Binding and internalization of Clostridium perfringens iota-toxin in lipid rafts
Binding and internalization of Clostridium perfringens iota-toxin in lipid rafts
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DOI：
10.1128/iai.72.6.3267-3275.2004
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发表时间：
2004-06-01
影响因子：
3.1
通讯作者：
Sakurai, J
中科院分区：
文献类型：
--
作者：
Nagahama, M;Yamaguchi, A;Sakurai, J
产气荚膜梭菌毒素是一种由酶成分（Ia）和结合成分（Ib）组成的二元毒素。在膜中形成的Ib低聚物诱导胞吞作用。我们用cy3标记的Ib检测了Ib的结合和内化。标记的Ib在MDCK细胞37度孵育60分钟后，在细胞质囊泡中检测到。为了确定Ib是否与脂筏相关，我们将MDCK细胞与Ib在4℃或37℃下孵育，并对triton不溶性膜进行分离。500 kDa的Ib复合物在37°c时定位于满足脂筏标准的不溶性组分，但在4°c时不形成。37℃孵育60 min后，筏状馏分中复合物的数量达到最大值。在4℃下用Ib预孵育的细胞在37℃下孵育时，筏分数中检测到复合物。甲基-p-环糊精处理MDCK细胞，降低了Ib复合物在筏上的定位和la + Ib诱导的细胞的圆角。当125 i标记的la与细胞在37℃Ib存在下孵育时，它定位在筏分数中。表面等离子体共振分析表明，la与Ib的低聚物结合。我们得出结论，Ib与膜中的受体结合，然后移动到筏中，la与筏中形成的Ib低聚物结合被内化。
Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). The oligomer of Ib formed in membranes induces endocytosis. We examined the binding and internalization of Ib by using Cy3-labeled Ib. Labeled Ib was retained at the membranes of MDCK cells for 60 min of incubation at 37degreesC, and later it was detected in cytoplasmic vesicles. To determine whether Ib associates with lipid rafts, we incubated MDCK cells with Ib at 4 or 37degreesC and fractionated the Triton-insoluble membranes. An Ib complex of 500 kDa was localized at 37degreesC to the insoluble fractions that fulfilled the criteria of lipid rafts, but it did not form at 4degreesC. The amount of complex in the raft fraction reached a maximum after 60 min of incubation at 37degreesC. When the cells that were preincubated with Ib at 4degreesC were incubated at 37degreesC, the complex was detected in the raft fraction. The treatment of MDCK cells with methyl-p-cyclodextrin reduced the localization of the Ib complex to the rafts and the rounding of the cells induced by la plus Ib. When 125 I-labeled la was incubated with the cells in the presence of Ib at 37degreesC, it was localized in the raft fraction. Surface plasmon resonance analysis revealed that la binds to the oligomer of Ib. We conclude that Ib binds to a receptor in membranes and then moves to rafts and that la bound to the oligomer of Ib formed in the rafts is internalized.