Expression and characterization of recombinant rat α3(IV)NC1 and its use in induction of experimental autoimmune glomerulonephritis

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Abstract
The autoantigen in Goodpasture's disease is known to be the non-collagenous domain of the alpha3 chain of type IV collagen, alpha 3(IV)NC1. There is mounting evidence that alpha 3(IV)NC1 is also a target of autoimmunity in experimental autoimmune glomerulonephritis (EAG). Sado et al. [Kidney Int 1998; 53, 664-671] have reported that recombinant human alpha 3(IV)NC1 and alpha4(IV)NC1 are nephritogenic in WKY rats. We have proposed that immunization with homologous antigen is more appropriate for detailed investigation of autoimmunity in EAG. To this end, we have cloned and sequenced rat alpha 3(IV)NC1 and expressed it in COS-7 cells. Recombinant rat alpha 3(IV)NC1, secreted into the COS-7 cell supernatant, was purified on an anti-M2 FLAG affinity column and characterized by western blotting. Recombinant antigen was then used to immunize WKY rats, in order to induce EAG. The recombinant material was antigenic as judged by binding to sera from patients with Goodpasture's disease and a mAb to alpha 3(IV)NC1. Immunization of WKY rats (n=5), with recombinant rat alpha 3(IV)NC1 in FCA at a dose of 1 mg/kg resulted in circulating anti-GBM antibodies directed towards alpha 3(IV)NC1, linear deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severe focal necrotizing glomerulonephritis with crescent formation, and glomerular influx of CD8+ T cells and macrophages. Western blot analysis demonstrated that sera from these rats bound strongly to recombinant rat alpha 3(IV)NC1, as well as to collagenase-solubilized human and rat GBM. The pattern of binding was indistinguishable from that of sera from patients with Goodpasture's disease. This purified recombinant rat alpha 3(IV)NC1, which is both antigenic and nephritogenic, will be of value in analysing autoimmune responses in experimental anti-GBM disease.