Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains.
Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains.
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DOI：
10.1083/jcb.141.4.905
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发表时间：
1998-05-18
期刊：
影响因子：
--
通讯作者：
Fishman PH
中科院分区：
文献类型：
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作者：
Orlandi PA;Fishman PH
霍乱毒素（CT）在胞内还原和随后激活腺苷酸环化酶之前从质膜内化的机制尚不清楚。神经节苷脂GM 1，CT的受体，主要是聚集在洗涤剂不溶性糖脂筏和小窝，无涂层，富含胆固醇的质膜上的内陷。在这项研究中，我们使用菲律宾，一种甾醇结合剂，破坏小窝和小窝样结构，探讨其在CaCo-2人肠上皮细胞的CT的内化和激活的作用。当毒素内化进行定量，只有33%的表面结合的毒素被菲律宾处理的细胞在1小时内内化，而在未处理的细胞中为79%。然而，CT激活确定其减少，形成A1肽和CT活性测定环AMP积累被抑制在菲律宾处理的细胞。另一种甾醇结合剂，2-羟基-β-环糊精，给出了类似的结果。阳离子两亲性药物氯丙嗪，网格蛋白依赖性，受体介导的内吞作用的抑制剂，但是，既不影响CT内化，激活，也不活动，其抑制作用白喉毒素的细胞毒性。由于菲律宾不抑制后者，这两种药物似乎区分小窝和包被坑介导的过程。除了其在表达低水平小窝蛋白的CaCo-2细胞中的作用外，菲律宾肽还抑制分别富含或缺乏小窝蛋白的人表皮样癌A431和Jurkat T淋巴瘤细胞中的CT活性。因此，菲律宾蛋白抑制与CT结合膜的生化特性的改变，由于菲律宾蛋白与胆固醇的相互作用，而不是与小窝蛋白和小窝结构的表达水平更密切相关。我们的研究结果表明，CT的内化和激活是依赖于和介导的，而不是通过一个特定的形态结构，这些糖脂微结构域的细胞膜上的胆固醇和糖脂丰富的微结构域具有必要的组件需要介导内吞作用。
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-β-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.