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 缺乏葡萄糖单元直接从UDP-GLC中葡萄糖基（即 在内质网中没有Dolichol衍生物作为中间体） 哺乳动物，植物，真菌和原生动物细胞的（ER），葡萄糖 随后将单位删除，可能是葡萄糖酶II。 最终 该提议的目的是为这些反应找到作用。 为了这 目的UDP-GLC：糖蛋白葡萄糖基转移酶（酶（一种酶） 显然将纯净地固定在ER的管腔上，将被纯化， 反对它的抗体将被提出，并将其定位 由免疫细胞化学建立。 葡萄糖酶II的双重定位 通过免疫金技术的葡萄糖基转移酶将表明是否是否 这两种酶共享相同的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 分离相应的基因。 它的破坏可能会提供有关 反应的作用。 我们还建议研究 糖基化糖蛋白的寡糖和肽部分 速度。 对于这些提出的糖肽含有可变的寡糖 部分（MAN7，8，9GLCNAC2）和相同的肽或 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 关于寡糖的不同特异性，这将 表明ER甘露糖苷酶的加工会影响 糖蛋白。 我们已经有证据表明在 接近寡糖的蛋白质结构域强烈影响 糖基化。 鉴定与 葡萄糖基转移酶将长期允许影响糖基化 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单元格，只有 从UDP-GLC转移到糖蛋白的葡萄糖单元将成为 标记，用某些药物抑制糖苷酶II并研究 使用针对已知糖蛋白的抗体，如果糖基化为 仅限于某些类型的糖蛋白（膜结合，分泌， 磨损，溶酶体等），或者如果它构成了机械的一部分 细胞将不同的糖蛋白从ER转移到高尔基 价格不同。