Goodpasture antigen: Expression of the full-length α3(IV) chain of collagen IV and localization of epitopes exclusively to the noncollagenous domain

Goodpasture antigen: Expression of the full-length α3(IV) chain of collagen IV and localization of epitopes exclusively to the noncollagenous domain.BackgroundTissue injury in Goodpasture (GP) syndrome (rapidly progressive glomerular nephritis and pulmonary hemorrhage) is mediated by antibasement membrane antibodies that are targeted to the α3(IV) chain of type IV collagen, one of five α(IV) chains that occur in the glomerular basement membrane. GP antibodies are known to bind epitopes within the carboxyl terminal noncollagenous domain (NC1) of the α3(IV) chain, termed the GP autoantigen. Whether epitopes also exist in the 1400-residue collagenous domain is unknown because studies to date have focused solely on the NC1 domain. A knowledge of GP epitopes is important for the understanding of the etiology and pathogenesis of the disease and for the development of therapeutic strategies.MethodsA cDNA construct was prepared for the full-length human α3(IV) chain. The construct was stably transfected into human embryonic kidney 293 cells. The purified full-length r-α3(IV) chain was characterized by electrophoresis and electron microscopy. The capacity of this chain for binding of GP antibodies from five patients was compared with that of the human r-α3(IV)NC1 domain by competitive enzyme-linked immunosorbent assay.ResultsThe r-α3(IV) chain was secreted from 293 cells as a single polypeptide chain that did not spontaneously undergo assembly into a triple-helical molecule. An analysis of GP-antibody binding to the full-length r-α3(IV) chain showed binding exclusively to the globular NC1 domain.ConclusionThe full-length human α3(IV) chain possesses the capacity to bind GP autoantibodies. The epitope(s) is found exclusively on the nontriple-helical NC1 domain of the α3(IV) chain, indicating the presence of specific immunogenic properties. The α3(IV) chain alone does not spontaneously undergo assembly into a triple-helical homotrimeric molecule, suggesting that coassembly with either the α4(IV) and/or the α5(IV) chain may be required for triple-helix formation