Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)
Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)
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DOI：
10.1073/pnas.091090798
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发表时间：
2001-04-24
影响因子：
11.1
通讯作者：
Ohno-Iwashita, Y
中科院分区：
文献类型：
--
作者：
Waheed, AA;Shimada, Y;Ohno-Iwashita, Y
越来越多的证据表明，质膜中存在富含鞘脂和胆固醇的微区（脂筏）。特定的蛋白质在这些膜区聚集，并在信号转导和许多其他细胞活动中发挥作用。胆固醇缺失会导致脂筏相关蛋白质解体，这表明胆固醇在脂筏的结构维持和功能方面起着至关重要的作用。然而，一直没有可用于检测和监测活细胞中脂筏胆固醇的工具。在此我们表明，产气荚膜梭菌溶素O（θ -毒素）的一种经蛋白酶切割且生物素化的衍生物（BCθ）能选择性地与完整细胞中富含胆固醇的微区结合，这些微区符合脂筏的标准。我们在蔗糖梯度上对与BCθ孵育后的未处理血小板和经Triton X - 100处理的血小板匀浆进行分级分离。BCθ主要定位于漂浮的低密度组分（FLDF）中，此处胆固醇、鞘磷脂和Src家族激酶含量丰富。免疫电子显微镜显示BCθ与FLDF中的一个囊泡亚群结合。用环糊精使血小板中的胆固醇减少35%，这伴随着FLDF中胆固醇减少76%，几乎完全消除了BCθ与FLDF的结合。在有和没有胆固醇缺失的人表皮样癌A431细胞中，BCθ和菲律宾菌素的染色模式表明BCθ与细胞表面的特定膜区结合，而菲律宾菌素与细胞胆固醇的结合是无差别的。此外，BCθ的结合不会对细胞膜造成任何损伤，这表明BCθ是一种用于检测活细胞中膜脂筏的有用探针。
There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BC theta) of perfringolysin O (theta -toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BC theta on a sucrose gradient. BC theta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BC theta binds to a subpopulation of vesicles in FLDF, Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BC theta binding to FLDF. The staining patterns of BC theta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BC theta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BC theta binding does not cause any damage to cell membranes, indicating that BC theta is a useful probe for the detection of membrane rafts in living cells.