MURINE MONOCLONAL-ANTIBODIES SPECIFIC FOR CONSERVED AND NONCONSERVED ANTIGENIC DETERMINANTS OF THE HUMAN AND MURINE KU AUTOANTIGENS

The Ku autoantigen is a DNA binding factor consisting of 70 and approx imately 80 kDa proteins (p70 and p80, respectively) which form a heter odimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates cert ain transcription factors in vitro. Previous studies have suggested th at Ku is abundant in primate cells, but undetectable in most non-prima te cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibod ies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these se ra for studying the distribution, abundance and function of Ku is limi ted by the polyclonal immune response to Ku and the presence of contam inating autoantibodies in most patients' sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodi es (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amoun ts of Ku could be detected in MOPC-315, but not L-929, cells by immuno precipitating with mAb 162. In addition, autoantibodies to Ku were ide ntified in the sera of approximately 1/3 of MRL/lpr mice. The murine a utoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Ch aracterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to dist inct epitopes of human p80 (amino acids 610-705, 8-221, and 1-374, res pectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 ( amino acids 506-541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblo ts, whereas 162 was completely unreactive with human or murine Ku on i mmunoblots, despite immunoprecipitating Ku efficiently. Studies with m Abs N3H10 and 162 suggest that the level of Ku is considerably lower i n nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein. These mAbs constitute a panel of immu nological reagents reactive with defined regions of the Ku autoantigen which should be useful for examining the assembly and function of Ku.