Experimental evidence of pathogenic role of IgG autoantibodies in IgA nephropathy.

BACKGROUND: IgA nephropathy is thought to be an autoimmune disease wherein galactose-deficient IgA1 (Gd-IgA1) is recognized by IgG autoantibodies, resulting in formation and renal accumulation of nephritogenic immune complexes. Although this hypothesis is supported by recent findings that, in renal immunodeposits of IgA nephropathy patients, IgG is enriched for Gd-IgA1-specific autoantibodies, experimental proof is still lacking. METHODS: IgG isolated from sera of IgA nephropathy patients or produced as a recombinant IgG (rIgG) was mixed with human Gd-IgA1 to form immune complexes. IgG from healthy individuals served as a control. Nude and SCID mice were injected with human IgG and Gd-IgA1, in immune complexes or individually, and their presence in kidneys was ascertained by immunofluorescence. Pathologic changes in the glomeruli were evaluated by quantitative morphometry and exploratory transcriptomic profiling was performed by RNA-Seq. RESULTS: Immunodeficient mice injected with Gd-IgA1 mixed with IgG autoantibodies from patients with IgA nephropathy, but not Gd-IgA1 mixed with IgG from healthy individuals, displayed IgA, IgG, and mouse complement C3 glomerular deposits and mesangioproliferative glomerular injury with hematuria and proteinuria. Un-complexed Gd-IgA1 or IgG did not induce pathological changes. Moreover, Gd-IgA1-rIgG immune complexes injected into immunodeficient mice induced histopathological changes characteristic of human disease. Exploratory transcriptome profiling of mouse kidney tissues indicated that these immune complexes altered gene expression of multiple pathways, in concordance with the changes observed in kidney biopsies of patients with IgA nephropathy. CONCLUSIONS: This study provides the first in vivo evidence for a pathogenic role of IgG autoantibodies specific for Gd-IgA1 in the pathogenesis of IgA nephropathy.

Quantitative assessment of successive carbohydrate additions to the clustered O-glycosylation sites of IgA1 by glycosyltransferases.

Mucin-type O-glycosylation occurs on many proteins that transit the Golgi apparatus. These glycans impact structure and function of many proteins and have important roles in cellular biosynthetic processes, signaling and differentiation. Although recent technological advances have enhanced our ability to profile glycosylation of glycoproteins, limitations in the understanding of the biosynthesis of these glycan structures remain. Some of these limitations stem from the difficulty to track the biosynthetic process of mucin-type O-glycosylation, especially when glycans occur in dense clusters in repeat regions of proteins, such as the mucins or immunoglobulin A1 (IgA1). Here, we describe a series of nano-liquid chromatography (LC)-mass spectrometry (MS) analyses that demonstrate the range of glycosyltransferase enzymatic activities involved in the biosynthesis of clustered O-glycans on IgA1. By utilizing nano-LC-MS relative quantitation of in vitro reaction products, our results provide unique insights into the biosynthesis of clustered IgA1 O-glycans. We have developed a workflow to determine glycoform-specific apparent rates of a human UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltrasnfersase (GalNAc-T EC 2.4.1.41) and demonstrated how pre-existing glycans affect subsequent activity of glycosyltransferases, such as core 1 galactosyltransferase and α2,3- and α2,6-specific sialyltransferases, in successive additions in the biosynthesis of clustered O-glycans. In the context of IgA1, these results have potential to provide insight into the molecular mechanisms implicated in the pathogenesis of IgA nephropathy, an autoimmune renal disease involving aberrant IgA1 O-glycosylation. In a broader sense, these methods and workflows are applicable to the studies of the concerted and competing functions of other glycosyltransferases that initiate and extend mucin-type core 1 clustered O-glycosylation.

Glycan Positioning Impacts HIV-1 Env Glycan-Shield Density, Function, and Recognition by Antibodies.

