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.     

O-glycosylation of serum IgD in IgA nephropathy

In IgA nephropathy (IgAN), serum IgA1 with abnormal O-glycosylation preferentially deposits in the glomerular mesangium. The control of O-glycosylation is poorly understood. Among Ig isotypes, only IgD, produced early in B cell development, and IgA1, produced by mature B cells, are O-glycosylated. For investigation of the stage of B cell maturation at which the defect seen in IgAN arises, the O-glycosylation of serum IgA1 and IgD was studied in IgAN and controls. Serum was obtained from 20 patients with IgAN and 20 control subjects. The O-glycosylation profiles of native and desialylated IgA1 and IgD were measured in an ELISA-type system using the lectins Helix aspersa and peanut agglutinin, which bind to alternative forms of O-glycan moieties. The lectin-binding patterns of the two immunoglobulins differed in all participants, with that of IgD suggesting that it is more heavily galactosylated than IgA1. Defective O-glycosylation of IgA1, probably taking the form of reduced galactosylation, was confirmed in IgAN in this study. This undergalactosylation was not shared by IgD; in contrast, IgD carried more galactosylated O-glycans in IgAN than controls. The contrasting lectin-binding patterns of IgA1 and IgD shows that Ig O-glycosylation is differentially controlled during B cell maturation. Compared with controls, O-glycosylation in IgAN is incomplete in IgA1 but more complete in IgD. These observations show that abnormal IgA1 O-glycosylation in IgAN is not due to an inherent defect in glycosylation mechanisms but arises only at a later stage in B cell development and may be secondary to aberrant immunoregulation.     

Heterogeneity of carbohydrate moieties of IgA1 molecules from IgA nephropathy patients and normal individuals

Summary: IgA nephropathy (IgAN) is characterized by the deposition of IgA1 in kidney mesangia and the presence of IgA1-containing immune complexes in the circulation. Structural studies of IgA1 isolated from sera of IgAN patients indicated a statistically significant decrease in the content of galactose (Gal). Using a combination of lectins specific for glycans in O- or N-linked glycan side chains, this Gal deficiency was restricted to O-linked glycans present in the hinge region of IgA1 molecules. Gal-deficient IgA1 displayed a significantly higher binding to mesangial cells through a putative non-internalizing receptor specific for N-acetyl galactosamine (GalNAc) in O-linked glycans. These data suggest that Gal deficiency results in diversion of IgA1 molecules from the usual degradative pathway and deposition of altered IgA1 in the mesangium.     

Analysis of IgA1 O-glycans in IgA nephropathy by fluorophore-assisted carbohydrate electrophoresis.

Abnormal O-glycosylation of IgA1 may contribute to pathogenic mechanisms in IgA nephropathy (IgAN). Observations of altered lectin binding to IgA1 in IgAN suggest that the O-glycan chains may be undergalactosylated, but precise structural definition of the defect has proved technically difficult, and it remains unconfirmed. This is the first study using fluorophore-assisted carbohydrate electrophoresis (FACE) to analyze IgA1 O-glycans in IgAN and controls. IgA1 was purified from serum, and the intact O-glycans were released by hydrazinolysis at 60 degrees C. After re-N-acetylation, the glycans were fluorophore-labeled and separated by polyacrylamide gel electrophoresis. Sequential exoglycosidase digestions of IgA1 allowed identification of the different O-glycan bands on FACE gels, and their relative frequencies in IgA1 samples were measured by ultraviolet densitometry. Lectin binding of the IgA1 samples was also measured. In some patients with IgAN, FACE analysis demonstrated a significant increase in the percentage of IgA1 O-glycan chains consisting of single N-acetyl galactosamine (GalNAc) units rather than the more usual galactosylated and sialylated forms. This finding was confirmed using both desialylated IgA1 and enzymatically released O-glycans. Good correlation was also found between O-glycan agalactosylation on FACE analysis and IgA1 lectin binding in IgAN, supporting the value of lectins as tools for detection of this abnormality. This is the first study in which all of the predicted O-glycan forms of IgA1 have been analyzed simultaneously, and demonstrates that in IgAN, the IgA1 Oglycan chains are truncated, with increased terminal GalNAc. This abnormality has the potential to significantly affect IgA1 behavior and handling with pathogenic consequences in IgAN.     

