TWO REGULATORY ENZYMES OF DOLICHOL-LINKED GLYCOSYLATION

多醇连接糖基化的两种调节酶

• 批准号: 3290819
• 负责人: SHARON S KRAG
• 金额: $ 15.78万
• 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 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3290819
• 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 共价附着在天冬酰胺残基上。 示例的例子 糖基化蛋白包括细胞器和细胞的成分 表面膜，分泌的蛋白质，例如激素和 酶，例如可溶性溶酶体水解酶。 这 这些寡糖的生物合成涉及多利科 - 链接的单糖和寡糖中间体以及最小 四十个步骤发生在三个不同的亚细胞室中。 了解这一复杂途径及其法规 内源性和外源性剂将需要净化 体外涉及和重构的酶。 作为 第一步，我们建议净化两种酶AD的基因 从中国仓鼠卵巢细胞中编码它们；两种蛋白质 催化生物合成方案中的早期反应并利用 糖核苷酸和磷酸二甲基糖都是底物。 两个都 酶，UDP-N-乙酰葡萄糖胺：Dolichyl磷酸盐N- 乙酰葡萄糖1-磷酸转移酶和 甘露磷酸二硫醇合酶是整体膜 蛋白质在内质中以少量存在 细胞的网状，并催化潜在的调节步骤 糖蛋白合成。 纯化编码的基因 因为这些酶将提供所需的蛋白质量 表征和重建研究。 我们将隔离 通过利用B4-- 2-1，中国野村卵巢突变细胞系，我们拥有 特征是缺乏合酶活性。 与父母相比 细胞，该突变体的甘露糖6-磷酸甘露糖少十倍 依赖外源溶酶体酶和十倍的摄取 与野生型细胞相比，内源性α-辅助酶活性较少。 表达合成酶基因的转染的细胞将被检测到 用于吸收标记的溶酶体的放射自显影屏 酶或用于α-双念珠菌活性的荧光筛选。 第二种酶，葡萄糖胺磷酸转移酶将是 从3E11细胞纯化，这是一种抗穿纹犬霉素的克隆 仓鼠卵巢细胞在此中分离并表征 实验室。 抗穿纹犬霉素种群的膜 该人口的克隆（例如3E11）有15次 转移的特定活性和膜上的膜一样 野生型细胞。 针对纯化酶的抗血清将是 生产以促进碎片的初始隔离，最后 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特定方面 连接的糖蛋白生物合成。