HIV-1 envelope (Env) N-glycosylation impact virus-cell entry and immune evasion. How each glycan interacts to shape the Env-protein-sugar complex and affects Env function is not well understood. Here, analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation sites affected the Env structure at distant locations and had a ripple effect on Env-wide glycan processing, virus infectivity, antibody recognition, and virus neutralization. Specifically, the N262 glycan, although not in the CD4-binding site, modulated Env binding to the CD4 receptor, affected Env recognition by several glycan-dependent neutralizing antibodies, and altered site-specific glycosylation heterogeneity, with, for example, N448 displaying limited glycan processing. Molecular-dynamic simulations visualized differences in glycan density and how specific oligosaccharide positions can move to compensate for a glycan loss. This study demonstrates how changes in individual glycans can alter molecular dynamics, processing, and function of the Env-glycan shield.

Glomerular Immunodeposits of Patients with IgA Nephropathy Are Enriched for IgG Autoantibodies Specific for Galactose-Deficient IgA1.

BACKGROUND: IgA nephropathy (IgAN) is the leading primary GN worldwide. The disease is thought to result from glomerular deposition of circulating immune complexes of IgG bound to galactose-deficient IgA1 (Gd-IgA1). However, routine immunofluorescence microscopy fails to detect IgG in many kidney biopsies from patients with IgAN and the specificity of IgG in immunodeposits has not been tested. METHODS: We used remnant frozen kidney-biopsy specimens from 34 patients with IgAN; 14 were IgG-positive and 20 were IgG-negative by routine immunofluorescence microscopy. Six patients with primary membranous nephropathy (MN) and eight with lupus nephritis (LN) served as controls. IgG in the kidney tissue was extracted and its amount determined by ELISA. IgG molecular integrity was assessed by SDS-PAGE immunoblotting. Antigenic specificity of extracted IgG was determined by ELISA using phospholipase A2 receptor (PLA2R) or Gd-IgA1 as antigen. In addition, ten other IgAN cases, six IgG-positive and four IgG-negative by routine immunofluorescence, were used for colocalization studies by confocal microscopy. RESULTS: IgG extracted from MN but not IgAN immunodeposits reacted with PLA2R. Conversely, IgG extracted from IgAN but not MN or LN immunodeposits reacted with Gd-IgA1. Even IgAN kidney-biopsy specimens without IgG by routine immunofluorescence microscopy had IgG specific for Gd-IgA1. Confocal microscopy confirmed the presence of IgG in the IgAN biopsies with colocalization of glomerular IgA and IgG. CONCLUSIONS: These results reveal for the first time that IgAN kidney biopsies, with or without IgG by routine immunofluorescence, contain Gd-IgA1-specific IgG autoantibodies. These findings support the importance of these autoantibodies in the pathogenesis of IgAN.

Correction: Galactose-deficient IgA1 and the corresponding IgG autoantibodies predict IgA nephropathy progression.

[This corrects the article DOI: 10.1371/journal.pone.0212254.].

Galactose-deficient IgA1 and the corresponding IgG autoantibodies predict IgA nephropathy progression.

BACKGROUND: IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, has serious outcomes with end-stage renal disease developing in 30-50% of patients. The diagnosis requires renal biopsy. Due to its inherent risks, non-invasive approaches are needed. METHODS: We evaluated 91 Czech patients with biopsy-proven IgAN who were assessed at time of diagnosis for estimated glomerular filtration rate (eGFR), proteinuria, microscopic hematuria, and hypertension, and then followed prospectively. Serum samples collected at diagnosis were analyzed for galactose-deficient IgA1 (Gd-IgA1) using new native-IgA1 and established neuraminidase-treated-IgA1 tests, Gd-IgA1-specific IgG autoantibodies, discriminant analysis and logistic regression model assessed correlations with renal function and Oxford classification (MEST score). RESULTS: Serum levels of native (P <0.005) and neuraminidase-treated (P <0.005) Gd-IgA1 were associated with the rate of eGFR decline. A higher relative degree of galactose deficiency in native serum IgA1 predicted a faster eGFR decline and poor renal survival (P <0.005). However, Gd-IgA1 has not differentiated patients with low vs. high baseline eGFR. Furthermore, patients with high baseline eGFR that was maintained during follow-up were characterized by low serum levels of Gd-IgA1-specific IgG autoantibodies (P = 0.003). CONCLUSIONS: Including levels of native and neuraminidase-treated Gd-IgA1 and Gd-IgA1-specific autoantibodies at diagnosis may aid in the prognostication of disease progression in Czech patients with IgAN. Future tests will assess utility of these biomarkers in larger patients cohorts from geographically distinct areas.

