ANTIBODY SPECIFICITIES OF THAI AND AUSTRALIAN SCLERODERMA SERA WITH TOPOISOMERASE-I RECOMBINANT FUSION PROTEINS

Autoantibodies that react with the nuclear enzyme topoisomerase I (Top o I) are used as a diagnostic marker of diffuse scleroderma. To better define immune reactivity to Topo I, antibody epitopes in two patient populations were analyzed using recombinant Topo I proteins. Two overl apping partial cDNA clones encoding the complete amino acid sequence o f Topo I were isolated from human placenta. Using the polymerase chain reaction, specific regions of Topo I were amplified and cloned into t he pGEX expression vectors. To map Topo I epitopes, recombinant fusion proteins were analyzed by immunoblotting with 66 anti-Topo I sera fro m Thai and Australian patients with diffuse scleroderma. Six distinct epitope regions were identified along the length of the 765 amino acid enzyme. Almost all sera contained antibodies that recognized the midr egion of Topo I (amino acids 453-560), as well as antibodies to one of more of the other epitope regions. Sixty percent of the sera containe d antibodies that recognized a COOH-terminal epitope region (amino aci ds 658-765) encompassing the active site of the enzyme. This subset of Topo I antibodies could be responsible for the inhibition of enzymati c activity previously reported in vitro. Heterogeneous patterns of rea ctivity with the six Topo I epitope regions were observed, although ov er half the sera could be assigned to one of six distinct patterns. In general, antibodies in the Thai sera reacted more strongly with the s ix epitope regions. Furthermore, two of the epitope regions reacted ex clusively with Thai sera, suggesting a degree of racial or geographica l specificity in the autoantibody response to Topo I. The identificati on of multiple epitopes in Topo I conforms with the polyclonal autoant ibody response to intracellular Ag found in other multisystem autoimmu ne diseases and is presumed to be driven by the presentation of multip le peptides from Topo I itself.