IgA1 production in IgA nephropathy

SUMMARY: IgA nephropathy (IgAN) is characterized by the mesangial deposition of polymeric IgA1 (plgA1). the original view that this plgA1 is derived from the mucosal immune system can no longer be sustained. Studies of duodenal mucosa and marrow indicate increased production of plgA1 in the marrow and decreased production in the mucosa. These changes are consistent with immunization studies showing exaggerated and prolonged plgA responses to systemic immunization, and reduced mucosal responses to mucosal neoantigens. However, the IgA1 and IgG systemic responses to mucosal antigen are increased in IgAN, a finding consistent with impairment in oral tolerance, the process by which systemic immune responses, to mucosal antigen challenge are normally suppressed. Both IgA1 production and the induction of oral tolerance are under T-cell control. T-cell populations involved in these processes include γδ T cells, Tr cells and T-helper (Th)3 cells; cytokines with a key role in the control of IgA production include interleukin (IL)-10 and transforming growth factor (TGF)-β. There is evidence of abnormal γδ T-cell V region usage in both mucosa and marrow in IgAN. Increased expression of relevant cytokines has also been reported in circulating T cells in IgAN. the increased O-glycosylation of circulating IgA1 in IgAN may also be further evidence of a shift in the production of mucosal-type plgA1 from the mucosa to marrow. These findings suggest that the specific lymphocyte homing mechanisms that normally maintain oral tolerance and control the site of IgA production require further study in IgAN.     

IgA肾病患者IgA1糖基化异常及其致病机制

IgA肾病(IgAN)是导致终末期肾病最常见的原发性肾小球疾病。其病理特点为IgA1在肾小球系膜区沉积,IgA1分子的异常糖基化是导致IgAN发病的关键因素。多种与IgAN相关的基因位点已经被发现。这些基因编码的细胞因子参与了IgA1糖基化异常的发病机制。此外糖基化酶缺乏、分子伴侣甲基化异常都可能导致IgA1异常糖基化。异常糖基化的IgA1可通过自我聚集或形成免疫复合物沉积于系膜区,进而刺激系膜细胞增殖、分泌系膜基质、细胞因子、趋化因子、生长因子等,导致肾小球损伤。对IgA1异常糖基化的深入研究有助于了解IgA肾病的发病机制并提供新的诊断与治疗措施。     

The pathogenic role of IgA1 O-linked glycosylation in the pathogenesis of IgA nephropathy

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.     

Contribution of macromolecular IgA1 to IgA abnormality in IgA nephropathy

To evaluate the contribution of macromolecular IgA₁ to IgA abnormality in childhood IgA nephropathy, serum samples from 29 healthy children and 26 patients with IgA nephropathy in different age-groups (7–9, 10–12, and 13–15 years) were each separated by sucrose density gradient ultracentrifugation and assayed for IgA₁ using an enzyme-linked immunosorbent assay. IgA₁ in fraction I (sedimentation coefficient >11.4s) was significantly greater in patients 7–15 years of age (median 36.3–57.0 mg/dl) than in the age-matched controls (median 8.8–10.4 mg/dl). IgA₁ in fraction II (11.4–9.3s) was significantly greater in patients 10–15 years of age (median 46.7–52.6 mg/dl) than in the controls (median 27.8–35.5 mg/dl), and IgA₁ in fraction III (<9.3s) was significantly greater in patients 13–15 years of age (median 156.9 mg/dl) than in the controls (median 120.7 mg/dl). The ratio of IgA₁ in fractions I–III was higher in the patients of each age-group (median 0.233–0.314) than in the controls (median 0.067–0.082), while the ratio of IgA₁ in fractions II–III was not significantly high in patients 7–12 years old (median 0.268 to 0.318) compared with the controls (median 0.182–0.264). Thus, IgA abnormality in childhood IgA nephropathy would be better represented by an increase in macromolecular IgA₁ of >11.4s than by an increase in IgA₁ in fractions of 11.4–9.3s or <9.3s. Received: 8 December 1999 / Revised: 2 March 2000 / Accepted: 2 May 2000     

Light-chain composition of serum IgA1 and in vitro IgA1 production in IgA nephropathy.

A predominant expression of IgA1 in mesangial deposits, serum, and bone marrow culture supernatants has been shown in IgA nephropathy (IgAN). Furthermore an excess of lambda light chains in both mesangial deposits and serum IgA has been observed. However, the origin of mesangial IgA remains controversial. In the present study, we have examined the IgA1 light chain type in IgAN. Total IgA1, IgA1 kappa and IgA1 lambda were measured by ELISA in serum and culture supernatants from spontaneous and pokeweed-mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMC). We observed an increase in IgA and IgA1 serum concentrations in IgA nephropathy patients, with a ratio of serum IgA1 to total serum IgA identical between patients and controls. The concentration of serum IgA kappa did not differ between patients and controls but patients had a significantly higher concentration of serum IgA lambda. The IgA1 kappa to IgA1 lambda ratio was 1.06 +/- 0.42 in IgAN patients versus 1.55 +/- 0.36 in controls (P less than 0.01). By contrast, the concentrations of IgA1 kappa and IgA1 lambda in PBMC culture supernatants, both spontaneous and PWM-stimulated, were identical in patients and controls. Therefore, there is a specific increase in IgA1 lambda in patients' sera. This contrasts with the normal IgA1 production by PBMC, which are derived from mucosal-associated lymphoid tissues. This suggests that IgA isotypic deregulation is confined to the bone marrow compartment and is not a generalised defect of the IgA system.     

