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Molecular biological and crystallographycal anaylsis of Clostridium perfringens iota-toxin

产气荚膜梭菌毒素的分子生物学和晶体学分析

基本信息

批准号：
14570251
负责人：
SAKURAI J.
金额：
$ 2.24万
依托单位：
Tokushima Bunri University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and binding component (Ib). Ib oligomerizes to form ion-permeable channels in membranes and the oligomer induces endocyosis. To elucidate the mode of action of iota-toxin, we examined the binding and internalization of Ib using Cy3-labeled Jib. The labeled Ib was retained at the plasma membrane of MDCK cells till 60 min of incubation at 37℃, and was detected in cytoplasmic vesicles within 120 min. Treatment of the cells with methyl-β-cyclodextrin (MβCD) resulted in a marked reduction in binding to and internalization into the cells of Ib. To determine whether Ib associates with lipid rafts, MDCK cells were incubated with Ib at 4 or 37 ℃ and the Triton-insoluble membranes were fractionated by sucrose density gradient centrifugation. An Ib complex of 500 kDa was localized at 37 ℃ to the insoluble fractions that fulfilled the criteria of lipid rafts, but did not form at 4 ℃. The amount of complex in the lipid raft fraction reached a maximum after 60 min of incubation at 37 ℃ and the complex disappeared within 120 min. When the cells preincubated with Ib at 4 ℃ for 30 min were incubated at 37 ℃ for 60 min, the complex was detected in the raft fraction. Treatment of MDCK cells with Mith MβCD reduced the localization of the Tb complex to the lipid rafts and rounding of the cells induced by Ia plus Ib. When ^<125> I-labeled Ia was incubated with the cells in the presence of Ib at 37 ℃, it was localized in the lipid raft fraction. Surface plasmon resonance analysis revealed that Ia binds to the oligomer of Ib, but not the monomer. We conclude that Ib binds to a receptor in plasma membranes, then moves to lipid rafts, and Ia bound to the oligomer of Ib formed in the rafts is internalized in the cells.

产气荚膜梭菌I毒素是由酶组分（Ia）和结合组分（Ib）组成的二元毒素。Ib寡聚化以在膜中形成离子渗透通道，并且寡聚体诱导胞吞作用。为了阐明iota毒素的作用模式，我们使用Cy 3标记的Jib检测Ib的结合和内化。在37℃孵育60 min后，标记的Ib仍保留在MDCK细胞的质膜上，120 min内可在胞浆囊泡中检测到。用甲基-β-环糊精（MβCD）处理MDCK细胞，可显著降低Ib与细胞的结合和内化。为了确定Ib是否与脂筏结合，将MDCK细胞与Ib在4或37 ℃下孵育，并通过蔗糖密度梯度离心分离Triton不溶性膜。在37 ℃下，500 kDa的Ib复合物定位于满足脂筏标准的不溶性组分，但在4 ℃下不形成。37 ℃孵育60 min后，脂筏组分中复合物的含量达到最大值，120 min内复合物消失。将4 ℃下与Ib预孵育30 min的细胞在37 ℃孵育60 min后，在脂筏组分中检测到复合物。用Mith MβCD处理MDCK细胞可减少Tb复合物在脂筏上的定位以及Ia和Ib诱导的细胞变圆。当<125>37 ℃下，在Ib存在的情况下，将^I标记的Ia与细胞一起孵育时，其定位于脂筏部分。表面等离子体共振分析表明，Ia结合的低聚体Ib，但不是单体。我们的结论是，Ib结合到质膜上的受体，然后移动到脂筏，Ia结合到形成的筏中的Ib的寡聚体在细胞中被内化。