A Randomized, Controlled Trial of Rituximab in IgA Nephropathy with Proteinuria and Renal Dysfunction.

IgA nephropathy frequently leads to progressive CKD. Although interest surrounds use of immunosuppressive agents added to standard therapy, several recent studies have questioned efficacy of these agents. Depleting antibody-producing B cells potentially offers a new therapy. In this open label, multicenter study conducted over 1-year follow-up, we randomized 34 adult patients with biopsy-proven IgA nephropathy and proteinuria >1 g/d, maintained on angiotensin-converting enzyme inhibitors or angiotensin receptor blockers with well controlled BP and eGFR<90 ml/min per 1.73 m2, to receive standard therapy or rituximab with standard therapy. Primary outcome measures included change in proteinuria and change in eGFR. Median baseline serum creatinine level (range) was 1.4 (0.8-2.4) mg/dl, and proteinuria was 2.1 (0.6-5.3) g/d. Treatment with rituximab depleted B cells and was well tolerated. eGFR did not change in either group. Rituximab did not alter the level of proteinuria compared with that at baseline or in the control group; three patients in each group had ≥50% reduction in level of proteinuria. Serum levels of galactose-deficient IgA1 or antibodies against galactose-deficient IgA1 did not change. In this trial, rituximab therapy did not significantly improve renal function or proteinuria assessed over 1 year. Although rituximab effectively depleted B cells, it failed to reduce serum levels of galactose-deficient IgA1 and antigalactose-deficient IgA1 antibodies. Lack of efficacy of rituximab, at least at this stage and severity of IgA nephropathy, may reflect a failure of rituximab to reduce levels of specific antibodies assigned salient pathogenetic roles in IgA nephropathy.

Differential glycosylation of envelope gp120 is associated with differential recognition of HIV-1 by virus-specific antibodies and cell infection.

BACKGROUND: HIV-1 entry into host cells is mediated by interactions between the virus envelope glycoprotein (gp120/gp41) and host-cell receptors. N-glycans represent approximately 50% of the molecular mass of gp120 and serve as potential antigenic determinants and/or as a shield against immune recognition. We previously reported that N-glycosylation of recombinant gp120 varied, depending on the producer cells, and the glycosylation variability affected gp120 recognition by serum antibodies from persons infected with HIV-1 subtype B. However, the impact of gp120 differential glycosylation on recognition by broadly neutralizing monoclonal antibodies or by polyclonal antibodies of individuals infected with other HIV-1 subtypes is unknown. METHODS: Recombinant multimerizing gp120 antigens were expressed in different cells, HEK 293T, T-cell, rhabdomyosarcoma, hepatocellular carcinoma, and Chinese hamster ovary cell lines. Binding of broadly neutralizing monoclonal antibodies and polyclonal antibodies from sera of subtype A/C HIV-1-infected subjects with individual gp120 glycoforms was assessed by ELISA. In addition, immunodetection was performed using Western and dot blot assays. Recombinant gp120 glycoforms were tested for inhibition of infection of reporter cells by SF162 and YU.2 Env-pseudotyped R5 viruses. RESULTS: We demonstrated, using ELISA, that gp120 glycans sterically adjacent to the V3 loop only moderately contribute to differential recognition of a short apex motif GPGRA and GPGR by monoclonal antibodies F425 B4e8 and 447-52D, respectively. The binding of antibodies recognizing longer peptide motifs overlapping with GPGR epitope (268 D4, 257 D4, 19b) was significantly altered. Recognition of gp120 glycoforms by monoclonal antibodies specific for other than V3-loop epitopes was significantly affected by cell types used for gp120 expression. These epitopes included CD4-binding site (VRC03, VRC01, b12), discontinuous epitope involving V1/V2 loop with the associated glycans (PG9, PG16), and an epitope including V3-base-, N332 oligomannose-, and surrounding glycans-containing epitope (PGT 121). Moreover, the different gp120 glycoforms variably inhibited HIV-1 infection of reporter cells. CONCLUSION: Our data support the hypothesis that the glycosylation machinery of different cells shapes gp120 glycosylation and, consequently, impacts envelope recognition by specific antibodies as well as the interaction of HIV-1 gp120 with cellular receptors. These findings underscore the importance of selection of appropriately glycosylated HIV-1 envelope as a vaccine antigen.

