SPECIFICITY OF GOODPASTURE AUTOANTIBODIES FOR THE RECOMBINANT NONCOLLAGENOUS DOMAINS OF HUMAN TYPE-IV COLLAGEN

Type IV collagen has recently emerged as a family composed of five kno wn chains (alpha1-alpha5), each of which contains a carboxyl-terminal noncollagenous domain (NC1) of approximately 230 amino acids. The NC1 domain of the alpha3(IV) chain is the probable target for autoantibodi es in patients with Goodpasture syndrome (GP), as evidenced from studi es employing bovine type IV collagen. In the present experiments, the specificity of GP antibodies for the five NC1 domains of human type IV collagen was determined by using recombinant NC1 domains as the antig en. cDNAs encoding each NC1 domain were expressed in E. coli as fusion proteins with a 6-histidine amino-terminal leader. The recombinant NC 1 monomers ralpha1(IV), ralpha2(IV), ralpha3(IV), ralpha4(IV), and ral pha5(IV) were purified by affinity chromatography to the fusion protei n using a nickel resin column, and then characterized by electrophores is and immunoblot analysis using chain-specific peptide antibodies. Th e specificity of GP antibodies from four patients to these recombinant proteins was then further evaluated by immunoblot analysis and enzyme -linked immunosorbent assay measurements. The GP antibodies reacted st rongly with the ralpha3(IV) NC1 domain but were not reactive when test ed against the other four recombinant monomers. In contrast, neither a ntisera from patients with two other forms of autoimmune disease (anti -tubular basement membrane disease and Wegener's syndrome) nor normal control sera bound to any of the recombinant NC1 moieties. These resul ts unambiguously establish that GP antibodies are specifically targete d to the NC1 domain of the alpha3(IV) chain of human type IV collagen. The findings also establish a methodology for large scale preparation of ralpha3(IV) NC1 domain for use in diagnostic tests and development of therapeutic procedures and offer a strategy for the elucidation of a more complete GP epitope by site-directed mutagenesis.