title: Non-linear epitopes of the large subunit of Ku autoantigen recognized by monoclonal and autoantibodies.
abstract: 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.
mesh_terms: 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
chemical_list: 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