Determination of severity of murine IgA nephropathy by glomerular complement activation by aberrantly glycosylated IgA and immune complexes.

The pathogenic roles of glomerular deposition of components of the complement cascade in IgA nephropathy (IgAN) are not completely clarified. To investigate the pathologic role of complement pathways in IgAN, two IgAN-prone mouse models were examined. Grouped ddY (gddY) mice showed significant high proteinuria, severe glomerular lesions, and extracellular matrix expansion compared with high serum IgA (HIGA) mice but with similar intensity of glomerular IgA deposition. Glomerular activation of the classical, lectin, and alternative pathways was demonstrated by significantly stronger staining for complement (C)3, C5b-9, C1q, C4, mannose-binding lectin (MBL)-A/C, MBL-associated serine protease-2, and factor B and properdin in gddY mice than in HIGA mice. Similarly, the serum levels of IgA-IgG2a/IgM and IgA-MBL-A/C immune complexes and polymeric IgA were significantly higher in gddY mice than in HIGA mice. Moreover, the serum levels of aberrantly glycosylated IgA characterized by the binding of Sambucus nigra bark lectin and Ricinus communis agglutinin I were significantly higher in gddY mice than in HIGA mice. This aberrancy in glycosylation was confirmed by monosaccharide compositional analysis of purified IgA using gas-liquid chromatography. This study is the first to demonstrate that aberrantly glycosylated IgA may influence the formation of macromolecular IgA including IgA-IgG immune complexes and subsequent complement activation, leading to full progression of IgAN.

Development of a model of early-onset IgA nephropathy.

ddY mice spontaneously develop IgA nephropathy (IgAN) with a variable age of disease onset. Establishing a model with early-onset IgAN could aid the investigation of mechanisms that underlie the pathogenesis of this disease. On the basis of histologic grading in serial biopsies, we previously classified ddY mice into early-onset, late-onset, and quiescent groups. Here, we selectively mated mice with the early-onset phenotype for >20 generations and established "grouped ddY" mice that develop IgAN within 8 weeks of age. Similar to human IgAN, the prognosis was worse for male mice than females. These mice homogeneously retained genotypes of four marker loci previously associated with the early-onset phenotype, confirming a close association of these loci with early-onset IgAN in ddY mice. Grouped ddY mice comprised two sublines, however, which had distinct genotypes at a susceptibility locus for high serum IgA levels, which maps within the Ig heavy-chain gene complex. The subline bearing the Igh-2(a) IgA allotype had a more rapid course of fatal disease and lower oligosaccharide content, suggesting that aberrant IgA glycosylation may promote the progression of murine IgAN. Taken together, these data indicate that grouped ddY mice may be a useful model for the identification of susceptibility genes and the underlying molecular mechanisms involved in the pathogenesis of human IgAN.

In vitro-generated immune complexes containing galactose-deficient IgA1 stimulate proliferation of mesangial cells.

IgA nephropathy (IgAN) patients have elevated serum levels of immune complexes consisting of IgA1 with galactose-deficient hinge-region O-glycans (Gd-IgA1) and anti-glycan IgG. These immune complexes deposit in the kidney and activate mesangial cells. To confirm that the activity of these immune complexes depends on the interaction of Gd-IgA1 with anti-glycan IgG, we generated in vitro analogous immune complexes using Gd-IgA1 myeloma protein and anti-glycan IgG from cord blood of healthy women. The Gd-IgA1 and anti-glycan IgG from cord-blood serum formed IgA1-IgG immune complexes that resembled those in sera of patients with IgAN. Furthermore, the ability to activate cellular proliferation was dependent on a heat-sensitive serum factor. In summary, we developed a new protocol for in-vitro formation of IgA1-IgG immune complexes, thus providing a new tool for studies of the pathogenesis of IgAN.

IgA1 immune complexes from pediatric patients with IgA nephropathy activate cultured human mesangial cells.

