Orlandi P A, Fishman P H
Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, The National Institutes of Health, Bethesda, Maryland 20892-4440, USA.
J Cell Biol. 1998 May 18;141(4):905-15. doi: 10.1083/jcb.141.4.905.
The mechanism by which cholera toxin (CT) is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside GM1, the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma membrane. In this study, we used filipin, a sterol-binding agent that disrupts caveolae and caveolae-like structures, to explore their role in the internalization and activation of CT in CaCo-2 human intestinal epithelial cells. When toxin internalization was quantified, only 33% of surface-bound toxin was internalized by filipin-treated cells within 1 h compared with 79% in untreated cells. However, CT activation as determined by its reduction to form the A1 peptide and CT activity as measured by cyclic AMP accumulation were inhibited in filipin-treated cells. Another sterol-binding agent, 2-hydroxy-beta-cyclodextrin, gave comparable results. The cationic amphiphilic drug chlorpromazine, an inhibitor of clathrin-dependent, receptor-mediated endocytosis, however, affected neither CT internalization, activation, nor activity in contrast to its inhibitory effects on diphtheria toxin cytotoxicity. As filipin did not inhibit the latter, the two drugs appeared to distinguish between caveolae- and coated pit-mediated processes. In addition to its effects in CaCo-2 cells that express low levels of caveolin, filipin also inhibited CT activity in human epidermoid carcinoma A431 and Jurkat T lymphoma cells that are, respectively, rich in or lack caveolin. Thus, filipin inhibition correlated more closely with alterations in the biochemical characteristics of CT-bound membranes due to the interactions of filipin with cholesterol rather than with the expressed levels of caveolin and caveolar structure. Our results indicated that the internalization and activation of CT was dependent on and mediated through cholesterol- and glycolipid-rich microdomains at the plasma membrane rather than through a specific morphological structure and that these glycolipid microdomains have the necessary components required to mediate endocytosis.
霍乱毒素（CT）在细胞内还原并随后激活腺苷酸环化酶之前从质膜内化的机制尚未完全清楚。神经节苷脂GM1是CT的受体，主要聚集在去污剂不溶性糖脂筏和小窝中，小窝是质膜上无包被、富含胆固醇的内陷结构。在本研究中，我们使用了菲律宾菌素（一种破坏小窝和类小窝结构的固醇结合剂）来探究它们在CaCo-2人肠上皮细胞中CT内化和激活过程中的作用。当对毒素内化进行定量时，与未处理细胞中的79%相比，菲律宾菌素处理的细胞在1小时内仅33%的表面结合毒素被内化。然而，通过还原形成A1肽来确定的CT激活以及通过环磷酸腺苷积累来测量的CT活性在菲律宾菌素处理的细胞中受到抑制。另一种固醇结合剂2-羟基-β-环糊精也得到了类似的结果。然而，阳离子两亲性药物氯丙嗪是网格蛋白依赖性、受体介导的内吞作用的抑制剂，与它对白喉毒素细胞毒性的抑制作用相反，它对CT的内化、激活或活性均无影响。由于菲律宾菌素并不抑制后者，这两种药物似乎可以区分小窝介导和有被小窝介导的过程。除了对表达低水平小窝蛋白的CaCo-2细胞有影响外，菲律宾菌素还抑制人表皮样癌A431细胞和Jurkat T淋巴瘤细胞中的CT活性，这两种细胞分别富含或缺乏小窝蛋白。因此，菲律宾菌素的抑制作用与由于菲律宾菌素与胆固醇相互作用导致的CT结合膜生化特性的改变更为密切相关，而不是与小窝蛋白的表达水平和小窝结构相关。我们的结果表明，CT的内化和激活依赖于质膜上富含胆固醇和糖脂的微结构域并通过其介导，而不是通过特定的形态结构，并且这些糖脂微结构域具有介导内吞作用所需的必要成分。
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