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: 3760202
• 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/3760202
• 关键词: G protein; adenylate cyclase; antifungal antibiotics; biological signal transduction; cholera toxin; gangliosides; gel electrophoresis; glycolipids; glycoproteins; glycosphingolipids; human tissue; receptor binding; receptor coupling; receptor mediated endocytosis; tissue /cell culture

Cholera toxin (CT) produced by Vibrio cholerae and the heat-labile enterotoxin (LT) produced by certain strains of Escherichia coli are the causative agents of cholera and traveler's diarrhea, respectively. The toxins are structurally, immunologically and functionally similar: each has a pentameric B subunit which binds to specific receptors on the intestinal mucosal cell and an A subunit which is involved in activation of adenylylcyclase. Whereas CT uses the ganglioside GM1 as its only receptor, LT appears to recognize G/M1 as well as other receptors on intestinal cells. In order to identify these alternate LT receptors, we used human intestinal CaCo-2 cells, which behave in culture as differentiated enterocytes, the natural target for the two toxins. CaCo-2 cells bound 8-fold more LT than CT, and LT binding was only partially inhibited by CT-B whereas CT binding was completely inhibited by LT-B. Although G/M1 was the only CaCo-2 glycolipid recognized by both CT and LT, a series of membrane galactoproteins (apparent molecular weights between 92 and 120 kDa) were recognized by LT but not by CT. By using specific glycosidases and transferrin and fetuin as model galactoproteins, we established that the binding determinant for LT on these galactoproteins was galactose (beta1-4)N-acetylglucosamine. Furthermore, we were able to show that these galactoproteins recognized by LT were immunoprecipitated by specific antisera against polyactosylated glycoproteins of the lacto-N-neotetraosyl type. Finally, some of these alternate galactoprotein receptors for LT appear to be functional as activation of adenylylcyclase by LT in CaCo-2 cells was only partially inhibited by CT-B. Thus, despite the high degree of homology between LT and CT, LT can recognize glycoconjugates with carbohydrate structures distinct from the oligosaccharide of GM/1 which is recognized by both toxins. Furthermore, the apparent ability of LT to utilize both glycolipids and glycoproteins as receptors raises the possibility that LT may use more than one pathway for cellular activation. In this regard, we had shown previously that CT bound to neoganglioproteins enters the cell through a different pathway than when bound to G/M1.

霍乱弧菌产生的霍乱毒素（CT）及其不耐热性 某些大肠杆菌菌株产生的肠毒素（LT）是 分别是霍乱和旅行者腹泻的病原体。 的 毒素在结构上、免疫学上和功能上是相似的： 具有一个五聚体B亚基，其结合到 肠粘膜细胞和参与激活的A亚单位 腺苷酸环化酶 而CT使用神经节苷脂GM 1作为其唯一的 受体，LT似乎识别G/M1以及其他受体上 肠细胞 为了鉴定这些替代LT受体，我们 使用人类肠道CaCo-2细胞，其在培养中表现为 分化的肠上皮细胞，这两种毒素的天然目标。 CaCo-2细胞与LT的结合是CT的8倍，而与LT的结合仅为CT的1倍。 CT-B部分抑制，而CT结合完全抑制 Lt-B。 虽然G/M1是唯一被两者识别的CaCo-2糖脂 CT和LT是一系列膜半乳糖蛋白（表观分子量 重量在92和120 kDa之间）被LT识别，但不被CT识别。 通过 使用特异性糖苷酶和转铁蛋白和胎球蛋白作为模型 半乳糖蛋白，我们建立了LT的结合决定簇 这些半乳糖蛋白是半乳糖（β 1 -4）N-乙酰葡糖胺。 此外，我们能够证明这些半乳糖蛋白识别 通过LT的特异性抗血清免疫沉淀， 乳糖-N-新四糖基型的多乳糖基化糖蛋白。 最后， LT的这些替代半乳糖蛋白受体中的一些似乎是 在CaCo-2细胞中，LT激活腺苷酸环化酶的功能是 仅部分被CT-B抑制。 因此，尽管高度 由于LT和CT之间的同源性，LT可以识别具有 糖结构不同于GM/1的寡糖， 两种毒素都能识别 此外，LT的明显能力 利用糖脂和糖蛋白作为受体， LT可能使用一种以上的细胞通路， activation. 在这方面，我们以前已经表明，CT结合到 新神经节蛋白通过不同的途径进入细胞， 与G/M1绑定。