BACKGROUND: Circulating immune complexes (CIC) containing galactose (Gal)-deficient IgA1 from adults with IgA nephropathy (IgAN) induce proliferation of cultured mesangial cells, but activities of CIC from pediatric patients with the disease have not been studied. METHODS: CIC of different sizes were isolated from sera of pediatric and adult IgAN patients and their effects on cultured human mesangial cells (MC) were assessed by measuring cellular proliferation, expression of IL-6 and IL-8 and laminin and phosphotyrosine signaling. RESULTS: Large CIC from pediatric IgAN patients (>800 kDa) containing Gal-deficient IgA1 stimulated cellular proliferation, whereas in some patients, smaller CIC were inhibitory. Addition of stimulatory and inhibitory CIC to MC differentially altered phosphorylation patterns of three major tyrosine-phosphorylated proteins of molecular mass 37, 60 and 115 kDa. The stimulatory CIC transiently increased tyrosine-phosphorylation of the 37-kDa protein and decreased phosphorylation of the other two proteins, whereas the inhibitory CIC increased phosphorylation of all three proteins. Furthermore, we investigated the influence of IgA1-containing CIC from sera of children with IgAN with clinically active disease (i.e., abnormal urinalysis and/or serum creatinine concentration) or inactive disease (i.e., normal urinalysis and serum creatinine concentration) on the expression of IL-6 and IL-8 genes by mesangial cells. Real-time reverse transcription-polymerase chain reaction results showed that the CIC from a patient with active disease stimulated MC to express the two cytokine genes at higher levels than did the CIC from a patient with inactive disease. Moreover, stimulatory CIC increased production of the extracellular matrix protein laminin. CONCLUSION: These data indicate that sera of pediatric IgAN patients contain biologically active CIC with Gal-deficient IgA1.

Glycosylation patterns of HIV-1 gp120 depend on the type of expressing cells and affect antibody recognition.

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.

Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity.

IgA nephropathy (IgAN) is characterized by circulating immune complexes composed of galactose-deficient IgA1 and a glycan-specific IgG antibody. These immune complexes deposit in the glomerular mesangium and induce the mesangioproliferative glomerulonephritis characteristic of IgAN. To define the precise specificities and molecular properties of the IgG antibodies, we generated EBV-immortalized IgG-secreting lymphocytes from patients with IgAN and found that the secreted IgG formed complexes with galactose-deficient IgA1 in a glycan-dependent manner. We cloned and sequenced the heavy- and light-chain antigen-binding domains of IgG specific for galactose-deficient IgA1 and identified an A to S substitution in the complementarity-determining region 3 of the variable region of the gene encoding the IgG heavy chain in IgAN patients. Furthermore, site-directed mutagenesis that reverted the residue to alanine reduced the binding of recombinant IgG to galactose-deficient IgA1. Finally, we developed a dot-blot assay for the glycan-specific IgG antibody that differentiated patients with IgAN from healthy and disease controls with 88% specificity and 95% sensitivity and found that elevated levels of this antibody in the sera of patients with IgAN correlated with proteinuria. Collectively, these findings indicate that glycan-specific antibodies are associated with the development of IgAN and may represent a disease-specific marker and potential therapeutic target.

IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1.

Aberrant glycosylation of IgA1 plays an essential role in the pathogenesis of IgA nephropathy. This abnormality is manifested by a deficiency of galactose in the hinge-region O-linked glycans of IgA1. Biosynthesis of these glycans occurs in a stepwise fashion beginning with the addition of N-acetylgalactosamine by the enzyme N-acetylgalactosaminyltransferase 2 and continuing with the addition of either galactose by beta1,3-galactosyltransferase or a terminal sialic acid by a N-acetylgalactosamine-specific alpha2,6-sialyltransferase. To identify the molecular basis for the aberrant IgA glycosylation, we established EBV-immortalized IgA1-producing cells from peripheral blood cells of patients with IgA nephropathy. The secreted IgA1 was mostly polymeric and had galactose-deficient O-linked glycans, characterized by a terminal or sialylated N-acetylgalactosamine. As controls, we showed that EBV-immortalized cells from patients with lupus nephritis and healthy individuals did not produce IgA with the defective galactosylation pattern. Analysis of the biosynthetic pathways in cloned EBV-immortalized cells from patients with IgA nephropathy indicated a decrease in beta1,3-galactosyltransferase activity and an increase in N-acetylgalactosamine-specific alpha2,6-sialyltransferase activity. Also, expression of beta1,3-galactosyltransferase was significantly lower, and that of N-acetylgalactosamine-specific alpha2,6-sialyltransferase was significantly higher than the expression of these genes in the control cells. Thus, our data suggest that premature sialylation likely contributes to the aberrant IgA1 glycosylation in IgA nephropathy and may represent a new therapeutic target.

