TRANSIENT GLUCOSYLATION OF GLYCOPROTEINS

糖蛋白的瞬时糖基化

• 批准号: 3303629
• 负责人: ARMANDO JOSE PARODI
• 金额: $ 6.05万
• 依托单位: LELOIR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-15 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303629
• 关键词: Golgi apparatus; antibody formation; carbohydrate sequence; endoplasmic reticulum; enzyme mechanism; glucose metabolism; glycopeptides; glycoprotein biosynthesis; glycoprotein structure; glycosyltransferase; immunocytochemistry; intracellular transport; laboratory rabbit; laboratory rat; mannosidase; molecular cloning; oligosaccharides; protein purification; site directed mutagenesis

We have described that protein-linked, high mannose-type oligosaccharides lacking glucose units are glucosylated directly from UDP-Glc (that is, without dolichol derivatives as intermediates) in the endoplasmic reticulum (ER) of mammalian, plant, fungal and protozoan cells and that the glucose units are subsequently removed, probably by glucosidase II. The ultimate aim of this proposal is to find a role for these reactions. For this purpose the UDP-Glc: Glycoprotein Glucosyltransferase (an enzyme apparently loosely attached to the lumen of the ER) will be purified, antibodies against it will be raised an d its localization will be established by immunocytochemistry. Double localization of glucosidase II and the glucosyltransferase by the immunogold technique will indicate if both enzymes share the same ER compartments. Purification of the enzyme and availability of monospecific antibodies will allow, in the long term, to isolate the corresponding gene. Its disruption may provide clues on the role of the reaction. We also propose to study the influence of the oligosaccharide and peptide moieties of glycoproteins on the glycosylation rate. For these propose glycopeptides containing variable oligosaccharide moieties (Man7, 8, 9GlcNAc2) and the same peptide or alternatively, the same oligosaccharide (Man9 GlcNAc2) but variable peptide moieties will be prepared and incubated with the enzyme. If the glucosyltransferase shows a different specificity with respect to the oligosaccharides, this will indicate that processing by ER mannosidases influences the glycosylation of glycoproteins. We already have evidence that some unknown feature in a protein domain close to the oligosaccharide strongly affects the rate of glycosylation. Identification of amino acids interacting with the glucosyltransferase will allow in the long term, to affect glycosylation of known glycoproteins by site-directed mutagenesis and thus obtain clues on the role of the glycosylation reaction. Finally, we propose to incubate intact cells with the appropriate precursor in such a way that only the glucose units transferred from UDP-Glc to glycoproteins will become labeled, to inhibit glucosidase II with certain drugs and to investigate, using antibodies against known glycoproteins, if glycosylation is restricted to certain types of glycoproteins (membrane bound, secreted, malfolded, lysosomal, etc.) or if it forms part of the machinery by which cells transport different glycoproteins from the ER to the Golgi at different rates.

我们已经描述了蛋白质连接的高甘露糖型寡糖 缺乏葡萄糖单位直接从 UDP-Glc 进行糖基化（即， 在内质网中不含多醇衍生物作为中间体 (ER) 哺乳动物、植物、真菌和原生动物细胞的葡萄糖 随后可能通过葡萄糖苷酶 II 去除单位。 终极 该提案的目的是找到这些反应的作用。 为了这 UDP-Glc 的用途：糖蛋白葡萄糖基转移酶（一种酶 显然松散地附着在 ER 管腔上）将被净化， 针对它的抗体将被产生并且其定位将被 通过免疫细胞化学建立。 葡萄糖苷酶 II 的双重定位 免疫金技术的葡萄糖基转移酶将表明是否 两种酶共享相同的 ER 隔室。 酶的纯化 从长远来看，单特异性抗体的可用性将允许 从而分离出相应的基因。 它的破坏可能提供线索 反应的作用。 我们还建议研究 糖蛋白的寡糖和肽部分对糖基化的影响 速度。 对于这些建议的糖肽含有可变寡糖 部分（Man7、8、9GlcNAc2）和相同的肽，或者， 相同的寡糖（Man9 GlcNAc2）但可变的肽部分将是 制备并与酶一起孵育。 如果葡萄糖基转移酶显示 寡糖的不同特异性，这将 表明 ER 甘露糖苷酶的加工影响糖基化 糖蛋白。 我们已经有证据表明某个未知特征 靠近寡糖的蛋白质结构域强烈影响 糖基化。 与氨基酸相互作用的鉴定 从长远来看，葡萄糖基转移酶将影响糖基化 通过定点诱变已知的糖蛋白，从而获得线索 糖基化反应的作用。 最后，我们建议孵化 具有适当前体的完整细胞，只有 从 UDP-Glc 转移到糖蛋白的葡萄糖单位将变成 标记，用某些药物抑制葡萄糖苷酶 II 并进行研究， 使用针对已知糖蛋白的抗体，如果糖基化是 仅限于某些类型的糖蛋白（膜结合的、分泌的、 错误折叠的、溶酶体的等）或者如果它构成了机器的一部分 细胞将不同的糖蛋白从内质网转运到高尔基体 不同的费率。