Goodpasture antigen: Expression of the full-length alpha 3(IV) chain of collagen IV and localization of epitopes exclusively to the noncollagenous domain

Background. Tissue injury in Goodpasture (GP) syndrome (rapidly progressive glomerular nephritis and pulmonary hemorrhage) is mediated by antibasement membrane antibodies that are targeted to the alpha 3(IV) chain of type IV collagen, one of five alpha(IV) chains that occur in the glomerular basemen t membrane. GP antibodies are known to bind epitopes within the carboxyl te rminal noncollagenous domain (NC1) of the alpha 3(IV) chain, termed the CTP autoantigen. Whether epitopes also exist in the 1400-residue collagenous d omain is unknown because studies to date have focused solely on the NC1 dom ain. A knowledge of GP epitopes is important for the understanding of the e tiology and pathogenesis of the disease and for the development of therapeu tic strategies. Methods. A cDNA construct was prepared for the full-length human alpha 3(IV ) chain. The construct was stably transfected into human embryonic kidney 2 93 cells. The purified full-length r-alpha 3(IV) chain was characterized by electrophoresis and electron microscopy. The capacity of this chain for bi nding of GP antibodies from five patients was compared with that of the hum an r-alpha 3(IV)NC1 domain by competitive enzyme-linked immunosorbent assay . Results. The r-alpha 3(IV) chain was secreted from 293 cells as a single po lypeptide chain that did not spontaneously undergo assembly into a triple-h elical molecule. An analysis of GP-antibody binding to the full-length r-al pha 3(IV) chain showed binding exclusively to the globular NC1 domain. Conclusion. The full-length human alpha 3(IV) chain possesses the capacity to bind GP autoantibodies. The epitope(s) is found exclusively on the nontr iple-helical NC1 domain of the alpha 3(IV) chain, indicating the presence o f specific immunogenic properties. The alpha 3(IV) chain alone does not spo ntaneously undergo assembly into a triple-helical homotrimeric molecule, su ggesting that coassembly with either the alpha 4(IV) and/or the alpha 5(IV) chain may be required for triple helix formation.