Antigenic determinants of the 70-kDa subunit of the Ku autoantigen.
Autoantibodies against Ku antigen were found in subsets of sera from patients with rheumatic diseases. The Ku autoantigen was characterized as a DNA-binding protein complex composed of two subunits, 70 and 86 kDa. In this study, we report the amino acid sequences of the 70-kDa subunit that are important for interactions with a monoclonal and autoimmune antibodies. Full-length cDNA and numerous 5' and 3' deletion mutants were expressed in bacteria and the immunoreactivity of the fusion proteins was analyzed by Western blotting. The reactivity of the monoclonal antibody depended on the region between Ile321 and Phe350. Ten autoimmune sera were tested for reactivity with deletion mutants in immunoblots. The reactivity of six sera strongly depended on the C-terminal amino acids and four sera did not show such dependence; however, these C-terminal sequences did not react with the sera when expressed alone. These results strongly suggest the conformational nature of the Ku autoepitopes. Interestingly, the DNA-binding activity of this Ku protein subunit analyzed by Southwestern blot depended on the same C-terminal amino acids that were involved in interactions with autoantibodies, indicating that anti-Ku autoantibodies are directed to conformationally intact Ku antigen. Reactivities of the autoimmune sera with Met1-Arg115, Met116-Val149, and Val149-Arg586 were also observed. These results demonstrate that different amino acid regions can be involved in interactions with autoimmune antibodies.
Abu-Elheiga
L
L
Department of Pharmacology, Baylor College of Medicine Houston, Texas 77030.
Yaneva
M
M
D000911:Antibodies, Monoclonal / Q000737:chemistry; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000939:Epitopes / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D001324:Autoantigens; D004268:DNA-Binding Proteins; D000939:Epitopes; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen