Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells
Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells
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DOI：
10.1074/jbc.m306562200
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发表时间：
2003-09-19
影响因子：
4.8
通讯作者：
Sakurai, J
中科院分区：
文献类型：
--
作者：
Nagahama, M;Hayashi, S;Sakurai, J
产气荚膜梭菌β毒素是坏死性肠炎的重要病原体。在测试的 10 个细胞系中，只有 HL 60 细胞系对 β 毒素敏感。毒素引起细胞肿胀和裂解。用毒素处理细胞导致 K+ 从细胞流出，Ca2+、Na+ 和 Cl- 流入。这些事件在细胞被毒素裂解之前达到最大值。细胞与毒素的孵育显示形成了约 191 和 228 kDa 的毒素复合物，这些复合物位于满足脂筏标准的结构域中。孵育后30分钟才观察到228kDa的复合物，60分钟后仅保留191kDa的复合物。用甲基-β-环糊精或胆固醇氧化酶处理细胞可阻断毒素与筏的结合以及毒素诱导的 K+ 流出和肿胀。毒素诱导的Ca2+内流和形态变化受到聚乙二醇流体动力学直径从200增加到400的抑制，并且被聚乙二醇600和1000显着或完全抑制。然而，这些聚乙二醇对毒素诱导的K+流出没有影响。该毒素诱导含有羧基荧光素的磷脂酰胆碱-胆固醇脂质体释放羧基荧光素，并以剂量依赖性方式形成228 kDa的寡聚物，但不与191 kDa复合物形成寡聚物。我们得出的结论是，该毒素通过与脂筏结合并形成 228 kDa 的功能性寡聚物来作用于 HL 60 细胞。
Clostridium perfringens beta toxin is an important agent of necrotic enteritis. Of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin. The toxin induced swelling and lysis of the cell. Treatment of the cells with the toxin resulted in K+ efflux from the cells and Ca2+, Na+, and Cl- influxes. These events reached a maximum just before the cells were lysed by the toxin. Incubation of the cells with the toxin showed the formation of toxin complexes of about 191 and 228 kDa, which were localized in the domains that fulfilled the criteria of lipid rafts. The complex of 228 kDa was observed until 30 min after incubation, and only the complex of 191 kDa remained after 60 min. Treatment of the cells with methyl-beta-cyclodextrin or cholesterol oxidase blocked binding of the toxin to the rafts and the toxin-induced K+ efflux and swelling. The toxin-induced Ca2+ influx and morphological changes were inhibited by an increase in the hydrodynamic diameter of polyethylene glycols from 200 to 400 and markedly or completely inhibited by polyethylene glycol 600 and 1000. However, these polyethylene glycols had no effect on the toxin-induced K+ efflux. The toxin induced carboxyfluorescein release from phosphatidylcholine-cholesterol liposomes containing carboxyfluorescein and formed an oligomer with 228 kDa in a dose-dependent manner but did not form an oligomer with the 191-kDa complex. We conclude that the toxin acts on HL 60 cells by binding to lipid rafts and forming a functional oligomer with 228 kDa.