Centre for Cancer Biomedicine and Institute for Cancer Research, Department of Biochemistry, Oslo University Hospital and University of Oslo, 0316 Oslo, Norway.
Cell Microbiol. 2010 Dec;12(12):1809-20. doi: 10.1111/j.1462-5822.2010.01512.x.
Clostridium botulinum C2 toxin is an ADP-ribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft-dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant-negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA-mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin-independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho-mediated uptake of C2 toxin. In conclusion, C2 toxin is endocytosed by dynamin-dependent mechanisms and we provide evidence for involvement of clathrin and Rho.
肉毒梭菌 C2 毒素是一种 ADP-核糖基转移酶，导致真核细胞中肌动蛋白细胞骨架的解聚。C2 毒素是一种由酶亚基 C2I 和结合亚基 C2II 组成的二元毒素。结合亚基的蛋白水解激活触发七聚体结构（C2IIa）的形成，该结构与细胞受体结合。C2I 能够与 C2IIa 寡聚体结合，有人提出整个复合物通过筏依赖机制被内化。在这里，我们分析了 C2 毒素是如何被内吞的。在表达显性失活 dynamin 突变体的 HeLa 细胞中，细胞毒性和 C2 毒素摄取被阻断。此外， flotillin 的 siRNA 介导敲低或 Arf6 功能的抑制，这些蛋白被认为参与 dynamin 独立的内吞作用，并不影响 C2 毒性。caveolin 的敲低并不抑制 C2 毒素的内吞作用，而 clathrin 功能的抑制减少了 C2 毒素的摄取并延迟了细胞毒性作用。最后，我们发现了 C2 毒素被 Rho 介导摄取的证据。总之，C2 毒素通过依赖 dynamin 的机制被内吞，并且我们提供了涉及网格蛋白和 Rho 的证据。
