BIOSYNTHESIS AND FUNCTION OF GLYCOSPHINGOLIPIDS AND OTHER GLYCOCONJUGATES

霍乱毒素（CT），由霍乱弧菌产生 霍乱的病原体是典型的A-B型 毒素B亚基与神经节苷脂GM 1在顶端结合 肠粘膜细胞表面的A亚基，而 被还原生成A1肽，激活腺苷酸环化酶 在基底外侧表面。A1是ADP-核糖基转移酶， 修饰刺激性G蛋白（Gs）的α亚基， 腺苷酸环化酶，从而阻断其内在的GT3活性， 保持环化酶持续激活。我们一直 研究细胞加工的详细机制， 激活CT，使用人肠CaCo-2细胞作为模型， 在培养中表现为分化的肠上皮细胞， CT目标。我们特别感兴趣的是滞后期间的事件 毒素结合和环化酶激活之间的时间。我们之前 显示，全毒素与细胞表面结合， 亚基背对膜，并通过 小窝一部分毒素逆行运输到 高尔基体和内质网 亚基被蛋白质二硫键异构酶还原形成小的 A1肽的量。然而，目前尚不清楚， Gs（α）的ADP-核糖基化发生在A1位点或其附近 形成或在基底外侧表面，从而需要进一步的 A1向环化酶复合物的运输。为了解决这个问题，我们 研究了A1和A2之间的时空关系， Gs（α）的ADP核糖基化和腺苷酸环化酶 activation.将在4 ℃下用CT处理的细胞洗涤并加温 在37 ℃下反应不同时间，然后制备粗膜 并测定。A1肽在升温20 min内检测到 而Gs（α）的ADP-核糖基化和腺苷酰的活化 环化酶直到30-35分钟才检测到， 同时发生。将细胞加热到30摄氏度， 总激活时间，但未解决这两起事件。 在Transwell细胞培养室中生长的极化细胞得到 当CT应用于根尖表面时，结果相似。水性 粗膜的两相分配表明， ADP-核糖基化和腺苷酸环化酶激活发生 主要存在于质膜部分，并反映了 用粗制膜观察时间过程。我们的研究结果表明 A1肽在形成时必须被转运到 细胞质面的基底外侧表面激活 腺苷酸环化酶复合物。

Cholera toxin (CT), produced by Vibrio cholerae and the causative agent of the disease cholera, is a typical A-B toxin. The B subunit binds to ganglioside GM1 on the apical surface of the intestinal mucosal cell whereas the A subunit after being reduced to generate the A1 peptide, activates adenylyl cyclase on the basolateral surface. A1 is an ADP-ribosyltransferase that modifies the alpha subunit of the stimulatory G protein (Gs) of adenylyl cyclase, thus blocking its intrinsic GTPase activity and keeping the cyclase persistently activated. We have been investigating the detailed mechanism of cellular processing and activation of CT, using as a model human intestinal CaCo-2 cells, that behave in culture as differentiated enterocytes, the natural target for CT. We are particularly interested in events during the lag period between toxin binding and cyclase activation. We previously showed that the holotoxin binds to the cell surface with the A subunit facing away from the membrane and is internalized through caveolae. A portion of the toxin undergoes retrograde transport to the Golgi apparatus and the endoplasmic reticulum where the A subunit is reduced by protein disulfide isomerase to form small amounts of A1 peptide. It is unclear, however, whether ADP-ribosylation of Gs(alpha) occurs at or near the site of A1 formation or at the basolateral surface, thereby requiring further trafficking of A1 to the cyclase complex. To address this issue, we examined the temporal and spatial relationship between A1 formation, ADP-ribosylation of Gs(alpha), and adenylyl cyclase activation. Cells treated with CT at 4 C were washed, and warmed to 37 C for different times; then crude membranes were prepared and assayed. A1 peptide was detected within 20 min of warming whereas ADP-ribosylation of Gs(alpha) and activation of adenylyl cyclase were not detectible until 30-35 min, and both appeared to occur concurrently. Warming the cells to only 30 C increased overall activation times, but did not resolve the two events. Polarized cells grown in Transwell cell culture chambers gave similar results when CT was applied to the apical surface. Aqueous two-phase partitioning of the crude membranes showed that ADP-ribosylation and adenylyl cyclase activation occurred predominantly in the plasma membrane fraction and mirrored the time course observed with crude membranes. Our results indicate that A1 peptide upon formation must be transported to the cytoplasmic face of the basolateral surface to activate the adenylylcyclase complex.