BIOSYNTHESIS AND FUNCTION OF GLYCOSPHINGOLIPIDS AND OTHER GLYCOCONJUGATES

霍乱弧菌产生的霍乱毒素(CT)是引起霍乱的病原体 霍乱。CT由与其结合的B亚基组成 肠粘膜细胞表面神经节苷脂GM1和A 参与腺酰环化酶激活的亚基。后者 该过程需要减少A亚基以产生A1 腺酰环化酶G蛋白(Gs)。此修改会阻止 Gs固有的GTP酶活性并使环化酶处于持续的 激活状态。我们一直在调查这一事件的详细机制 CT的细胞处理和激活。作为一个模特，我们使用的是人类 肠上皮细胞，CT的天然靶点。我们特别感兴趣的是 在毒素结合和环化酶之间的滞后期发生的事件 激活。我们之前已经证明，全息毒素与 A亚基背离细胞膜的细胞表面 滞后期细胞产生少量的A1肽，并且 循环酶被激活。我们现在证明，CT减少了一个 我们已鉴定为蛋白质二硫化物的细胞还原酶活性 异构酶(PDI)。尽管在内质腔中发现了PDI 网状结构，一些存在于细胞表面。使用单元格 不参与CT降低，因此A1的产生发生在 胞内位置。有人提出，CT通过以下方式内化 微域富含胆固醇、糖脂、 小窝及其功能。我们观察到非利平治疗阻断了 CT激活腺酰环化酶能力强，见效快， 哪个CT通过小窝进入细胞，并接受PDI的还原 在细胞内的隔间中产生A1肽。

Cholera toxin (CT) produced by Vibrio cholerae is the causative agent of the disease cholera. CT consists of a B subunit that binds to ganglioside GM1 on the surface of the intestinal mucosal cell and an A subunit that is involved in activation of adenylyl cyclase. The latter process requires that the A subunit be reduced to generate the A1 G protein (Gs) of adenylyl cyclase. This modification blocks the intrinsic GTPase activity of Gs and keeps the cyclase in a persistently activated state. We have been investigating the detailed mechanism of cellular processing and activation of CT. As a model, we are using human enterocytes, the natural target for CT. We are particularly interested in events during the lag period between toxin binding and cyclase activation. We previously have shown that the holotoxin binds to the cell surface with the A subunit facing away from the membrane and that lag period small amounts of A1 peptide are generated by the cells, and the cyclase becomes activated. We now show that CT is reduced by a cellular reductase activity which we have identified as protein disulfide isomerase (PDI). Although PDI is found in the lumen of the endoplasmic reticulum, some is present on the cell surface. Using cell not involved in CT reduction, and thus generation of A1 occurs at an intracellular site. It has been proposed that CT is internalized through microdomains are enriched in cholesterol, glycolipids, caveolae and their function. We observed that filipin treatment blocks the ability of CT to activate adenylyl cyclase and the effect is rapid, which CT enters the cell through caveolae, and undergoes reduction by PDI in an intracellular compartment to generate the A1 peptide.