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

Objective. To characterize an immunodominant epitope recognized by anti-DNA topoisomerase I (topo I) antibody, a major autoantibody in sera of patient s with systemic sclerosis (SSc), Methods. Topo I fragments were generated as fusion proteins using a bacteri al expression system as well as polypeptides translated in vitro using a eu karyotic expression system. Reactivities to the 2 preparations of recombina nt topo I polypeptides in anti-topo I-positive sera from SSc patients of va ried ethnic backgrounds were examined by immunoblotting, immunoprecipitatio n, 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-termi nal deletion studies as well as competitive inhibition assays using topo I synthetic peptides showed that a region of greater than or equal to 52 amin o acids (512-563) was necessary for recognition by anti-topo I antibodies. The minimum epitope region and conformation required for this reactivity we re variable among sera from Caucasian, African American, Japanese, and Choc taw SSc patients. Conclusion. An immunodominant epitope recognized by anti-topo I autoantibod y 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 regio n by anti-topo I-positive sera is heterogeneous and is influenced by ethnic background.