Auto-catalytic cleavage of Clostridium difficile toxins a and B depends on cysteine protease activity
Auto-catalytic cleavage of Clostridium difficile toxins a and B depends on cysteine protease activity
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DOI：
10.1074/jbc.m703062200
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发表时间：
2007-08-31
影响因子：
4.8
通讯作者：
Aktories, Klaus
中科院分区：
文献类型：
--
作者：
Egerer, Martina;Giesemann, Torsten;Aktories, Klaus
艰难梭菌毒素 A 和 B 的作用取决于催化葡萄糖基转移酶结构域的加工和易位到 Rho GTP 酶被修饰的靶细胞的胞质溶胶中。在这里我们研究了毒素的加工过程。二硫苏糖醇和β-巯基乙醇诱导纯化的天然毒素A和毒素B自动裂解成类似于250/210-和类似于63-kDa的片段。通过质谱分析将相似的63-kDa片段鉴定为N端葡萄糖基转移酶结构域。该裂解被 N-乙基马来酰亚胺或碘乙酰胺阻断。毒素 B 的半胱氨酸 698、组氨酸 653 或天冬氨酸 587 的交换可防止全长重组毒素 B 和覆盖残基 1-955 的 N 末端片段的裂解，并抑制全长毒素 B 的细胞毒性。二硫苏糖醇可协同增强肌醇六磷酸的作用，据报道，六磷酸肌醇可促进毒素 B 的自动裂解（Reineke, J.、Tenzer, S.、Rupnik, M.、Koschinski, A.、Hasselmayer, O.、Schrattenholz, A.、Schild, H. 和 Von Eichel-Streiber, C. (2007) Nature 446, 415-419）。 N-乙基马来酰亚胺阻断了添加肌醇六磷酸诱导的自裂解，表明半胱氨酸残基对于梭菌糖基化毒素的加工至关重要。我们的数据表明，梭菌糖基化细胞毒素具有与霍乱弧菌 RTX 毒素的半胱氨酸蛋白酶相关的固有半胱氨酸蛋白酶活性，后者负责糖基化毒素的自动裂解。
The action of Clostridium difficile toxins A and B depends on processing and translocation of the catalytic glucosyltransferase domain into the cytosol of target cells where Rho GTPases are modified. Here we studied the processing of the toxins. Dithiothreitol and beta-mercaptoethanol induced auto-cleavage of purified native toxin A and toxin B into similar to 250/210- and similar to 63-kDa fragments. The similar to 63-kDa fragment was identified by mass spectrometric analysis as the N-terminal glucosyltransferase domain. This cleavage was blocked by N-ethylmaleimide or iodoacetamide. Exchange of cysteine 698, histidine 653, or aspartate 587 of toxin B prevented cleavage of full-length recombinant toxin B and of an N-terminal fragment covering residues 1-955 and inhibited cytotoxicity of full-length toxin B. Dithiothreitol synergistically increased the effect of myo-inositol hexakisphosphate, which has been reported to facilitate auto-cleavage of toxin B (Reineke, J., Tenzer, S., Rupnik, M., Koschinski, A., Hasselmayer, O., Schrattenholz, A., Schild, H., and Von Eichel-Streiber, C. ( 2007) Nature 446, 415-419). N-Ethylmaleimide blocked auto-cleavage induced by the addition of myo-inositol hexakisphosphate, suggesting that cysteine residues are essential for the processing of clostridial glucosylating toxins. Our data indicate that clostridial glucosylating cytotoxins possess an inherent cysteine protease activity related to the cysteine protease of Vibrio cholerae RTX toxin, which is responsible for auto-cleavage of glucosylating toxins.