IgA nephropathy: characterization of IgG antibodies specific for galactose-deficient IgA1.

The circulating immune complexes in IgA nephropathy (IgAN) are composed of galactose (Gal)-deficient IgA1 bound to IgG or IgA1 antibodies specific for hinge-region O-linked glycans of Gal-deficient IgA1. To analyze properties of the anti-glycan antibodies, we determined the binding of serum IgG and IgG secreted by Epstein-Barr virus (EBV)- immortalized B cells from patients with biopsy-proven IgAN (n = 12) and healthy controls (n = 5) to a panel of antigens coated on ELISA plates. These antigens were: (1) enzymatically desialylated and degalactosylated IgA1 myeloma protein (dd-IgA1), (2) Fab fragment of Gal-deficient IgA1 containing part of the hinge region with O-glycans (Fab-IgA1), (3) synthetic hinge-region peptide linked to bovine albumin (HR-BSA), and (4) synthetic hingeregion glycopeptide with three GalNAc residues linked to BSA (HR-GalNAc-BSA). IgG-secreting EBV-immortalized cell lines were subcloned by limiting dilution. The concentration of total IgG and distribution of IgG subclasses were measured by ELISA. The levels of IgG in sera and supernatants directed against dd-IgA1 and Fab-IgA1 were significantly higher in IgAN patients than in controls (p < 0.01). IgG from IgAN patients exhibited strong reactivity with HR-GalNAc-BSA, but not with HR-BSA. The IgG-secreting cell lines produced antibodies specific to dd-IgA1; the antigen-specific IgG was most frequently of the IgG2 subclass. In summary, sera and supernatants from IgG-secreting cell lines from patients with IgAN were characterized by high levels of IgG antibodies with specificity to the Gal-deficient O-linked glycans of IgA1. The immortalized cell lines will provide a stable and convenient source of IgG for molecular studies of antibodies specific to the aberrant O-glycans in IgA1.

IgA nephropathy and Henoch-Schoenlein purpura nephritis: aberrant glycosylation of IgA1, formation of IgA1-containing immune complexes, and activation of mesangial cells.

IgA1 in the circulation and glomerular deposits of patients with IgA nephropathy (IgAN) is aberrantly glycosylated; the hinge-region O-linked glycans are galactose-deficient. The circulating IgA1 of patients with Henoch-Schoenlein purpura nephritis (HSPN) has a similar defect. This aberrancy exposes N-acetylgalactosamine-containing neoepitopes recognized by naturally occurring IgG or IgA1 antibodies resulting in formation of immune complexes. IgA1 contains up to six O-glycosylation sites per heavy chain; it is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We sought to define the aberrant glycosylation of a galactose-deficient IgA1 myeloma protein and analyze the formation of the immune complexes and their biological activities. Supplementation of serum or cord-blood serum with this IgA1 protein resulted in formation of new IgA1 complexes. These complexes stimulated proliferation of cultured human mesangial cells, as did the naturally-occurring IgA1-containing complexes from sera of patients with IgAN and HSPN. Uncomplexed IgA1 did not affect cellular proliferation. Using specific proteases, lectin Western blots, and mass spectrometry, we determined the O-glycosylation sites in the hinge region of the IgA1 myeloma protein and IgA1 proteins from sera of IgAN patients. The IgA1 myeloma protein had galactose-deficient sites at residues 228 and/or 230 and 232. These sites reacted with IgG specific to galactose-deficient IgA1. IgA1 from the IgAN patients had galactose-deficient O-glycans at the same residues. In summary, we identified the neoepitopes on IgA1 responsible for formation of the pathogenic immune complexes. These studies may lead to development of noninvasive diagnostic assays and future disease-specific therapy.

