TWO REGULATORY ENZYMES OF DOLICHOL-LINKED GLYCOSYLATION

多醇连接糖基化的两种调节酶

• 批准号: 3290820
• 负责人: SHARON S KRAG
• 金额: $ 14.8万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3290820
• 关键词: antibody; dolichol; glycosylation; glycosyltransferase; hamsters; laboratory rabbit; mannose; membrane proteins; messenger RNA; molecular genetics; mutant; oligosaccharides; ovary; phosphotransferases; protein biosynthesis; tissue /cell culture

Many proteins in eukaryotic cells have oligosaccharide moieties covalently attached to asparagine residues. Examples of glycosylated proteins include components of organellar and cell surface membranes, secreted proteins such as hormones, and enzymes such as the soluble lysosomal hydrolases. The biosynthesis of these oligosaccharide sidechains involves dolichol- linked mono- and oligosaccharide intermediates and a minimum of forty steps occurring in three different subcellular compartments. An understanding of this complex pathway and its regulation by endogenous and exogenous agents will require purification of the enzymes involved and reconstitution of the system in vitro. As a first step, we propose to purify two enzymes ad the genes which encode them from Chinese hamster ovary cells; both proteins catalyze early reactions in the biosynthetic scheme and utilize both sugar nucleotide and dolichyl phosphate as substrates. Both enzymes, UDP-N-acetylglucosamine:dolichyl phosphate N- acetylglucosamine-1-phosphate transferase and mannosylphosphoryldolichol synthase, are integral membrane proteins, are present in small amounts in the endoplasmic reticulum of cells, and catalyze potential regulatory steps in glycoprotein synthesis. Purification of the genes which encode for these enzymes will provide the amounts of protein needed for characterization and reconstitution studies. We will isolate the gene for the mannosylphosphoryldolichol synthase by utilizing B4- 2-1, a Chinese hamester ovary mutant cell line which we have characterized as lacking synthase activity. Compared to parental cells, this mutant has ten-fold less mannose 6-phosphate- dependent uptake of exogenous lysosomal enzymes and ten-fold less endogenous alpha-iduronidase activity than wild-type cells. Transfected cells expressing the synthase gene will be detected by an autoradiographic screen for uptake of labelled lysosomal enzymes or a fluorescent screen for alpha-iduronidase activity. The second enzyme, glucosamine phosphate transferase, will be purified from 3E11 cells, a clone of tunicamycin-resistant Chinese hamster ovary cells which were isolated and characterized in this laboratory. Membranes for a tunicamycin-resistant population and clones (such as 3E11) from that population had fifteen times the specific activity of the transferse as did membranes from wild-type cells. Antiserum against the purified enzyme will be produced to facilitate the initial isolation of fragments and finally the entire gene for the transferase. Finally, 3E11 and B4-2-1 will be used to study specific aspects of the regulation of asparagine- linked glycoprotein biosynthesis.

真核细胞中的许多蛋白质都含有寡糖部分 共价连接到天冬酰胺残基上。举例 糖基化蛋白包括细胞器成分和细胞成分 表面膜，分泌的蛋白质，如激素，和 酶，如可溶性溶酶体水解酶。这个 这些低聚糖侧链的生物合成涉及二羟基乙醇- 连接的单糖和低聚糖中间体和至少 在三个不同的亚细胞隔间中发生了40个台阶。 对这一复杂途径的理解及其调控 内源性和外源性因素将需要提纯 酶的参与和体外系统的重建。作为一名 第一步，我们建议提纯两种酶和它们的基因 从中国仓鼠卵巢细胞中编码它们；两种蛋白质 在生物合成方案中催化早期反应并利用 以糖核苷酸和磷酸双酯为底物。两者都有 酶，UDP-N-乙酰氨基葡萄糖：磷酸二氢呋喃-N- 乙酰氨基葡萄糖-1-磷酸转移酶和 甘露糖硫磷二醇合成酶，是一种完整的膜 蛋白质，以少量存在于内质中 ，并催化潜在的调控步骤 糖蛋白合成。编码基因的纯化 因为这些酶将提供所需的蛋白质 表征和重建研究。我们将分离出 利用B4-对甘露糖硫代磷酸二醇合成酶基因的研究 2-1，我们已有的中国人卵巢突变细胞系 以缺乏合成酶活性为特征。与父代相比 细胞，这个突变体的甘露糖6-磷酸减少了十分之一- 外源溶酶体酶的依赖摄取和10倍 内源性α-艾杜糖苷酶活性低于野生型细胞。 表达合成酶基因的转基因细胞将被检测到 一种摄取标记溶酶体的放射自显影屏幕 酶或用于检测α-艾杜糖醛酸酶活性的荧光屏幕。 第二种酶，氨基葡萄糖磷酸转移酶，将是 从3E11细胞中纯化衣霉素抗性中国人克隆 仓鼠卵巢细胞的分离和鉴定 实验室。用于衣霉素耐药人群的膜 而来自该种群的克隆(如3E11)有15次 转移剂的比活性和膜的活性一样 野生型细胞。针对纯化的酶的抗血清将被 以便于片段的初始分离，并最终 转移酶的整个基因。最后，3E11和B4-2-1将 用于研究天冬酰胺调节的具体方面- 连接糖蛋白的生物合成。