The Goodpasture Autoantigen

Abstract

The family of type IV collagen comprises six chains numbered ␣1 through ␣6. The ␣3(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 ␣3 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 ␣3(IV) NC1 monomer by nearly 80%, with the helixjunction region of the ␣3 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 ␣3(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 ␣3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the ␣3(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 ␣3 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 ␣3 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 ␣3 chain. These sites are subsequently concealed by NC1 hexamer assembly of type IV collagen.

Extracellular Matrix-derived Peptide Binds to αvβ3 Integrin and Inhibits Angiogenesis

“…SDS-PAGE and Coomassie Blue Staining-Tum-5, the recombinant deletion mutant of tumstatin, was analyzed by SDS-PAGE and Coomassie Blue staining as previously described (8,25).…”

Two RGD-independent αvβ3 Integrin Binding Sites on Tumstatin Regulate Distinct Anti-tumor Properties

“…Immunoblotting-Recombinant tumstatin and deletion mutants were analyzed by SDS-PAGE and immunoblotting as described previously (18). Rabbit antibody raised against C-terminal 36 amino acids of tumstatin (tum-4 antibody) was prepared as described previously (19).…”

Quaternary Organization of the Goodpasture Autoantigen, the α3(IV) Collagen Chain

“…Sequestration of Goodpasture Autoepitopes-Previous studies have established that the epitopes of GP autoantibodies are sequestered within the NC1 hexamers and hence remain inaccessible for autoantibody binding unless the hexamer dissociates (4,11,12). However, the molecular basis of this cryptic property has remained unknown.…”

“…Unmasking the GP epitopes is thought to be critical for etiology and pathogenesis of GP disease, but the molecular basis for the epitope sequestration is not known (12). Based on the identification of hydrophobic residues in the epitope of the immunodominant GP A autoantibodies, it has been proposed that the epitope is buried at the interface between interacting NC1 domains (13).…”