Deletion analysis of the Clostridium perfringens enterotoxin
Deletion analysis of the Clostridium perfringens enterotoxin
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DOI：
10.1128/iai.65.3.1014-1022.1997
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发表时间：
1997-03-01
影响因子：
3.1
通讯作者：
McClane, BA
中科院分区：
文献类型：
--
作者：
KokaiKun, JF;McClane, BA
为了进一步了解产气荚膜梭菌肠毒素（CPE）的结构-功能关系和作用机制，采用重组DNA方法构建了一系列含有N端和c端CPE缺失片段的重组CPE （rCPE）物种，每个rCPE物种的特点是能够完成CPE作用的前四个早期步骤，推测顺序为特异性结合，即结合后的物理变化。这些研究表明(i)至少有44个氨基酸可以从CPE的N端去除而不损失细胞毒性，（ii）从CPE的N端去除前53个氨基酸产生的片段似乎没有细胞毒性，因为它不能经历CPE作用中的结合后物理变化步骤。（iii）从CPE的C端去除5个氨基酸会产生一个没有细胞毒性的片段，缺乏受体结合活性；（iv）缺乏天然CPE的前44个n端氨基酸的片段形成了两倍大的复合物，细胞毒性是天然CPE的两倍。从这些结构-功能结果来看，最小尺寸的细胞毒性CPE片段大约包含天然CPE的45到319个残基。这些缺失片段研究的结果也有助于我们理解CPE的作用(i)独立支持先前的建议，即结合，结合后的物理变化步骤和大复合物的形成是CPE细胞毒性的重要步骤；（ii）提供独立的证据证实CPE作用中这些早期事件的假定顺序。
To further our knowledge of the structure-function relationship and mechanism of action of the Clostridium perfringens enterotoxin (CPE), a series of recombinant CPE (rCPE) species containing N- and C-terminal CPE deletion fragments was constructed by recombinant DNA approaches, Each rCPE species was characterized for its ability to complete the first four early steps in the action of CPE, putatively ordered as specific binding, a postbinding physical change to bound CPE, large-complex formation, and induction of alterations in small-molecule membrane permeability, These studies demonstrated that (i) at least 44 amino acids can be removed from the N terminus of CPE without loss of cytotoxicity, (ii) removal of the first 53 amino acids from the N terminus of CPE produces a fragment that appears to be noncytotoxic because it cannot undergo the postbinding physical change step in CPE action, (iii) removal of as few as five amino acids from the C terminus of CPE produces a noncytotoxic fragment lacking receptor binding activity, and (iv) a fragment lacking the first 44 N-terminal amino acids of native CPE formed twice as much large complex and was twice as cytotoxic as native CPE, From these structure-function results, it appears that the minimum-size cytotoxic CPE fragment comprises approximately residues 45 to 319 of native CPE, Results from these deletion fragment studies have also contributed to our understanding of CPE action by (i) independently supporting previous suggestions that binding, the postbinding physical change step, and large-complex formation represent important steps in CPE cytotoxicity and (ii) providing independent evidence confirming the putative sequential order of these early events in CPE action.