Antibody specificities of Thai and Australian scleroderma sera with topoisomerase I recombinant fusion proteins.
Autoantibodies that react with the nuclear enzyme topoisomerase I (Topo 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 overlapping partial cDNA clones encoding the complete amino acid sequence of Topo I were isolated from human placenta. Using the polymerase chain reaction, specific regions of Topo I were amplified and cloned into the pGEX expression vectors. To map Topo I epitopes, recombinant fusion proteins were analyzed by immunoblotting with 66 anti-Topo I sera from 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 midregion 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 contained antibodies that recognized a COOH-terminal epitope region (amino acids 658-765) encompassing the active site of the enzyme. This subset of Topo I antibodies could be responsible for the inhibition of enzymatic activity previously reported in vitro. Heterogeneous patterns of reactivity with the six Topo I epitope regions were observed, although over half the sera could be assigned to one of six distinct patterns. In general, antibodies in the Thai sera reacted more strongly with the six epitope regions. Furthermore, two of the epitope regions reacted exclusively with Thai sera, suggesting a degree of racial or geographical specificity in the autoantibody response to Topo I. The identification of multiple epitopes in Topo I conforms with the polyclonal autoantibody response to intracellular Ag found in other multisystem autoimmune diseases and is presumed to be driven by the presentation of multiple peptides from Topo I itself.
Cram
D S
DS
Burnet Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.
Fisicaro
N
N
McNeilage
L J
LJ
Coppel
R L
RL
Harrison
L C
LC
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001315:Australia; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000276:immunology*; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012595:Scleroderma, Systemic / Q000201:enzymology* / Q000276:immunology*; D013785:Thailand
D001323:Autoantibodies; D001324:Autoantigens; D017931:DNA Primers; D018076:DNA, Complementary; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D004264:DNA Topoisomerases, Type I