Structural heterogeneity of glycans in IgA molecules: Implications for IgA nephropathy

Summary: The carbohydrate moieties on glycoproteins, including immunoglobulins (Ig), are involved in a broad spectrum of biological functions. As revealed by enzymatic or chemical removal of carbohydrate moieties, inhibition of glycosylation, or site-directed mutagenesis of asparagine residues to prevent N-linked glycosylation, carbohydrates on Ig have been shown to participate in binding, internalization and catabolism by hepatocytes or other cells, binding to Fc receptors on phagocytic cells, activation of complement, and opsonization. the structure of human IgA1 is unique among all Ig. the heavy chain contains a hinge region with a characteristic primary structure not seen in any other Ig, and which contains five short O-linked oligosaccharide side-chains composed of serine-linked N-acetylgalactosamine (GalNAc) and βl-3-linked galactose (Gal). Both of these monosaccharides may be sialylated. In contrast to ubiquitous N-linked side-chains, O-linked carbohydrate moieties are found rarely among human serum glycoproteins. We have demonstrated that IgA1 proteins from the sera of patients with IgA nephropathy (IgAN) are galactosylated to a lesser extent than those from healthy controls. Decreased content of Gal and decreased reactivity of IgA from IgAN patients with lectins specific for GalNAc indicate that these structural changes occur on glycans located in the hinge region of IgA1. Thus, in addition to rheumatoid arthritis, systemic lupus erythmatosus, inflammatory bowel disease and other disorders, IgA nephropathy may represent another example of a chronic disease in which aberrancies of carbohydrates are observed and may participate in aetiopathogenesis.     

Autoantibodies targeting galactose-deficient IgA1 associate with progression of IgA nephropathy

Mesangial and circulating IgA1 with aberrantly glycosylated hinge region O-glycans characterize IgA nephropathy (IgAN). Unlike healthy individuals, some IgA1 is galactose deficient in patients with IgAN, leaving terminal N-acetylgalactosamine residues in the hinge region exposed. Circulating autoantibodies that recognize such galactose-deficient IgA1 as an autoantigen, or the levels of the autoantigen itself, may allow prediction of disease progression. Here, we analyzed serum samples obtained at diagnosis for autoantigen and autoantibodies from 97 patients with IgAN selected from our prospective cohort according to their absolute renal risk for progression to dialysis or death (0, very low; 1, low; 2, high; 3, very high). We also analyzed samples from controls comprising 30 healthy volunteers and 30 patients with non-IgAN disease. The mean follow-up was 13.8 years. We found that mean serum levels of total autoantigen, normalized IgG autoantibody, and total IgA autoantibody were significantly higher in patients than in the combined controls (all P≤0.01). Furthermore, increasing levels correlated with worse clinical outcomes. In Cox regression and Kaplan-Meier analyses, IgG autoantibody levels ≥1.33 predicted dialysis or death (both P≤0.01). In conclusion, these data suggest that serum levels of IgG and IgA autoantibodies strongly associate with the progression of IgAN nephropathy.     

