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

Abstract

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.Conflict of interest: The authors have declared that no conflict of interest exists.Nonstandard abbreviations used: CDR3, complementarity-determining region 3; dd-IgA1, desialylated and degalactosylated IgA1; DH, diversity region of IGH gene; Fab-IgA1, Fab fragment of Gal-deficient IgA1 containing the N-terminal part of the hinge region with O-glycans; Gal, galactose; GalNAc, N-acetylgalactosamine; HAA, Helix aspersa agglutinin; HR-BSA, synthetic IgA1 hinge-region peptide linked to BSA; HR-GalNAc-BSA, synthetic IgA1 hinge-region glycopeptide linked to BSA with 3 Gal-NAc residues; IgAN, IgA nephropathy; JH, joint region of IGH gene; rIgG, recombinant human IgG; ROC, receiver operating characteristic curve; UP/Cr, urinary protein/urinary creatinine (ratio); VH, variable region of IGH gene.