BIOSYNTHESIS AND FUNCTION OF GLYCOSPHINGOLIPIDS AND OTHER GLYCOCONJUGATES

鞘糖脂和其他糖复合物的生物合成和功能

• 批准号: 6111811
• 负责人: P H FISHMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6111811
• 关键词: ADP ribosylation; G protein; adenine phosphoribosyltransferase; adenylate cyclase; biological signal transduction; cell membrane; chemical binding; cholera toxin; enzyme activity; gangliosides; glycolipids; glycoproteins; glycosphingolipids; guanosinetriphosphatases; human tissue; isomerase; lipid biosynthesis; peptides; polyenes; protein structure function; tissue /cell culture

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.

霍乱毒素 (CT)，由霍乱弧菌产生 霍乱的病原体是典型的 A-B 毒素。 B 亚基与顶端的神经节苷脂 GM1 结合 肠粘膜细胞表面，而 A 亚基后 被还原生成 A1 肽，激活腺苷酸环化酶 位于基底外侧表面。 A1 是一种 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 GTPase 活性 保持环化酶持续激活。我们曾经 研究细胞处理的详细机制和 CT 的激活，使用人肠 CaCo-2 细胞作为模型， 在培养物中表现为分化的肠细胞，天然的 CT 目标。我们对滞后期间的事件特别感兴趣 毒素结合和环化酶激活之间的时期。我们之前 表明全毒素通过 A 与细胞表面结合 亚基背向膜并通过内化 小穴。一部分毒素逆行运输到 高尔基体和内质网，其中 A 亚基被蛋白质二硫键异构酶还原形成小亚基 A1肽的量。然而，尚不清楚是否 Gs(α) 的 ADP-核糖基化发生在 A1 位点处或附近 形成或在基底外侧表面，因此需要进一步 将 A1 转运至环化酶复合物。为了解决这个问题，我们 检查A1之间的时间和空间关系 Gs(α) 的形成、ADP-核糖基化和腺苷酸环化酶 激活。洗涤并加温在 4°C 下用 CT 处理的细胞 37°C 不同时间；然后制备粗膜 并进行了测定。升温 20 分钟内检测到 A1 肽 而 Gs(α) 的 ADP-核糖基化和腺苷酸的激活 直到 30-35 分钟才检测到环化酶，并且两者似乎都 同时发生。将细胞加热至仅 30°C 会增加 总体激活时间，但没有解决两个事件。 在 Transwell 细胞培养室中生长的极化细胞给出 当CT应用于根尖表面时，得到类似的结果。水性 粗膜的两相分配表明 发生 ADP-核糖基化和腺苷酸环化酶激活 主要存在于质膜部分并反映了 用粗膜观察到的时间进程。我们的结果表明 A1肽在形成后必须被转运至 基底外侧表面的细胞质面激活 腺苷酸环化酶复合物。