Molecular biological and crystallographycal anaylsis of Clostridium perfringens iota-toxin

产气荚膜梭菌毒素是一种由酶组分(Ia)和结合组分(Ib)组成的二元毒素。IB寡聚在膜上形成离子通透性通道，寡聚体诱导内吞。为了阐明IOTA-毒素的作用方式，我们用Cy3标记的Jib检测了Ib的结合和内化。37℃孵育60min后，标记的Ib主要分布在细胞膜上，120min内可在胞浆内检测到Ib的存在。用甲基-β-环糊精(M-β-CD)处理细胞后，与Ib细胞的结合和内化明显减少。为了确定Ib是否与脂筏结合，将MDCK细胞与Ib在4或37℃孵育，并用蔗糖密度梯度离心法分离不溶于Triton的膜。Ib复合体在37℃时定位于符合脂筏标准的不溶性组分，但在4℃时未形成。37℃孵育60min后，脂筏组分中的复合体数量最多，120min内复合体消失。当Ib在4℃预孵育30min，37℃孵育60min时，在RAFT组分中可检测到该复合体。MITH-M-β-CD处理MDCK细胞后，减少了TB复合体在脂筏上的定位，减少了Ia+Ib诱导的细胞圆形。当~(125)I标记的Ia在37℃的Ib存在下与细胞孵育时，它定位于脂筏组分。表面等离子体共振分析表明，Ia与Ib的齐聚物结合，但不与单体结合。我们得出结论，Ib与质膜上的受体结合，然后移动到脂筏上，Ia与脂筏中形成的Ib低聚体结合在细胞内。

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.