TRANSIENT GLUCOSYLATION OF GLYCOPROTEINS

糖蛋白的瞬时糖基化

• 批准号: 2459411
• 负责人: ARMANDO JOSE PARODI
• 金额: $ 5.77万
• 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 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459411
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; Schizosaccharomyces pombe; Trypanosoma cruzi; calcium binding protein; endoplasmic reticulum; enzyme activity; enzyme mechanism; enzyme model; gene mutation; glycoprotein biosynthesis; glycoproteins; glycosylation; hexosyltransferase; hydropathy; laboratory rabbit; laboratory rat; molecular chaperones; molecular cloning; nucleic acid sequence; oligosaccharides; protein folding; uridine diphosphate glucose

Protein-linked, high mannose-type oligosaccharides devoid of glucose residues are glucosylated directly from UDP-Glc in the lumen of the endoplasmic reticulum (ER) to form monoglucosylated derivatives. In vivo the glucose units are immediately removed by glucosidase Il. The glucosylating enzyme (UDP-Glc:glycoprotein glucosyltransferase) has the unique property of glucosylating denatured but not native glycoproteins in cell-free assays. We have purified to homogeneity the enzyme from rat liver and the yeast Schizosaccharomyces pombe and cloned, sequenced and disrupted the gene coding for it in the last organism. It has been proposed that the enzyme participates, together with an ER chaperone that binds monoglucosylated oligosaccharides (calnexin) in the quality control mechanism of glycoprotein folding by which only species having the correct tertiary structures are transported to the Golgi apparatus. As the participation of the glucosyltransferase is solely based in the above mentioned property but not in in vivo performed experiments, we propose to confirm or disprove the proposed involvement of the enzyme in the quality control mechanism by performing the following experiments: a) To characterize, sequence and disrupt the gene encoding the glucosyltransferase in Trypanosoma cruzi and to study glycoprotein folding, secretion and association with calnexin in the genetically modified protozoon. Trypanosomatids are the only organisms known so far that transfer unglucosylated oligosaccharides in protein N-glycosylation and therefore, the only mechanism by which they glucosylate proteins is that involving the glucosyltransferase. b) To study calnexin-association, folding and secretion of glycoproteins in S. porn be glucosyltransferase minus cells. As these cells but not calnexin minus ones proved to be viable, to study if disruption of another gene in addition to that encoding the glucosyltransferase renders S. pombe cells non-viable. c) As we have determined that the glucosyltransferase binds hydrophobic amino acids, to study the possibility that in vivo the enzyme behaves as a chaperone and to further characterize the hydrophobic domains recognized by the glucosyltransferase in denatured glycoproteins.

不含葡萄糖的蛋白连接高甘露糖型寡糖 残基直接从 UDP-Glc 在管腔中进行糖基化 内质网（ER）形成单葡萄糖基化衍生物。体内 葡萄糖单位立即被葡萄糖苷酶II除去。这 葡萄糖基化酶（UDP-Glc：糖蛋白葡萄糖基转移酶）具有 糖基化变性而非天然糖蛋白的独特性质 无细胞测定。我们已将大鼠的酶纯化至均质 肝脏和酵母裂殖酵母，并进行克隆、测序和 破坏了最后一个生物体中编码它的基因。它一直 提出该酶与 ER 伴侣一起参与 在质量控制中结合单葡萄糖基化寡糖（钙连接蛋白） 糖蛋白折叠的机制，只有物种具有正确的折叠 三级结构被运输到高尔基体。作为 葡萄糖基转移酶的参与仅基于上述 提到的属性但不是在体内进行的实验中，我们建议 证实或反驳所提议的酶与质量的关系 通过执行以下实验来控制机制：a） 表征、测序和破坏编码基因 克氏锥虫中的葡萄糖基转移酶并研究糖蛋白 折叠、分泌以及与遗传中的钙联蛋白的关联 修饰的原生动物。锥虫是迄今为止唯一已知的生物 在蛋白质 N-糖基化中转移非糖基化寡糖 因此，它们糖基化蛋白质的唯一机制是 涉及葡萄糖基转移酶。 b) 研究钙联接蛋白关联， S. Porn中糖蛋白的折叠和分泌是葡萄糖基转移酶 减去细胞。因为这些细胞（但不是钙联蛋白负细胞）被证明是 可行，研究除此之外的另一个基因是否被破坏 编码葡萄糖基转移酶使粟酒裂殖酵母细胞无法存活。 c) 作为 我们已经确定葡萄糖基转移酶结合疏水性氨基 酸，以研究该酶在体内发挥作用的可能性 分子伴侣并进一步表征所识别的疏水域 通过变性糖蛋白中的葡萄糖基转移酶。