The Goodpasture Autoantigen

The family of type IV collagen comprises six chains numbered α1 through α6. The α3(IV) NC1 domain is the primary target antigen for autoantibodies from patients with anti-basement membrane disease and Goodpasture syndrome. Earlier peptide studies suggested that the last 36 amino acids of the α3 NC1 domain probably contains one recognition site for Goodpasture autoantibodies, and an algorithm analysis of secondary structure from a later study predicted a second possible upstream epitope near the triple helix junction. We have used several analytic approaches to evaluate the likelihood of two immunologic epitopes for the Goodpasture antigen. In our first set of studies, peptide antibodies directed against these two putative regions co-inhibited Goodpasture autoantibodies binding to denatured human α3(IV) NC1 monomer by nearly 80%, with the helix-junction region of the α3 NC1 domain contributing 26% of the binding sites and the C-terminal region contributing the remaining 50%. Second, both of these candidate regions are normally sequestered within the associated α3(IV) NC1 hexamer but become more visible for binding by anti-peptide antibodies upon their dissociation, a property that is shared by the Goodpasture autoantibodies. Third, segment deletions of recombinant α3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the α3(IV) NC1 domain. Since the lysines in that region have been previously advanced as possible contact sites, we created several substitutions within the C-terminal epitope of the α3 NC1 domain. Substitution of lysines to alanines revealed lysines 219 and 229 as essential for antibody binding to this distal site; no lysines were present in the NC1 part of the helix-NC1 junction region. Substitutions involving arginine and cysteines to alanines in the same C-terminal region did not produce significant reductions in antibody binding. In summary, our findings characterize two Goodpasture epitopes confined to each end of the α3 NC1 domain; one is lysine-dependent, and the other is not. We propose, as a hypothetical model, that these two immunologically privileged regions fold to form an optimal pathogenic structure within the NC1 domain of the α3 chain. These sites are subsequently concealed by NC1 hexamer assembly of type IV collagen.