title: Point mutations of single amino acids abolish ability of alpha3 NC1 domain to elicit experimental autoimmune glomerulonephritis in rats.
abstract: We previously showed concordance between Goodpasture syndrome antibody binding and production of experimental glomerulonephritis using human chimeric proteins. We now examine a more limited amino-terminal region of alpha3(IV) non-collagenous domain (NC1) and the impact of single amino acid (AA) mutations of this region on glomerulonephritis induction. Rats were immunized with collagenase-solubilized glomerular basement membrane (csGBM), D3, an alpha1(IV)NC1 chimeric protein with 69 AA of alpha3(IV)NC1 (binds Goodpasture sera), D4, the D3 construct shortened by 4 AA (non-binding), P9, P10, single AA mutants (non-binding), and S2, alpha1(IV)NC1 with 9 AA of alpha3(IV)NC1 (binding). All rats immunized with csGBM and S2 and 50% of D3 rats developed glomerulonephritis. csGBM rats had intense GBM-bound IgG deposits, but S2 and D3 rats had minimal deposits. None of the D4, P9, or P10 rats developed glomerulonephritis. Lymphocytes from nephritic rats proliferated with csGBM, S2, and D3, but not with D4, P9, or P10. Discrete segments of alpha3(IV)NC1 within the alpha1(IV)NC1 backbone can induce glomerulonephritis. Single AA mutations within that epitope render the antigen unresponsive to Goodpasture sera and incapable of inducing glomerulonephritis. These studies support the concordance of glomerulonephritis inductivity and Goodpasture serum binding. Further, they define a critical limited AA sequence within alpha3(IV)NC1 of nine or fewer AA, which confers nephritogenicity to the nonnephritogenic alpha1(IV)NC1 without in vivo antibody binding. This region may be a T-cell epitope responsible for induction of glomerulonephritis in this model in rats and Goodpasture syndrome in man.
authors.affiliation: Department of Nephrology, Lund University Hospital, S-22185 Lund, Sweden.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hellmark
authors.forename: Lanlin
authors.initials: L
authors.lastname: Chen
authors.forename: Sophie
authors.initials: S
authors.lastname: Ohlsson
authors.forename: Jörgen
authors.initials: J
authors.lastname: Wieslander
authors.forename: Warren Kline
authors.initials: WK
authors.lastname: Bolton
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019867:Anti-Glomerular Basement Membrane Disease / Q000209:etiology / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001324:Autoantigens / Q000008:administration & dosage / Q000235:genetics* / Q000494:pharmacology; D001327:Autoimmune Diseases / Q000209:etiology / Q000235:genetics; D001485:Basement Membrane / Q000276:immunology; D024141:Collagen Type IV / Q000008:administration & dosage / Q000235:genetics* / Q000494:pharmacology; D005921:Glomerulonephritis / Q000209:etiology* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G; D017354:Point Mutation; D051381:Rats; D011993:Recombinant Fusion Proteins
chemical_list: D001324:Autoantigens; D024141:Collagen Type IV; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; C057112:type IV collagen alpha3 chain