Aberrantly glycosylated serum IgA1 are closely associated with pathologic phenotypes of IgA nephropathy

BACKGROUND: IgA nephropathy (IgAN) is the most common glomerulonephritis with various histologic and clinical phenotypes. The mechanisms underlying the pathogenesis of IgAN remained unclear. But now altered O-glycosylation of serum IgA1 observed in these patients was considered to be a key contributory factor. The aim of the current study is to investigate whether aberrantly glycosylated IgA1 was associated with pathologic phenotypes of IgAN. METHODS: Sera from 107 patients with IgAN recently diagnosed were collected. Fifty patients were with mild mesangial proliferative IgAN, the others were with focal proliferative and sclerosing IgAN. Sera from 22 normal blood donors were used as normal controls. Biotinylated lectins were used in enzyme-linked immunosorbent assay (ELISA) to examine different glycans on IgA1 molecules. The alpha2,6 sialic acid was detected by elderberry bark lectin (SNA), the exposure of terminal galactose (Gal) and N-acetylgalactosamine (GalNAc) were detected by arachis hypogaea [peanut agglutinin (PNA)] and vilsa villosa lectin (VVL), respectively. The serum IgA1 glycans levels corrected by serum IgA1 concentrations were compared between patients and controls. RESULTS: Reduced terminal alpha2,6 sialic acid (1.16 +/- 0.21 vs. 0.98 +/- 0.31) (P= 0.008) and galactosylation (0.30 +/- 0.29 vs. 0.16 +/- 0.19) (P= 0.029) increased exposure of (GalNAc) (0.00 vs. 0.03) (P= 0.024) were demonstrated in serum IgA1 from patients with IgAN as compared with those in controls. More important, the exposures of 2,6 sialic acid and Gal were significantly decreased, especially in patients with focal proliferative and sclerosing IgAN compared with that in patients with mild mesangial proliferative IgAN (0.91 +/- 0.34 vs. 1.05 +/- 0.25) (P= 0.014) (0.108 +/- 0.137 vs. 0.221 +/- 0.219) (P= 0.018). However, no significant difference was found between patients with mild mesangial proliferative IgAN and normal controls (P > 0.05). The exposure of GalNAc of serum IgA1 from patients with focal proliferative and sclerosing IgAN was significantly higher than that of controls (P= 0.017), but had no statistical difference with that of patients with mild mesangial proliferative IgAN. CONCLUSION: The desialylation and degalactosylation of IgA1 in sera of patients with IgAN were closely associated with pathologic phenotypes.     

Structural features of IgA molecules which contribute to IgA nephropathy.

IgA nephropathy (IgAN) is characterised by the mesangial deposition of polymeric IgA1 (pIgA1). pIgA1 production is reduced in the mucosal immune system in IgAN and increased in the marrow; this switch may be secondary to a defect in gammadeltaT cell control of IgA production. However this does not explain the mechanism by which pIgA1 deposits in the mesangium. There is no direct evidence that classical immune complex deposition occurs in IgAN and alternative mechanisms resulting from physicochemical abnormalities of the IgA1 molecule, particular altered glycosylation, have been proposed. IgA1 has a distinctive hinge region which is a site for O-glycosylation. There is reduced terminal galactose on the hinge region O-glycans of circulating IgA1 in IgAN, perhaps due to a defect in B cell beta1,3 galactosyltransferase. A concomitant O-glycan defect in mesangial IgA1 has not yet been proven. Altered hinge O-glycosylation may have substantial impact on the quaternary structure of the IgA1 molecule influencing its capacity to interact with matrix proteins, IgA receptors on mesangial cells and leucocytes, and complement; it may therefore play a key role in the pathogenesis of mesangial deposition of IgA1 and subsequent glomerular injury in IgAN.     

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.     

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.     

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.     

Role of aberrant glycosylation of IgA1 molecules in the pathogenesis of IgA nephropathy

