60-kDa Ro protein autoepitopes identified using recombinant polypeptides

The human Ro ribonucleoprotein is a clinically important yet poorly understood autoantigen. The contribution of Ro autoantibodies to pathogenesis of autoimmune disease remains unclear, as do the stimuli that initiate and maintain the response. Recent evidence suggests that patient anti-Ro responses target individual proteins, including a 60- and a 52-kDa species, within the complex in a disease-specific manner. However, Ro antisera retain considerable heterogeneity in their recognition of both continuous and discontinuous epitopes on the protein components. Previous characterization of Ro autoepitopes has primarily involved solid phase assays, which are of limited value in identifying discontinuous epitopes. To address the heterogeneity of Ro and the issue of discontinuous autoepitopes, we have generated 10 overlapping recombinant polypeptides of the human 60-kDa Ro protein and compared their reactivities using a soluble immunoprecipitation assay. Seven different epitopes, both continuous and discontinuous, were distinguished and seven distinct patterns of reactivity were discerned among the sera from 12 patients. These patterns of reactivity showed no relationship to clinical diagnosis but did correlate with the titer of Abs against recombinant 60-kDa Ro and with the concomitant presence of Abs directed against recombinant 52-kDa Ro protein. Sera that immunoprecipitated only the full-length 60-kDa protein had low or undetectable anti-60 kDa titers by ELISA and immunoblot using recombinant Ag, demonstrating a predominant recognition of discontinuous epitopes. These data indicate that autoantibody responses to the 60-kDa Ro Ag can preferentially target discontinuous epitopes and that the ability to recognize continuous epitopes is accompanied by the appearance of 52-kDa Ro autoantibodies.