Milan Tomana

Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies

Circulating immune complexes (CICs) isolated from sera of patients with IgA nephropathy (IgAN) consist of undergalactosylated, mostly polymeric, and J chain-containing IgA1 and IgG antibodies specific for N-acetylgalactosamine (GalNAc) residues in O-linked glycans of the hinge region of IgA1 heavy chains. Antibodies with such specificity occur in sera of IgAN patients, and in smaller quantities in patients with non-IgA proliferative glomerulonephritis and in healthy controls; they are present mainly in the IgG (predominantly IgG2 subclass), and less frequently in the IgA1 isotype. Their specificity for GalNAc was determined by reactivity with IgA1 myeloma proteins with enzymatically removed N-acetylneuraminic acid (NeuNAc) and galactose (Gal); removal of the O-linked glycans of IgA1 resulted in significantly decreased reactivity. Furthermore, IgA2 proteins that lack the hinge region with O-linked glycans but are otherwise structurally similar to IgA1 did not react with IgG or IgA1 antibodies. The re-formation of isolated and acid-dissociated CICs was inhibited more effectively by IgA1 lacking NeuNAc and Gal than by intact IgA1. Immobilized GalNAc and asialo-ovine submaxillary mucin (rich in O-linked glycans) were also effective inhibitors. Our results suggest that the deficiency of Gal in the hinge region of IgA1 molecules results in the generation of antigenic determinants containing GalNAc residues that are recognized by naturally occurring IgG and IgA1 antibodies.

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 Glycosylation and IgA Immune Complexes in the Pathogenesis of IgA Nephropathy

Circulating immune complexes containing aberrantly glycosylated IgA1 play a pivotal role in the pathogenesis of IgA nephropathy (IgAN). A portion of IgA1 secreted by IgA1-producing cells in patients with IgAN is galactose-deficient and consequently recognized by anti-glycan IgG or IgA1 antibodies. Some of the resultant immune complexes in the circulation escape normal clearance mechanisms, deposit in the renal mesangium, and induce glomerular injury. Recent studies of the origin of these aberrant molecules, their glycosylation profiles, and mechanisms of biosynthesis have provided new insight into the autoimmune nature of the pathogenesis of this common renal disease. An imbalance in the activities of the pertinent glycosyltransferases in the IgA1-producing cells favors production of molecules with galactose-deficient O-linked glycans at specific sites in the hinge region of the alpha heavy chains. By using sophisticated analytic methods, it may be possible to define biomarkers for diagnostic purposes and identify new therapeutic targets for a future disease-specific therapy.

Receptor-mediated binding and uptake of immunoglobulin a by human liver

We have studied the molecular mechanisms of the binding and uptake of secretory and serum immunoglobulin A (IgA) of both subclasses (1 and 2) and molecular forms (monomer and polymer) by the particulate fraction of human liver homogenate and by a human hepatoma cell line (HepG2). Inhibition by asialoorosomucoid and the requirement for the presence of calcium indicated that the binding of secretory IgA and polymeric IgA1 was mediated by the asialoglycoprotein receptor. Secretory component, which functions as a receptor for polymeric IgA in several animal species, was detected in the epithelial cells of bile ducts, but not in hepatocytes. Secretory IgA and all molecular forms and subclasses of serum IgA were bound by HepG2 cells, which do not express secretory component. The requirement for the presence of calcium, the presence of a terminal galactose residue in IgA, and the molecular weight of the major plasma membrane protein responsible for binding (41,700 daltons) indicated the involvement of asialoglycoprotein receptor. Immunoglobulin A proteins bound by HepG2 cells were endocytosed and catabolized.

Isolation, characterization and immunolocalization of a 53-kDal dentin sialoprotein (DSP).

We isolated a sialic-rich protein from rat dentin extracts and have named it dentin sialoprotein, DSP (formerly called 95K glycoprotein). DSP is rich in aspartic acid, glutamic acid, glycine and serine, but contains no cysteine or phosphate. The 30% carbohydrate content includes about 9% sialic acid and indicates that several N-glycosides and O-glycosides are present. Sedimentation equilibrium analysis gave a M(r) of 52,570. Based on this molecular weight we calculated that DSP contains about 350-amino acids and 75 monosaccharides. With automated Edman degradation the sequence of the first 8-amino acids was shown to be: Ile-Pro-Val-Pro-Gln-Leu-Val-Pro. The initial 3 residues of this sequence are identical to the first 3 in human osteopontin (OPN) and are closely similar to the Leu-Pro-Val sequences of OPN from other species, as well as at the beginning of bone acidic glycoprotein-75 (BAG-75). On Western immunoblots, purified polyclonal antibodies reacted only with DSP in dentin extracts and with none of the proteins from bone. Similarly, immunolocalization experiments showed the presence of DSP in dentin but not in enamel or alveolar bone. Along with immunohistochemical localization data reported elsewhere, these observations suggest that DSP may be an important marker for cells in the odontoblast lineage.

Determination of Aberrant O-Glycosylation in the IgA1 Hinge Region by Electron Capture Dissociation Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy.

Progress in molecular and genetic studies of IgA nephropathy.

Several new findings emerged recently from biochemical, genetic, and molecular studies of patients with IgA nephropathy. It appears that immunoglobulin A1-secreting cells of IgA nephropathy patients produce increased amounts of aberrantly glycosylated IgA1 in which the O-linked glycans in the hinge region are deficient in the content of galactose. The galactose-deficient IgA1 in the circulation is recognized by naturally occurring antibodies with anti-glycan specificity, and immune complexes are formed. These circulating immune complexes escape hepatic degradation and eventually are deposited in the kidney mesangium. Resident mesangial cells bind the IgA-containing immune complexes with the involvement of a novel IgA receptor and become activated. A familial form of IgA nephropathy has been linked to chromosome 6q22-23. Recent progress in molecular analyses of IgA nephropathy thus defines this disease as an autoimmune process with a novel IgA mesangial receptor and certain genetically determined traits.

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model.

Streptococcus parasanguis is a primary colonizer of the tooth surface and plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis are important in mediating adhesion to saliva‐coated hydroxylapatite (SHA), an in vitro tooth adhesion model. The Fap1 adhesin has been identified as the major fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated the presence of rhamnose, glucose, galactose, N‐acetylglucosamine and N‐acetylgalactosamine. A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide was similar to that of the intact molecule. The functionality of the glycan moiety was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein. These antibodies were grouped into two categories based on their ability to block adhesion of S. parasanguis to SHA and their corresponding specificity for either protein or glycan epitopes of the Fap1 protein. ‘Non‐blocking’ MAb epitopes were mapped to unique protein sequences in the N‐terminus of the Fap1 protein using non‐glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed in Escherichia coli. In contrast, the ‘blocking’ antibodies did not bind to the recombinant Fap1 proteins, and were effectively competed by the binding to the purified glycopeptide. These data suggest that the ‘blocking’ antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.

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.