Studies of the properties of immune complexes (IC) in the circulation, urine, and mesangium of IgA nephropathy (IgAN) patients have provided data relevant to the pathogenesis of this disease. IC contain predominantly polymeric IgA1 molecules which are deficient in galactose (Gal) residues on O-linked glycan chains in the hinge region (HR) of their heavy (H) chains. As a result of this aberrancy, a novel antigenic determinant(s) involving N-acetylgalactosamine (GalNAc) and perhaps sialic acid (SA) of O-linked glycans is generated and recognized by naturally occurring GalNAc-specific antibodies. Thus, IC in IgAN consist of Gal-deficient IgA1 molecules as an antigen, and GalNAc-specific IgG and/or IgA1 as an antibody. IgG antibodies to Gal-deficient IgA1 are probably induced by cross-reactive microbial antigens; they are present at variable levels not only in humans with or without IgAN but also in many phylogenetically diverse vertebrate species. Incubation of human mesangial cells with IC from sera of IgAN patients indicated that stimulation of cellular proliferative activity was restricted to the large (>800 kDa) complexes. These findings suggest that experimental approaches that prevent the formation of large Gal-deficient IgA1-IgG IC may be applied ultimately in an immunologically mediated therapy.     

血清低半乳糖化IgA1测定对鉴别IgA肾病的临床价值

目的 确定血清低半乳糖化IgA1对鉴别诊断IgA肾病的临床价值。 方法 以原发性肾小球疾病患者91例为研究对象，接受肾活检并留取血清；以健康体检者20例血清作为对照。血清标本先用装有耦联蚕豆凝集素的微球进行微量离心柱法分离并洗脱，获得低半乳糖化IgA1。再以凝集素HAA（Helix aspersa）用ELISA法定量检测异常糖基化IgA1（HAA-IgA1）。分析血清低半乳糖IgA1升高在鉴别诊断IgA肾病方面的临床价值。 结果 48例IgA肾病患者HAA-IgA1水平[（83.7±41.0） U]高于健康对照组[（52.6±22.9） U]及43例其他原发性肾小球疾病患者组[（49.2±27.3） U]（均P < 0.01）。而该43例中，非IgA系膜增殖性肾炎患者22例（51%）的HAA-IgA1水平[（47.6±21.5） U]亦显著低于IgA肾病患者。以肾穿刺病理诊断为金标准，所绘制ROC曲线面积为0.797，面积的标准误为0.047（P < 0.01）；鉴别诊断IgA肾病的灵敏度为72.9%，特异度为72.1%，准确度为72.5%。 结论 应用微量离心柱法联合ELISA法检测IgA肾病患者血清低半乳糖IgA1对于鉴别诊断IgA肾病具有一定临床价值。     

Integrin expression and IgA nephropathy: in vitro modulation by IgA with altered glycosylation and macromolecular IgA

BACKGROUND: Signal transduction by mesangial cell (MC) integrins regulates cell growth and survival, extracellular matrix production, and organization. The aim of the study was to investigate human MC integrin modulation by differently glycosylated IgA and macromolecular IgA, which are thought to play a pathogenetic role in IgA nephropathy (IgAN). METHODS: MCs were incubated with purified human polymeric IgA, heat-aggregated IgA, IgA glycoforms generated by enzymatic hydrolysis of saccharide residues and serum fractions from IgAN patients, and controls isolated by lectin affinity and containing IgA with peculiar glycan patterns. Integrins were quantitated by flow cytometry. RESULTS: Cultured MCs highly expressed alphavbeta3 and some alpha3beta1; alphavbeta3 was up-regulated by matrix components (P < 0.02). In vitro desialylated and degalactosylated polymeric human IgA enhanced alphavbeta3 expression on cultured MCs (P < 0.001). Serum IgA glycoforms isolated from IgAN patients with high exposure of internal sugars, GalNAc, Neu5Ac2,6GalNAc, and Man enhanced alphav expression on cultured MCs more than healthy controls. CONCLUSIONS.: These data support the hypothesis that IgA glycation plays a role in modulating the cell-matrix interaction, and that this mechanism can be operating in IgAN.     