Identification and characterization of CMP-NeuAc:GalNAc-IgA1 alpha2,6-sialyltransferase in IgA1-producing cells.

Glycosylation defects occur in several human diseases. In IgA nephropathy, IgA1 contains O-glycans that are galactose-deficient and consist mostly of core 1 alpha2,6 sialylated N-acetylgalactosamine, a configuration suspected to prevent beta1,3 galactosylation. We confirmed the same aberrancy in IgA1 secreted by the human DAKIKI B cell line. Biochemical assays indicated CMP-NeuAc:GalNAc-IgA1 alpha2,6-sialyltransferase activity in this cell line. However, a candidate enzyme, ST6-GalNAcI, was not transcribed in DAKIKI cells, B cells isolated from blood, or Epstein-Barr virus (EBV)-immortalized IgA1-producing cells from the blood of IgAN patients and healthy controls. Instead, ST6-GalNAcII transcription was detected at a high level. Expression of the ST6-GalNAcII gene and activity of the CMP-NeuAc:GalNAc-IgA1 alpha2,6-sialyltransferase were higher in IgA1-producing cell lines from IgAN patients than in such cells from healthy controls. These data are the first evidence that human cells that lack ST6-GalNAcI can sialylate core 1 GalNAc-Ser/Thr.

Reactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteins.

Lectins are proteins with specificity of binding to certain monosaccharides or oligosaccharides. They can detect abnormal glycosylation patterns on immunoglobulins in patients with various chronic inflammatory diseases, including rheumatoid arthritis and IgA nephropathy (IgAN). However, lectins exhibit binding heterogeneity, depending on their source and methods of isolation. To characterize potential differences in recognition of terminal N-acetylgalactosamine (GalNAc) on IgA1, we evaluated the binding characteristics of several commercial preparations of GalNAc-specific lectins using a panel of IgA1 and, as controls, IgA2 and IgG myeloma proteins. These lectins originated from snails Helix aspersa (HAA) and Helix pomatia (HPA), and the plant Vicia villosa (VV). Only HAA and HPA bound exclusively to IgA1, with its O-linked glycans composed of GalNAc, galactose, and sialic acid. In contrast, VV reacted with sugars of both IgA subclasses and IgG, indicating that it also recognized N-linked glycans without GalNAc. Furthermore, HAA and HPA from several manufacturers differed in their ability to bind various IgA1 myeloma proteins and other GalNAc-containing glycoproteins in ELISA and Western blot. For serum samples from IgAN patients, HAA was the optimal lectin to study IgA1 glycosylation in ELISA and Western blot assays, including identification of the sites of attachment of the aberrant glycans. The galactose-deficient glycans were site-specific, localized mostly at Thr228 and/or Ser230. Because of the heterogeneity of GalNAc-specific lectins, they should be carefully characterized with appropriate substrates before undertaking any study.

Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals.

BACKGROUND: The IgG from sera of patients with chronic inflammatory diseases of autoimmune character or some chronic microbial infections is frequently deficient in galactose on N-linked glycans. However, this phenomenon has not been investigated at length in human viral infections. OBJECTIVES: To evaluate the glycosylation of serum IgG in HIV-1-positive patients. METHODS: Psathyrella velutina lectin was used in enzyme-linked immunosorbent and Western blot assays to determine glycosylation. In addition, gas-liquid chromatography and mass spectrometry were utilized to confirm the galactose deficiency observed in the lectin-binding assays. RESULTS: HIV-1-infected individuals had significantly higher levels of galactose-deficient IgG than healthy controls. In fact, the galactose deficiency of the N-linked glycans observed in other diseases was even more profound in HIV-1 infection. This deficiency was primarily restricted to IgG when total serum glycoproteins were evaluated and IgG1 was the subclass most affected in all patients. Also, a significant increase in lectin binding was observed on IgG2 and IgG4 from HIV-1-positive females compared with HIV-1-negative females. CONCLUSIONS: Identification of deficient galactosylation of serum IgG from HIV-1-infected patients extended the spectrum of diseases in which this phenomenon has been observed. In addition, the results suggest yet another aspect of immune dysfunction as a result of HIV-1 infection.