Singh U, Van Itallie C M, Mitic L L, Anderson J M, McClane B A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
J Biol Chem. 2000 Jun 16;275(24):18407-17. doi: 10.1074/jbc.M001530200.
The previous model for the action of Clostridium perfringens enterotoxin (CPE) proposed that (i) CPE binds to host cell receptor(s), forming a small ( approximately 90 kDa) complex, (ii) the small complex interacts with other eucaryotic protein(s), forming a large ( approximately 160 kDa) complex, and (iii) the large complex triggers massive permeability changes, thereby inducing enterocyte death. In the current study, Western immunoblot analysis demonstrated that CPE bound to CaCo-2 human intestinal cells at 37 degrees C forms multiple large complex species, with apparent sizes of approximately 200, approximately 155, and approximately 135 kDa. These immunoblot experiments also revealed that occludin, an approximately 65-kDa tight junction protein, is present in the approximately 200-kDa large complex but absent from the other large complex species. Immunoprecipitation studies confirmed that occludin physically associates with CPE in large complex material and also indicated that occludin is absent from small complex. These results strongly suggest that occludin becomes associated with CPE during formation of the approximately 200-kDa large complex. A postbinding association between CPE and occludin is consistent with the failure of rat fibroblast transfectants expressing occludin to bind CPE in the current study. Those occludin transfectants were also insensitive to CPE, strongly suggesting that occludin expression is not sufficient to confer CPE sensitivity. However, the occludin-containing, approximately 200-kDa large complex may contribute to CPE-induced cytotoxicity, because nontoxic CPE point mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could be important for CPE-induced pathophysiology.
先前关于产气荚膜梭菌肠毒素（CPE）作用的模型提出：（i）CPE与宿主细胞受体结合，形成一个小的（约90 kDa）复合物；（ii）小复合物与其他真核蛋白相互作用，形成一个大的（约160 kDa）复合物；（iii）大复合物引发大量通透性变化，从而诱导肠上皮细胞死亡。在本研究中，蛋白质免疫印迹分析表明，在37℃时与CaCo-2人肠道细胞结合的CPE形成了多种大复合物种类，其表观大小约为200 kDa、约155 kDa和约135 kDa。这些免疫印迹实验还显示闭合蛋白（一种约65 kDa的紧密连接蛋白）存在于约200 kDa的大复合物中，但在其他大复合物种类中不存在。免疫沉淀研究证实，闭合蛋白在大复合物物质中与CPE发生物理结合，并且还表明在小复合物中不存在闭合蛋白。这些结果强烈表明，在约200 kDa大复合物形成过程中，闭合蛋白与CPE发生了结合。CPE与闭合蛋白在结合后的关联与本研究中表达闭合蛋白的大鼠成纤维细胞转染子无法结合CPE的现象一致。那些闭合蛋白转染子对CPE也不敏感, 强烈表明仅表达闭合蛋白不足以赋予细胞对CPE的敏感性。然而, 含有闭合蛋白的约200 kDa大复合物可能参与了CPE诱导的细胞毒性作用，因为无毒的CPE点突变体没有形成任何大复合物种类。通过证明大复合物物质由几种种类组成（一种含有闭合蛋白）, 本研究表明CPE的作用比之前认为的更为复杂，并且还为CPE与紧密连接蛋白的相互作用提供了额外证据，这可能对CPE诱导的病理生理学很重要。
