Brand S R, Bernstein R M, Mathews M B
Department of Rheumatology, University of Manchester, UK.
J Immunol. 1994 Oct 1;153(7):3070-8.
The proliferating cell nuclear Ag (PCNA) is a DNA replication factor postulated to function as a sliding clamp around DNA. PCNA is also a target for autoimmunity in systemic lupus erythematosus. The autoantigenicity of PCNA is highly conformation-dependent, and reaction with most anti-PCNA sera requires a nearly full-length PCNA molecule. Here we describe the use of gel filtration and glycerol gradient sedimentation to analyze the native structure and size of PCNA. PCNA from three sources was studied (PCNA from HeLa cells, PCNA purified after its overexpression in bacteria, and PCNA produced in the wheat germ cell-free translation system) as well as mutant forms of PCNA translated in vitro. In each case, full-length PCNA behaved as a trimer. Analysis of mutant proteins revealed a correlation between the trimeric form and binding to the common type of human anti-PCNA autoantibody, suggesting that the Abs are specific for the active form of the protein. These findings are consistent with the idea that autoantibodies are generated as a response to native Ag and provide experimental support for the hypothesis that PCNA serves its processive function in DNA replication as a trimeric ring structure.
增殖细胞核抗原（PCNA）是一种DNA复制因子，被认为可作为围绕DNA的滑动夹发挥作用。PCNA也是系统性红斑狼疮自身免疫反应的靶点。PCNA的自身抗原性高度依赖构象，与大多数抗PCNA血清的反应需要近乎全长 的PCNA分子。在此，我们描述了使用凝胶过滤和甘油梯度沉降法来分析PCNA的天然结构和大小。我们研究了来自三种来源的PCNA（来自HeLa细胞的PCNA、在细菌中过表达后纯化的PCNA以及在小麦胚无细胞翻译系统中产生的PCNA）以及体外翻译的PCNA突变形式。在每种情况下，全长PCNA均表现为三聚体。对突变蛋白的分析揭示了三聚体形式与结合常见类型的人抗PCNA自身抗体之间的相关性，这表明这些抗体对该蛋白的活性形式具有特异性。这些发现与自身抗体作为对天然抗原的反应而产生的观点一致，并为PCNA作为三聚体环结构在DNA复制中发挥其持续合成功能的假说提供了实验支持。
