TWO REGULATORY ENZYMES OF DOLICHOL-LINKED GLYCOSYLATION

多醇连接糖基化的两种调节酶

• 批准号: 3290818
• 负责人: SHARON S KRAG
• 金额: $ 5.02万
• 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/3290818
• 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具有寡糖部分 共价连接到天冬酰胺残基上。 的实例 糖基化蛋白质包括细胞器和细胞 表面膜，分泌的蛋白质如激素，和 酶如可溶性溶酶体水解酶。 的 这些寡糖侧链的生物合成涉及多萜醇， 连接的单糖和寡糖中间体和最少的 在三个不同的亚细胞区室中发生的四十个步骤。 了解这一复杂的途径及其调控， 内源性和外源性试剂将需要纯化 参与的酶和体外系统的重建。 作为 第一步，我们提出纯化两种酶和基因， 从中国仓鼠卵巢细胞中编码它们;这两种蛋白质 催化生物合成方案中的早期反应， 以糖核苷酸和长链磷酸酯作为底物。 两 酶，UDP-N-乙酰葡糖胺：N-磷酸长链甘油酯 乙酰葡糖胺-1-磷酸转移酶和 甘露糖基磷酸多萜醇合酶，是整合膜 蛋白质，存在于少量的内质 细胞的网状结构，并催化潜在的调节步骤， 糖蛋白合成 纯化编码 因为这些酶将提供所需的蛋白质量， 表征和重构研究。 我们将隔离 甘露糖基磷酸多萜醇合酶的基因， 2-1，我们获得的中国仓鼠卵巢突变细胞系， 其特征在于缺乏合酶活性。 相较于亲代 细胞中，这种突变体的甘露糖6-磷酸- 外源性溶酶体酶的依赖性摄取， 内源性α-艾杜糖醛酸酶活性低于野生型细胞。 表达合酶基因的转染细胞将通过 放射自显影筛选标记的溶酶体摄取 酶或荧光屏检测α-艾杜糖醛酸酶活性。 第二种酶，葡糖胺磷酸转移酶，将是 从中国人衣霉素抗性克隆3E 11细胞中纯化 仓鼠卵巢细胞，分离和表征，在此 实验室 用于衣霉素抗性群体的膜 来自该群体的克隆（如3E 11） 转移酶的比活性， 野生型细胞。 抗纯化酶的抗血清将被 产生以促进片段的初始分离， 转移酶的整个基因 最后，3E 11和B4-2-1将 用于研究天冬酰胺调节的特定方面- 连接糖蛋白生物合成。