Clostridium perfringens β-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity
Clostridium perfringens β-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity
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DOI：
10.1016/s0925-4439(99)00026-5
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发表时间：
1999-05-31
影响因子：
6.2
通讯作者：
Sakurai, J
中科院分区：
文献类型：
--
作者：
Nagahama, M;Kihara, A;Sakurai, J
从产气荚膜梭菌B型分离的β-毒素基因在大肠杆菌中表达为谷胱甘肽S-转移酶（GST）融合基因。从大肠杆菌中纯化GST-β毒素融合蛋白。大肠杆菌E2细胞无致死性。重组β毒素（r毒素）的N端氨基酸序列为G-S-N-D-I-G-K-T-T-T。r毒素的生物学活性和分子量与从C. C型产气荚膜杆菌通过定点突变将Cys-265替换为丙氨酸或丝氨酸导致活性几乎没有损失。用N-乙基马来酰亚胺（NEM）处理C265 A，使r毒素和n毒素的致死活性失活，但活性没有损失。酪氨酸或组氨酸取代Cys-265显著降低致死活性。此外，用乙氧甲酸酐处理C265 H，它特异性地修饰组氨酰残基，导致致死活性的显着降低，但与该代理的r毒素没有。这些结果表明，用具有大尺寸侧链的氨基酸替换位置265处的半胱氨酸残基或在该位置引入官能团导致毒素的致死活性丧失。Tyr-266、Leu-268或Trp-275的替代导致致命活性完全丧失。同时给予r毒素和W275 A导致β-毒素的致死活性降低。这些观察结果表明，该网站的活动是必不可少的接近半胱氨酸残基。(C)1999 Elsevier Science B. V.保留所有权利。
The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue. (C) 1999 Elsevier Science B.V. All rights reserved.