BIOSYNTHESIS AND FUNCTION OF GLYCOSPHINGOLIPIDS AND OTHER GLYCOCONJUGATES

霍乱弧菌产生的霍乱毒素（CT）是霍乱的病原体， 霍乱 CT由一个B亚单位组成， 肠粘膜细胞表面的神经节苷脂GM1和A 参与腺苷酸环化酶激活的亚基。 后者 这一过程需要还原A亚基以产生A1 腺苷酸环化酶G蛋白（Gs）。 此修改将阻止 GS的内在GT3活性，并保持环化酶持续 激活状态。我们一直在研究 CT的细胞加工和激活。 作为一个模型，我们使用人类 肠上皮细胞，CT的天然靶点。 我们特别感兴趣 在毒素结合和环化酶之间的滞后期期间的事件中， activation. 我们以前已经证明，全毒素结合到 细胞表面，A亚基背向膜， 滞后期细胞产生少量A1肽， 环化酶被激活。 我们现在表明，CT减少了一个 细胞还原酶活性，我们已经确定为蛋白质二硫化物 异构酶（PDI）。 尽管PDI存在于内质网的内腔中， 网织，一些存在于细胞表面。使用细胞 不参与CT减少，因此A1的产生发生在 胞内位点。 有人提出，CT是通过 微区富含胆固醇，糖脂， Caveolae及其功能。 我们观察到菲律宾治疗阻断了 CT激活腺苷酸环化酶的能力且作用迅速， CT通过小窝进入细胞，并被PDI还原 以产生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.