Non-linear epitopes of the large subunit of Ku autoantigen recognized by monoclonal and autoantibodies.
Non-linear epitopes of the large subunit of Ku autoantigen recognized by monoclonal and autoantibodies.
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单克隆抗体和自身抗体识别的 Ku 自身抗原大亚基的非线性表位。
DOI：
10.1016/0161-5890(92)90216-k
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发表时间：
1992
影响因子：
3.6
通讯作者：
Yaneva,M
中科院分区：
文献类型：
--
作者：
Wen,J;Yaneva,M
某些SLE、硬皮病和其他自身免疫性疾病患者的血清与Ku蛋白的两个亚基反应：86和70 kDa。先前的实验表明，在氨基酸667和708之间的86 kDa亚基的C-末端附近的40个氨基酸的区域对于单克隆抗体和一些自身免疫抗体的结合是关键的。在本研究中，在关键区域产生了一系列额外的5 '缺失和位点特异性突变，并检查了重组蛋白的免疫反应性。ELISA和免疫印迹分析表明，三个非竞争性的单克隆抗体特异性的86 kDa的亚基需要的氨基酸显着长于40个氨基酸的反应性，这表明抗原被识别在一个折叠状态，也许有一个以上的接触点。24份抗Ku抗体阳性自身免疫血清中有12份的反应性依赖于与单克隆抗体结合所需的相同氨基酸序列，位点特异性突变以类似的方式降低单克隆抗体和自身抗体的反应性。天然Ku蛋白与单克隆抗体的预孵育转移Ku蛋白-DNA复合物的电泳迁移率，表明这些单克隆抗体结合到天然Ku蛋白表面上的表位。总之，来自缺失和定点诱变的结果证明单克隆抗体和自身抗体都识别86 kDa多肽的非线性表位。这些发现表明，在大部分患者中，抗Ku自身免疫应答与小鼠中对Ku抗原的正常免疫应答相似。
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 theC-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.