Nagahama M, Kihara A, Miyawaki T, Mukai M, Sakaguchi Y, Ochi S, Sakurai J
Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770, Japan.
Biochim Biophys Acta. 1999 May 31;1454(1):97-105. doi: 10.1016/s0925-4439(99)00026-5.
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.
从产气荚膜梭菌B型分离得到的β毒素基因在大肠杆菌中作为谷胱甘肽S-转移酶（GST）融合基因表达。从大肠杆菌转化细胞中纯化得到的GST-β毒素融合蛋白没有致死性。通过凝血酶切割融合蛋白分离得到的重组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产气荚膜梭菌C型纯化得到的天然β毒素（n毒素）没有区别。通过定点诱变将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C265A，NEM使r毒素和n毒素的致死活性失活，但该处理并未导致活性丧失。用酪氨酸或组氨酸替换Cys-265显著降低了致死活性。此外，用特异性修饰组氨酸残基的乙氧基甲酸酐处理C265H导致致死活性显著降低，但用该试剂处理r毒素则没有这种情况。这些结果表明，用侧链较大的氨基酸替换265位的半胱氨酸残基或在该位置引入官能团会导致毒素致死活性丧失。替换Tyr-266、Leu-268或Trp-275会导致致死活性完全丧失。同时给予r毒素和W275A会导致β毒素的致死活性降低。这些观察结果表明，活性必需位点靠近半胱氨酸残基。
