Determination of functional regions of Clostridium perfringens enterotoxin through deletion analysis.
Determination of functional regions of Clostridium perfringens enterotoxin through deletion analysis.
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DOI：
10.1086/516246
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发表时间：
1997-09-01
影响因子：
11.8
通讯作者：
McClane, BA
中科院分区：
文献类型：
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作者：
KokaiKun, JF;McClane, BA
图1.描述产气荚膜梭菌肠毒素（CPE）分子作用前四步的图解模型。有关每个步骤的说明，请参见文本。从BBM中诱导释放）。观察到仅天然CPE rCPEI_319和含有两个最大N-末端CPE缺失片段rCPE 37_319和rCPE 45_319的rCPE种类能够对链霉蛋白酶诱导的BBM释放产生抗性（即，仅这些rCPE种类发生结合后物理变化）。因此，所有含有小于rCPE 45_319的N-末端CPE缺失片段的rCPE物种显然不能进行CPE分子作用中的第二步。还观察到，在链霉蛋白酶处理之前或之后，含有C-末端CPE缺失片段的rCPE种类不与BBM相关联。这些结果独立地支持了我们的竞争结合研究，表明含有三个C-末端CPE缺失片段中的任何一个的rCPE种类都缺乏受体结合活性;因此，由于这些C-末端CPE缺失片段不能与BBM结合，它们不能经历CPE分子作用的第二步。为了确定我们的任何rCPE物质是否可以形成CPE大复合物（CPE分子作用的第三步），对与rCPE物质孵育的BBM进行”大复合物凝胶”的蛋白质免疫印迹分析[5]。当与BBM一起孵育时，三种rCPE物质（rCPEI_319、rCPE37_319和rCPE 45 -319）能够形成较大分子量的物质，其与由与BBM一起孵育的天然CPE形成的大复合物共迁移。然而，我们的较小的N-末端CPE缺失片段不能诱导大复合物的形成。此外，含有C-末端CPE缺失片段的rCPE物质均不能诱导CPE大复合物的形成，鉴于结果表明这些rCPE物质不能与BBM结合，这是预期的。最后，在这些实验中还注意到，当将等摩尔量的rCPE物质添加到BBM中时，由rCPE 37_319或rCPE 45_319形成的大复合物是由天然CPE或rCPEi_319形成的大复合物的两倍。通过使用完善的86 Rb释放测定评价每种rCPE物质的细胞毒性活性[1，3]以检测由这些物质诱导的膜中的任何小分子渗透性改变。这些实验的结果表明，仅rCPE 1 -319、rCPE 37 -319和rCPE 45_319显示出细胞毒性活性。与含有rCPEI_319、rCPE 37_319或rCPE 45 -319的rCPE物质制备物相关的所有细胞毒性活性均是由于制备物中存在该rCPE物质所致，因为这些制备物与抗CPE mAb 3C 9（一种中和CPE细胞毒性活性的单克隆抗体）预孵育完全消除了与这些制备物相关的细胞毒性活性。对每种细胞毒性rCPE物质生成的细胞毒性曲线的定量分析表明，在摩尔基础上，需要两倍于rCPE 37_319或rCPE 45_319的天然CPE或rCPE i 319才能导致50%的86 Rb标记释放（即，两个较小的CPE片段在摩尔基础上的细胞毒性约为天然CPE的两倍）。这四种细胞毒性rCPE物质的动力学比较证实了这一结论，证明rCPE 37 -319或rCPE 45_319诱导50%的细胞毒性rCPE。
Figure 1. A model diagrammatically depicting the first four steps in the molecular action of Clostridium perfringens enterotoxin (CPE). See text for a description of each step. induced release from BBMs). It was observed that only native CPE, rCPEI_319, and the rCPE species containing the two largest N-terminal CPE deletion fragments, rCPE37_319 and rCPE45_319, were able to develop resistance to pronase-induced release from BBMs (ie, only these rCPE species underwent the postbinding physical change). Thus, all rCPE species containing a N-terminal CPE deletion fragment smaller than rCPE45_319 were apparently unable to perform the second step in the molecular action of CPE. It was also observed that rCPE species containing C-terminal CPE deletion fragments did not associate with BBMs, before or after pronase treatment. These results independently support our com-petitive binding studies indicating that rCPE species containing any of the three C-terminal CPE deletion fragments lack receptorbinding activity; therefore, because these C-terminal CPE deletion fragments cannot bind to BBMs, they are unable to undergo the second step in the molecular action of CPE. To determine whether any of our rCPE species could form CPE large complex (the third step in the molecular action of CPE), BBMs incubated with rCPE species were subjected to western immunoblot analysis of" large complex gels"[5]. When incubated with BBMs, three rCPE species (rCPEI_319, rCPE37_319, and rCPE45-319) were able to form a larger-molecular-weight species that comigrated with the large complex formed by native CPE incubated with BBMs. However, our smaller N-terminal CPE deletion fragments were unable to induce the formation of large com-plex. In addition, no rCPE species containing a C-terminal CPE deletion fragment were able to induce the formation of CPE large complex, as would be expected given the results indicating that these rCPE species cannot bind to BBMs. Finally, it was also noted in these experiments that when equimolar amounts of rCPE species were added to BBMs, twice as much large complex was formed by rCPE37_319 or rCPE45_319 than by either native CPE or rCPEi_319.The cytotoxic activity of each rCPE species was evaluated by using a well-established 86Rb-release assay [1, 3] to detect any small-molecule permeability alterations in membranes that were induced by these species. Results from these experiments indicated that only rCPE1-319, rCPE37-319, and rCPE45_319 display cytotoxic activity. All cytotoxic activity associated with preparations of rCPE species containing rCPEI_319, rCPE37_319, or rCPE45-319 was specifically due to the presence of that rCPE species in the preparation since preincubation of these preparations with mAb 3C9 to CPE (a monoclonal antibody that neutralizes the cytotoxic activity of CPE) completely abrogated the cytotoxic activity associated with these preparations. Quantitative analysis of the cytotoxicity curves generated for each of the cytotoxic rCPE species indicated that, on a molar basis, twice as much native CPE or rCPEi 319 than rCPE37_319 or rCPE45_319 was needed to cause a 50% release of 86Rb-label (ie, the two smaller CPE fragments appear to be about twice as cytotoxic as native CPE on a molar basis). Kinetic comparisons of these four cytotoxic rCPE species confirmed this conclusion by demonstrating that rCPE37-319 or rCPE45_319 induced 50%