Ohsaki Yuki, Sugimoto Yuko, Suzuki Michitaka, Kaidoh Toshiyuki, Shimada Yukiko, Ohno-Iwashita Yoshiko, Davies Joanna P, Ioannou Yiannis A, Ohno Kousaku, Ninomiya Haruaki
Department of Neurobiology, Tottori University Faculty of Medicine, 683-8503, Yonago, Japan.
Histochem Cell Biol. 2004 Apr;121(4):263-72. doi: 10.1007/s00418-004-0643-7. Epub 2004 Apr 7.
Theta-toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(-) that lacked Niemann-Pick C1 showed reduced sensitivity to theta-toxin, compared with wild-type (wt) cells. BCtheta is a derivative of theta-toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BCtheta, both on the cell surface and in the extracellular space of these cells. BCtheta binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4 degrees C and recovery of bound BCtheta in floating low-density fractions on sucrose density gradient fractionation. BCtheta-labeled vesicles were abolished when NPC1(-) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, theta-toxin sensitivity of NPC1(-) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to theta-toxin.
θ毒素（产气荚膜梭菌溶素O）与细胞表面胆固醇结合并形成寡聚孔，导致膜损伤。在细胞毒性和细胞存活试验中，与野生型（wt）细胞相比，缺乏尼曼-匹克C1的突变中国仓鼠卵巢细胞系NPC1(-)对θ毒素的敏感性降低。BCθ是θ毒素的衍生物，保留胆固醇结合活性但缺乏细胞毒性。共聚焦显微镜和电子显微镜显示，在这些细胞的细胞表面和细胞外空间存在多个与BCθ结合的囊泡。通过BCθ在4℃下对1% Triton X-100的抗性以及在蔗糖密度梯度分级分离中漂浮的低密度级分中回收结合的BCθ，验证了BCθ与筏微结构域的结合。当NPC1(-)细胞耗尽脂蛋白时以及用Rho相关激酶抑制剂Y-27632处理时，BCθ标记的囊泡消失。此外，在用孕酮处理的wt细胞中也观察到类似的囊泡。与这些结果一致，当细胞耗尽脂蛋白或用Y-27632处理时，NPC1(-)细胞对θ毒素的敏感性增加，而wt细胞的敏感性则因孕酮而降低。我们的研究结果表明，毒素被隔离到富含筏的细胞表面囊泡可能是NPC1缺陷细胞对θ毒素敏感性降低的基础。
