The combined repetitive oligopeptides of clostridium difficile toxin A counteract premature cleavage of the glucosyl-transferase domain by stabilizing protein conformation.
The combined repetitive oligopeptides of clostridium difficile toxin A counteract premature cleavage of the glucosyl-transferase domain by stabilizing protein conformation.
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DOI：
10.3390/toxins6072162
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发表时间：
2014-07-22
期刊：
影响因子：
4.2
通讯作者：
Gerhard R
中科院分区：
文献类型：
--
作者：
Olling A;Hüls C;Goy S;Müller M;Krooss S;Rudolf I;Tatge H;Gerhard R
艰难梭菌毒素A （TcdA）和毒素B （TcdB）通过受体介导的内吞作用进入宿主细胞。适当的毒素作用的先决条件是由固有的半胱氨酸蛋白酶在细胞内释放葡萄糖基转移酶结构域，该酶被六磷酸肌醇（IP6）变构激活。我们在体外实验中发现，与全长TcdA相比，c端截断的TcdA1-1065在ip6诱导的切割中更有效。我们假设位于c端的组合重复寡肽（庄稼）与毒素的n端部分相互作用，从而阻止了自身蛋白水解。谷胱甘肽s -转移酶（GST）下拉试验和微尺度热电泳分别证实了作物与TcdA的葡萄糖基转移酶（TcdA1-542）或中间结构域（TcdA1102-1847）之间的结合。在TcdB中没有发现N端和c端之间的相互作用。功能分析显示TcdB更容易被ip6诱导的细胞外切割失活。然而，在体外，TcdA的自动加工和失活显著增加，要么是由于周围环境的酸化，要么是TcdB的同源CROP结构域交换了TcdA的CROP结构域。因此，除了作为主要的受体结合域外，TcdA作物还有助于毒素构象的稳定和保护。
Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1–1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1–542) or intermediate (TcdA1102–1847) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.
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