Leucocyte beta 1,3 galactosyltransferase activity in IgA nephropathy

BACKGROUND: Reduced galactosylation of the O-linked glycans of the IgA1 hinge region in IgAN has recently been described. To investigate the underlying defect resulting in this abnormality, we have measured the activity of beta 1,3 galactosyltransferase, the enzyme responsible for galactosylation of O-linked sugars. METHODS: A galactose-acceptor substrate was prepared from degalactosylated hinge region fragments of normal IgA1, and incubated with the T cell, B cell, and monocyte lysates from patients with IgAN and controls for acceptor regalactosylation. The extent of acceptor galactosylation was then measured with biotinylated Vicia villosa lectin (VV), which is specific for ungalactosylated moieties. Lectin binding of serum IgA from the same subjects was also measured. RESULTS: T cell and monocyte beta 1,3 galactosyltransferase activities did not differ between IgAN and control, but B cell lysates in IgAN showed significantly lower beta 1,3 galactosyltransferase activity than control (6.2 +/- 0.71 vs. 9.5 +/- 1.03 AU/microgram, P = 0.018). Furthermore, B cell beta 1,3 galactosyltransferase activity showed a negative correlation (r = - 0.87, P = 0.002) with VV lectin binding of serum IgA in IgAN, but not controls. CONCLUSIONS: These data indicate that altered IgA1 O- galactosylation in IgAN results from a B cell-restricted reduction of beta 1,3 galactosyltransferase activity. This enzyme defect may be a fundamental pathogenic abnormality in IgAN.      

Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels

Immunoglobulin A (IgA) nephropathy is the most prevalent form of glomerulonephritis worldwide. A renal biopsy is required for an accurate diagnosis, as no convenient biomarker is currently available. We developed a serological test based upon the observation that this nephropathy is characterized by undergalactosylated IgA1 in the circulation and in mesangial immune deposits. In the absence of galactose, the terminal saccharide of O-linked chains in the hinge region of IgA1 is terminal or sialylated N-acetylgalactosamine. A lectin from Helix aspersa, recognizing N-acetylgalactosamine, was used to develop an enzyme-linked immunosorbent assay that measures galactose-deficient IgA1 in serum. The median serum lectin-binding IgA1 level was significantly higher for 153 Caucasian adult patients with IgA nephropathy without progression to end-stage renal disease as compared with that for 150 healthy Caucasian adult controls. As the lectin-binding IgA1 levels for the controls were not normally distributed, the 90th percentile was used for determination of significant elevation. Using a value of 1076 U/ml as the upper limit of normal, 117 of the 153 patients with IgA nephropathy had an elevated serum lectin-binding IgA1 level. The sensitivity as a diagnostic test was 76.5%, with specificity 94%; the positive predictive value was 88.6% and the negative predictive value was 78.9%. We conclude that this lectin-binding assay may have potential as a noninvasive diagnostic test for IgA nephropathy.     

Tonsillectomy and IgA nephritis

IgA nephritis (IgAN) is an autoimmune disease characterized by deposits of IgA in the glomerular mesangium. Clinically, the disease may be punctuated by episodes of macroscopic haematuria often associated with pharingotonsillitis or may be oligosyntomatic with microscopic haematuria and mild proteinuria. The natural course of IgAN may be indolent and benign; however, some 30-50% of patients may progress to end-stage renal disease when follow-up is extended to ≥20 years. In patients with IgAN, circulating IgA1 molecules have an aberrant structure of O-glycans in the hinge region, which is characterized by abbreviated glycans composed of N-acetylgalactosamine, with or without sialic acid. These aberrant IgA1 trigger the production of autoantibodies, with formation of immune complexes that deposit in the mesangium causing inflammation and production of extracellular matrix. A number of experimental and clinical data outlined a possible pathogenetic role of tonsillitis. As a consequence, tonsillectomy has been frequently performed in Japan. Observational studies, made in patients with normal renal function and mild proteinuria, reported that tonsillectomy could reduce the episodes of macrohaematuria as well as the entity of microhaematuria and proteinuria. However, the available studies had short-term follow-up and could not asses the role of tonsillectomy in protecting from renal function deterioration. In a longitudinal retrospective study, Isseki et al. compared the outcome of tonsillectomized patients with IgAN with that of IgAN patients who did not receive tonsillectomy. Tonsillectomized patients had a higher number of remissions and a better slope of glomerular filtration rate in comparison with controls. These data are interesting and suggest that tonsillectomy may prevent renal dysfunction in patients with IgAN and normal renal function. However, the retrospective nature of the study and the presence of some confounding factors require further investigations to confirm these promising data.     

