TRANSIENT GLUCOSYLATION OF GYLCOPROTEINS

糖蛋白的瞬时糖基化

• 批准号: 2182540
• 负责人: ARMANDO JOSE PARODI
• 金额: $ 7.13万
• 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 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182540
• 关键词: Schizosaccharomyces pombe; animal tissue; cell free system; enzyme activity; gene mutation; glycoprotein biosynthesis; glycoproteins; glycosylation; hydropathy; laboratory rabbit; laboratory rat; molecular cloning; nucleic acid probes; nucleic acid sequence; nucleotide metabolism; polymerase chain reaction; posttranslational modifications; protein denaturation; protein folding; recombinant proteins; secretion; tissue /cell culture; uridine diphosphate glucose

We have described that protein-linked, polymannose-type oligosaccharides lacking glucose units are glucosylated directly from UDP-Glc in the lumen of the endoplasmic reticulum (ER) of mammalian, plant, fungal and protozoan cells and that the glucose units are immediately removed, probably by glucosidase II. The long term goal of this application is to find a role for these reactions. The UDP-Glc:glycoprotein glucosyltransferase has been purified to homogeneity from rat liver. Cell-free assays showed that the enzyme glucosylates much more efficiently denatured than native glycoproteins and that this is caused by the interaction of protein determinants exposed in denatured but not in native conformations with the glucosyltransferase. It was suggested that in vivo only unfolded, partially folded and misfolded glycoproteins are glucosylated and that glucosylation stops once glycoproteins adopt their proper tertiary structures. In order to test the possible involvement of the glucosylation reaction in the mechanism by which cells dispose of misfolded structures in the ER or in that by which glycoproteins fold correctly in the same subcellular location we propose: a) to clone and sequence the DNA coding for the UDP-Glc:glycoprotein glucosyltransferase in rat liver and Schizosaccharomyces pombe; b) to disrupt the coding gene in S. pombe and if possible also in mammalian cells and to determine if the absence of the glucosyltransferase modifies the rate of secretion of glycoproteins in those cells. The rate of folding has been recognized to be one of the main pace limiting steps in the transit of proteins from the ER to the ultimate subcellular locations; c) to study if the glucosyltransferase influences the rate of folding of glycoproteins synthesized in a cell-free transcription- translation system; d) to study if the pure enzyme influences the thermal denaturation of glycoproteins; e) as peptides and denatured, deglycosylated glycoproteins were found to inhibit cell-free glucosylation, we propose to synthesize peptides of defined length and random composition, to determine the size, composition and sequence of those showing maximum inhibitory effect, and in this way determine the nature of the domain, exposed in all denatured but not in native glycoproteins, that interacts with the glucosyltransferase.

我们已经描述了蛋白质连接的多曼糖型寡糖 缺少葡萄糖单元直接从管腔中的UDP-GLC直接葡萄糖基化 哺乳动物，植物，真菌和真菌的内质网（ER） 原生动物细胞并立即去除葡萄糖单元， 可能是通过葡萄糖酶II。 该应用程序的长期目标是 找到这些反应的作用。 UDP-GLC：糖蛋白 葡萄糖基转移酶已被纯化为大鼠肝脏的同质性。 无细胞的测定表明葡萄糖基酶越多 比本地糖蛋白有效地变性，这是引起的 通过在变性中暴露的蛋白质决定因素的相互作用，但不是 在与葡萄糖基转移酶的天然构象中。 有人建议 体内只展开，部分折叠和错误折叠的糖蛋白 被葡萄糖基化，一旦采用糖蛋白，葡萄糖基化就会停止 它们适当的三级结构。 为了测试可能 葡萄糖基化反应参与细胞的机制 在急诊室或在其上处置错误折叠的结构 糖蛋白在我们建议的同一亚细胞位置正确折叠： a）克隆并对UDP-GLC的DNA进行测序：糖蛋白 大鼠肝脏和精神分裂症的葡萄糖基转移酶Pombe； b）到 破坏S. pombe中的编码基因，如果可能的话，也会在哺乳动物中 细胞并确定不存在葡萄糖基转移酶是否会改变 这些细胞中糖蛋白的分泌速率。 速率 折叠已被认为是主要速度限制步骤之一 蛋白质从ER到最终亚细胞的过渡 位置； c）研究葡萄糖基转移酶是否影响 在无细胞转录中合成的糖蛋白折叠 - 翻译系统； d）研究纯酶是否影响热酶 糖蛋白的变性； e）作为肽和变性， 发现脱糖基化的糖蛋白抑制无细胞 葡萄糖基化，我们建议合成定义长度和 随机组成，以确定大小，组成和序列 那些表现出最大抑制作用的人，以此方式确定 域的性质，在所有变性中暴露但不暴露在本地 糖蛋白与葡萄糖基转移酶相互作用。