Pathogenetic significance of aberrant glycosylation of IgA1 in IgA nephropathy

IgA nephropathy (IgAN), the most common form of primary glomerulonephritis worldwide, is defined by predominant IgA1 deposits in the glomerular mesangium. Among abnormalities of the IgA immune system reported so far in IgAN, aberrant O-linked glycosylation in the hinge region of IgA1 is the most consistent finding. IgA1 molecules bearing abnormal glycosylation have been found in serum, in tonsillar lymphocytes, and in eluate from mesangial deposits, and characterized by decreased O-linked N-acetylgalactosamine residues with or without alteration in the terminal sialylation of the O-linked sugars. IgA1 with incomplete galactosylation has a tendency to accumulate in glomerular mesangium by self-aggregation or immune complex formation. Glomerular mesangial cells exposed to immune complexes of these IgA1 can proliferate and secrete cytokines, chemokines, growth factors, and extracellular matrix components promoting inflammatory reactions in the glomeruli. Although genes encoding enzymes involved in the O-glycosylation process, such as C1GALT1, have been reported to be responsible for susceptibility to IgAN, recent evidence suggests that the abnormality is restricted to a small fraction of B cell populations and arises from dysregulated IgA1 production and secretion in mucosal immune system. This review will focus on and discuss the role of incompleteness of IgA1 O-galactosylation in the pathogenesis of IgAN and propose a possible mechanism in which abnormal IgA1 occurs in IgAN. Presented at the 37th Eastern Regional Meeting of the Japanese Society of Nephrology.     

Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy

BACKGROUND: The IgA1 molecule, which is predominantly deposited in glomeruli in IgA nephropathy (IgAN), is a unique serum glycoprotein because it has O-glycan side chains in its hinge region. Our study was conducted to investigate the O-glycan structure in the glomerular IgA1 in IgAN. METHODS: The IgA1 was separated from 290 renal biopsy specimens of 278 IgAN patients and from four serum IgA1 samples (IgAN, 2; control, 2). The variety of O-glycan glycoform was determined by estimating the precise molecular weights of the IgA1 hinge glycopeptides using matrix-assisted laser desorption ionization time of flight mass spectrometry. RESULTS: The peak distribution of IgA1 hinge glycopeptides clearly shifted to lesser molecular weights in both glomerular and serum IgA1 in IgAN compared with the serum IgA1 of controls. In the five major peaks of IgA1 hinge glycopeptides in each sample, the numbers of carbohydrates composing O-glycans (GalNAc, Gal, and NANA) in the deposited and serum IgA1 in IgAN patients were significantly fewer than those in the serum IgA1 in the control groups. CONCLUSION: The O-glycan side chains in the hinge of the glomerular IgA1 were highly underglycosylated in IgAN. These results indicate that the decreased sialylation and galactosylation of the IgA1 hinge glycopeptides play a crucial role in its glomerular deposition in IgAN.     

IgA1分子异常糖基化在IgA肾病发病机制中的研究进展

自1968年IgA肾病(IgAN)首次报道,已40余年,其发病机制目前尚不清楚.Andre等[1]首次报道IgAN患者血清IgA1分子铰链区糖链结构异常,随后,在IgAN患者血清、扁桃体及肾脏组织中均检测到IgA1分子糖链结构异常.很多研究对参与IgA1糖基化过程的各种酶及分子伴侣的表达、活性及基因水平进行了分析,同时利用体外培养的细胞或动物模型进行研究,以寻找IgA1分子糖基化异常在IgAN发病及疾病进展中的可能作用.本文综述近年来IgAN中有关IgA1分子糖基化异常的研究进展,并介绍本研究所的相关研究.     

Methodological approaches to the analysis of IgA1 O-glycosylation in IgA nephropathy.

IgA nephropathy (IgAN) is a common form of glomerulonephritis in which IgA1 molecules deposit in the renal mesangium, leading to progressive glomerular inflammatory injury in a significant proportion of patients. The mechanisms underlying the pathogenesis of IgAN remain poorly understood, but altered O-glycosylation, a physicochemical abnormality of IgA1 observed in these patients, may be a contributory factor. Although many studies have reported aberrant IgA1 O-glycosylation in IgAN, the precise structural nature of the defect remains to be fully characterised, and analysis of IgA1 O-glycans has proved technically challenging. Three main strategies have been employed: lectin binding to the O-glycans in situ on the whole IgA1 molecule; mass spectroscopy of isolated O-glycosylated glycopeptides; and size/charge separation of free O-glycans released from IgA1. In this review, the basic principles, strengths and weaknesses of each of these methodological approaches are considered, together with a summary of the data obtained from their use. One of the common criticisms of many studies of IgA1 O-glycosylation is the method of IgA1 purification employed, and therefore, this issue is also critically discussed.