Non-linear epitopes of the large subunit of Ku autoantigen recognized by monoclonal and autoantibodies.
Sera from certain patients with SLE, scleroderma and other autoimmune diseases react with the two subunits of the Ku protein: 86 and 70 kDa. Previous experiments indicated that a region of 40 amino acids near the C-terminus of the 86 kDa subunit between amino acids 667 and 708 was critical for binding of monoclonal and some autoimmune antibodies. In the present study, a series of additional 5' deletions and site-specific mutations in the critical region were produced and the immunoreactivities of the recombinant proteins were examined. ELISA and immunoblot analyses showed that three non-competing monoclonal antibodies specific for the 86 kDa subunit require stretches of amino acids significantly longer than 40 amino acids for reactivity, suggesting that the antigen is recognized in a folded state with perhaps more than one contact point. The reactivities of 12 of 24 anti-Ku positive autoimmune sera screened depended on the same amino acid sequences required for binding of the monoclonal antibodies, site-specific mutations reduced the reactivities of monoclonal and autoantibodies in a similar way. Preincubation of native Ku protein with the monoclonal antibodies shifted the electrophoretic mobility of Ku protein-DNA complex, suggesting that these monoclonal antibodies bind to epitopes on the surface of the native Ku protein. Taken together, the results from the deletion and site-directed mutagenesis demonstrate that both monoclonal and autoantibodies recognize non-linear epitopes of the 86 kDa polypeptide. These findings indicate that in a large portion of patients the anti-Ku autoimmune response is similar to the normal immune response to the Ku antigen in mice.
D000911:Antibodies, Monoclonal / Q000276:immunology; D034961:Antigens, Nuclear; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000276:immunology*; D001327:Autoimmune Diseases / Q000276:immunology; D015153:Blotting, Western; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D000072200:Ku Autoantigen; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D001323:Autoantibodies; D001324:Autoantigens; D004268:DNA-Binding Proteins; D000939:Epitopes; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen