Shibata S, Muryoi T, Saitoh Y, Brumeanu T D, Bona C A, Kasturi K N
Department of Microbiology, Mount Sinai School of Medicine, New York 10029-6574.
J Clin Invest. 1993 Aug;92(2):984-92. doi: 10.1172/JCI116675.
Autoantibodies against nuclear proteins like RNA polymerase I (RNA pol I) are produced in a number of rheumatic autoimmune diseases. Production of antibodies specific for the 190-kD subunit of RNA pol I appears to be characteristic in the patients with systemic sclerosis. Previous investigations have shown that the tight skin (TSK) mouse is an experimental model for systemic sclerosis. In the present study we show that the TSK mice produce high titers of anti-RNA pol I antibodies, both of IgM and IgG classes. To characterize the immunochemical properties of these antibodies we obtained a large panel of hybridomas from these mice. Analysis of these hybridomas revealed that clonal frequency of autoreactive B cells specific for RNA pol I are higher in the TSK mice that in the controls. mAbs obtained from the TSK mice were specific for the 190-kD subunit and cross-reacted with Escherichia coli and phage T7 RNA polymerases (155-, 150-, and 107-kD polypeptides). We have also demonstrated that these antibodies bind better to the phosphorylated enzymes. The anti-RNA pol I mAbs were divided into three groups in terms of their functional property. The first group of antibodies increased the catalytic activity of the enzyme whereas the antibodies of the second group inhibited the enzymatic activity. Competitive inhibition RIAs showed that these two groups of antibodies bound to distinct epitopes. The third group of antibodies was neutral and had no activity on the enzyme function. These results suggest that TSK mouse anti-RNA pol I antibodies recognize three or more conserved epitopes. To understand the molecular basis of the generation of such autoreactive antibodies we analyzed their V gene repertoire. Northern analysis of RNAs of 14 TSK hybridomas showed that the VH genes encoding these antibodies were mainly from VH J558 family. It is possible that these genes were derived from a single germline gene or from a set of related genes of a single subgroup.
在多种风湿性自身免疫疾病中会产生针对核蛋白（如RNA聚合酶I，RNA pol I）的自身抗体。针对RNA pol I 190-kD亚基的特异性抗体产生似乎是系统性硬化症患者的特征。先前的研究表明，紧皮（TSK）小鼠是系统性硬化症的实验模型。在本研究中，我们发现TSK小鼠产生高滴度的抗RNA pol I抗体，包括IgM和IgG类。为了表征这些抗体的免疫化学特性，我们从这些小鼠中获得了大量杂交瘤。对这些杂交瘤的分析表明，TSK小鼠中针对RNA pol I的自身反应性B细胞的克隆频率高于对照组。从TSK小鼠获得的单克隆抗体对190-kD亚基具有特异性，并与大肠杆菌和噬菌体T7 RNA聚合酶（155-kD、150-kD和107-kD多肽）发生交叉反应。我们还证明这些抗体与磷酸化酶的结合更好。抗RNA pol I单克隆抗体根据其功能特性分为三组。第一组抗体增加了酶的催化活性，而第二组抗体抑制了酶活性。竞争性抑制放射免疫分析表明，这两组抗体结合到不同的表位。第三组抗体是中性的，对酶功能无活性。这些结果表明，TSK小鼠抗RNA pol I抗体识别三个或更多保守表位。为了理解此类自身反应性抗体产生的分子基础，我们分析了它们的V基因库。对14个TSK杂交瘤的RNA进行Northern分析表明，编码这些抗体的VH基因主要来自VH J558家族。这些基因可能来自单个种系基因或单个亚组的一组相关基因。
