Wang J, Chou C H, Blankson J, Satoh M, Knuth M W, Eisenberg R A, Pisetsky D S, Reeves W H
Department of Medicine, University of North Carolina, Chapel Hill 27599-7280.
Mol Biol Rep. 1993 Jun;18(1):15-28. doi: 10.1007/BF01006891.
The Ku autoantigen is a DNA binding factor consisting of 70 and approximately 80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factors in vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients' sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (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 amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of approximately 1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610-705, 8-221, and 1-374, respectively). 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 immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Ku自身抗原是一种DNA结合因子，由70 kDa和大约80 kDa的蛋白质（分别为p70和p80）组成，二者形成异源二聚体。p70/p80二聚体对于一种350 kDa的DNA依赖性蛋白激酶（DNA-PK）的功能似乎至关重要，该激酶在体外可使某些转录因子磷酸化。先前的研究表明，Ku在灵长类细胞中含量丰富，但在大多数非灵长类细胞中无法检测到。然而，尚不清楚这是反映了非灵长类细胞中Ku含量低（以及可能的DNA-PK活性低）、缺乏与非灵长类Ku蛋白交叉反应的抗体，还是两者皆有。Ku最初是通过人类自身免疫血清鉴定出来的，但这些血清用于研究Ku的分布、丰度和功能的适用性受到对Ku的多克隆免疫反应以及大多数患者血清中存在污染性自身抗体的限制。在本研究中，我们使用细胞Ku以及重组人源和鼠源Ku抗原确定了鼠抗Ku单克隆抗体（mAb）的特异性。免疫荧光研究证实了先前的观察结果，即大多数非灵长类细胞中无法检测到Ku。然而，通过用mAb 162进行免疫沉淀，在MOPC-315细胞中可检测到少量Ku，但在L-929细胞中未检测到。此外，在大约1/3的MRL/lpr小鼠血清中鉴定出了针对Ku的自身抗体。鼠源自身抗体也从MOPC-315细胞裂解物中免疫沉淀出少量Ku（与用162观察到的相当），但从L-929细胞裂解物中未沉淀出。用Ku融合蛋白通过免疫印迹分析对mAb特异性进行表征，结果显示mAb 111、S10B1和N9C1分别结合到人p80的不同表位（分别为氨基酸610 - 705、8 - 221和1 - 374）。所有这三种mAb与鼠p80均无反应。mAb NOH10和S5C11紧邻p70的DNA结合位点（氨基酸506 - 541）结合。在免疫印迹上，只有N3H10对人源和鼠源p70显示出相当的反应性，但它对鼠Ku的免疫沉淀效果较差。S5C11在免疫印迹上与鼠p70的交叉反应较弱，而162尽管能有效地免疫沉淀Ku，但在免疫印迹上与人源或鼠源Ku均无反应。用mAb N3H10和162进行的研究表明，非灵长类细胞中Ku的水平比灵长类来源的细胞低得多，并且L-929细胞几乎不表达或不表达Ku蛋白。（摘要截短于400字）