Glycosylation of circulating IgA in patients with IgA nephropathy modulates proliferation and apoptosis of mesangial cells

Abnormalities in circulating IgA1 have been demonstrated in patients with IgA nephropathy (IgAN). This study addresses the question of the functional significance of this alteration in creating mesangial injury. Biologic effects of selected IgA glycoforms isolated from serum of IgAN patients and controls and in vitro deglycosylated normal IgA were tested on cultured human mesangial cells (MC). IgA glycoforms, ranging from 250 to 500 kD molecular weight, were isolated by lectin affinity chromatography followed by HPLC. IgA and IgG content was measured by enzyme-linked immunosorbent assay. HPLC fractions were incubated with MC to evaluate proliferation and apoptosis rates and nitric oxide synthesis. Moreover, MC were conditioned with in vitro desialylated and degalactosylated normal IgA. Patients with IgAN displayed increased levels of IgA glycoforms exposing sialic acid in alpha2,6 linkage with N-acetylgalactosamine (Neu5Acalpha2,6GalNAc) (P < 0.02) and GalNAc (P < 0.05), indicating truncation of O-linked glycans of IgA1. Moreover, IgA glycoforms with increased exposure of mannose were observed (P < 0.03), suggesting a defective N-linked glycosylation. No modification in IgG glycosylation was detected. When incubated with MC, the IgA glycoforms isolated from patients with increased exposure of GalNAc, Neu5Acalpha2,6GalNAc, or mannose, significantly depressed the proliferation and increased the apoptotic rate and nitric oxide synthesis activity of cultured MC, in comparison with fractions isolated from controls. Similarly, in vitro desialylated and degalactosylated IgAs significantly depressed the proliferation and enhanced the apoptosis rates of MC. In conclusion, a significant modulation of several human MC functions exerted by serum IgA with increased exposure of GalNAc, Neu5Acalpha2,6GalNAc, and mannose residues isolated from IgAN patients is reported for the first time.     

Differential glycosylation of polymeric and monomeric IgA: a possible role in glomerular inflammation in IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA1 (pIgA1) and complement. Complement activation via mannose-binding lectin and the lectin pathway is associated with disease progression. Furthermore, recent studies have indicated a possible role for secretory IgA. IgAN is associated with abnormalities in circulating IgA, including aberrant O-linked glycosylation. This study characterized and compared functional properties and N-linked glycosylation of highly purified monomeric IgA (mIgA) and pIgA from patients with IgAN and control subjects. Total serum IgA was affinity-purified from patients (n = 11) and control subjects (n = 11) followed by size separation. pIgA but not mIgA contained secretory IgA, and its concentration was significantly higher in patients with IgAN than in control subjects. Both in patients with IgAN and in control subjects, IgA binding to the GalNAc-specific lectin Helix Aspersa and to mannose-binding lectin was much stronger for pIgA than for mIgA. Furthermore, binding of IgA to mesangial cells largely was restricted to polymeric IgA. Binding of pIgA to mesangial cells resulted in increased production of IL-8, predominantly with IgA from patients with IgAN. Quantitative analysis of N-linked glycosylation of IgA heavy chains showed significant differences in glycan composition between mIgA and pIgA, including the presence of oligomannose exclusively on pIgA. In conclusion, binding and activation of mesangial cells, as well as lectin pathway activation, is a predominant characteristic of pIgA as opposed to mIgA. Furthermore, pIgA has different N-glycans, which may recruit lectins of the inflammatory pathway. These results underscore the role of pIgA in glomerular inflammation in IgAN.