BIOSYNTHESIS AND FUNCTION OF GLYCOSPHINGOLIPIDS AND OTHER GLYCOCONJUGATES

神经节苷脂GM1是霍乱毒素(CT)的细胞表面受体。这个 CT的五聚体B亚基与GM1结合，而CT的A亚基与GM1结合 参与腺酰环化酶的激活。A亚基被简化为 ADP-核糖化刺激G蛋白(Gs)的A1肽 循环酶。CT结合时的定位，结合的途径 A亚基进入细胞并被还原为A1，以及后者是如何获得的 与G(S)的联系尚未建立。我们已经成功地 利用人类肠道Caco-2细胞阐明了其中的一些方面， 在培养中表现为分化的肠道细胞，自然的靶细胞 做CT检查。 CT定位：我们能够在细胞表面组装活性CT 通过顺序地将细胞暴露于不活跃的B和A亚基。基座 根据已知的CT结构，我们得出结论：CT通过其自身的功能与细胞结合 背离细胞膜的一个亚单位。使用A1和B的抗体， 我们能够证明这两个亚基都被细胞内化。 CT的胞内处理：追求CT的进一步处理 内化CT，我们使用了特定的阻滞剂。氯喹和氯喹 莫能菌素，通过包衣抑制受体介导的内吞作用 对CT活动无明显影响。相比之下，灯盏花素A会导致 高尔基体的解体是CT作用的一个强有力的阻滞剂。 虽然灯盏花素A没有阻止CT的内化，但它确实阻止了CT的内化 阻止其转化为A1肽。因为后者是必不可少的 在CT的作用步骤中，灯盏花素A可能是一种有用的探针 勾勒出CT的细胞内加工过程。

Ganglioside GM1 is the cell surface receptor for cholera toxin (CT). The pentameric B subunit of CT binds to GM1 whereas the A subunit of CT is involved in activation of adenylyl cyclase. The A subunit is reduced to the A1 peptide which ADP-ribosylates the stimulatory G protein (Gs) of the cyclase. The orientation of CT when it binds, the pathway by which the A subunit enters cells and is reduced to A1, and how the latter gains access to G(s) have not yet been established . We have succeeded in clarifying some of these aspects using human intestinal Caco-2 cells, which behave in culture as differentiated enterocytes, the natural target for CT. Orientation of CT: We were able to assemble active CT at the cell surface by sequentially exposing cells to the inactive B and A subunits. Based on the known structure of CT, we conclude that CT binds to cells with its A subunit facing away from the membrane. Using antibodies to A1 and B, we were able to demonstrate that both subunits are internalized by cells. Intracellular Processing of CT: To pursue the further processing of the internalized CT, we employed specific blockers. Chloroquine and monensin, which inhibit receptor-mediated endocytosis through coated pits, had no effect on CT action. By contrast, brefeldin A, which causes disassembly of the Golgi apparatus, was a potent blocker of CT action. Although brefeldin A did not prevent the internalization of CT, it did prevent its conversion to the A1 peptide. As the latter is an essential step in the action of CT, brefeldin A may be a useful probe for delineating the intracellular processing of CT.