An immunodominant epitope on DNA topoisomerase I is conformational in nature: Heterogeneity in its recognition by systemic sclerosis sera

Abstract

Objective To characterize an immunodominant epitope recognized by anti–DNA topoisomerase I (topo I) antibody, a major autoantibody in sera of patients with systemic sclerosis (SSc). Methods Topo I fragments were generated as fusion proteins using a bacterial expression system as well as polypeptides translated in vitro using a eukaryotic expression system. Reactivities to the 2 preparations of recombinant topo I polypeptides in anti–topo I–positive sera from SSc patients of varied ethnic backgrounds were examined by immunoblotting, immunoprecipitation, and/or enzyme‐linked immunosorbent assay. Results The fragment encoding amino acids 489–573 of topo I was recognized by 98 of 100 anti–topo I–positive SSc sera. Both carboxyl‐ and amino‐terminal deletion studies as well as competitive inhibition assays using topo I synthetic peptides showed that a region of ≥52 amino acids (512–563) was necessary for recognition by anti–topo I antibodies. The minimum epitope region and conformation required for this reactivity were variable among sera from Caucasian, African American, Japanese, and Choctaw SSc patients. Conclusion An immunodominant epitope recognized by anti–topo I autoantibody is located in the region of amino acids 489–573 of the topo I protein and is largely conformational in nature. The recognition pattern of this region by anti–topo I–positive sera is heterogeneous and is influenced by ethnic background.