GLYCOPROTEIN MANNOSYLTRANSFERASES

糖蛋白甘露糖基转移酶

• 批准号: 3568437
• 负责人: INDER K VIJAY
• 金额: $ 8.88万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3568437
• 关键词: active sites; affinity chromatography; affinity labeling; antibody; carbohydrate biosynthesis; carbohydrate structure; complementary DNA; dolichol; glycosylation; hexosyltransferase; high performance liquid chromatography; immunoprecipitation; laboratory rabbit; mammary gland; mannose; microsomes; molecular cloning; nucleic acid probes; nucleic acid sequence; oligosaccharides; polymerase chain reaction; protein purification; protein sequence; western blottings

The ultimate objective of the proposed research program is to delineate the molecular basis for gene expression during animal growth and development. The focus of the investigation is to purify and characterize the (3DP-Mannose-requiring mannosyltransferases of the dolichol cycle in the rat mammary gland and develop molecular reagents, viz, monospecific polyclonal antibodies against the purified enzymes and cDNAs for the same. These investigations are being proposed in the context of earlier studies from the P.I.'s laboratory showing that several ER-localized enzymes for protein N-glycosylation are modulated during the reproductive cycle for the growth and differentiation of the mammary gland until they reach peak levels at mid-lactation. The combined action of insulin, prolactin, and hydrocortisone appears to regulate their expression above the basal level. A working hypothesis has been formulated according to which the dolichol- linked assembly and the polypeptide-linked maturation of the oligosaccharide precursor for N-glycosylation are coordinately upregulated by the synergistic action of insulin, prolactin and hydrocortisone during the lactogenic differentiation of the mammary gland. Asparagine-linked glycoproteins comprise the largest class of glycoproteins and are involved in a myriad of phenomena that are fundamental to biological recognition. Alterations in glycoprotein metabolism are associated with a variety of pathologies, e.g., malignancy, atherosclerosis, numerous genetic disorders and the initial host-parasite interaction leading to AIDS. A concert of seventeen glycosyltransferases and two glycosidases, viz., glucosidase I and II is minimally required for the assembly of all N-linked glycoproteins. Among these, five GDP-Man- requiring mannosyltransferases appear to constitute a family of enzymes. Because of their membrane-bound characteristic, extreme instability and the lack of well-defined acceptor substrates, these enzymes have consistently defied the efforts of many laboratories to purify them. In the proposed investigation, these enzymes will be purified and characterized, employing novel photoidentification and active site -SH tagging methodologies developed in the P.I.'s laboratory. A specific goal will be to identify a potential "mannosyl motif" within the active site of the enzymes. An experimental strategy is outlined to obtain polyclonal antibodies against individual enzymes even if they resist purification to homogeneity. Standard and PCR-based technologies will be employed to obtain cDNA probes for the mannosyltransferases. The availability of these reagents should open the door for future investigations on the regulation of protein N-glycosylation in the mammary gland and other tissues. The mammary gland offers a unique model to the investigator for studying glycoprotein biosynthesis at the biochemical and molecular-biological level. It is intensely modulated by a variety of hormones for its growth and differentiation throughout the reproductive life of the female. It can potentially serve as an excellent bioreactor in transgenic animals to harvest large quantities of biomedically significant glycoproteins in its secretion, i.e., milk. Preliminary successes with the secretion of alpha- antitrypsin, tissue plasminogen activator, and blood clotting factors appear very promising.

拟议的研究计划的最终目标是描述 动物生长过程中基因表达的分子基础 发展。调查的重点是净化和表征 （3dp-mannose征用Dolichol循环的甘露糖基转移酶） 大鼠乳腺并产生分子试剂，即单特异性 相同的纯化酶和cDNA的多克隆抗体。 这些调查是在早期研究的背景下提出的 从P.I.的实验室中，显示了几种ER定位酶 蛋白质N-糖基化在生殖循环中调节 乳腺的生长和分化直到它们达到峰 中间级别的水平。胰岛素，催乳素和 氢化可的松似乎调节其表达高于基础水平。 根据dolichol-提出了一个工作假设 链接的组件和多肽连接的成熟 N-糖基化的寡糖前体是协同上调的 通过胰岛素，催乳素和氢化可的松的协同作用 乳腺的乳酸分化。 与天冬酰胺相关的糖蛋白是最大的一类 糖蛋白，并参与多种现象 生物学识别的基础。糖蛋白的改变 代谢与多种病理有关，例如恶性肿瘤， 动脉粥样硬化，众多遗传疾病和最初的宿主寄生虫 相互作用导致艾滋病。一场17个糖基转移酶的音乐会 和两个糖苷酶，即葡萄糖酶I和II是最低的 所有N连接糖蛋白的组装。其中，五个GDP-Man- 需要甘露糖基转移酶似乎构成了一个酶家族。 由于它们的膜结合特征，极端不稳定和 缺乏明确定义的受体底物，这些酶具有 一贯违反许多实验室净化它们的努力。在 拟议的调查，这些酶将被纯化，并且 表征，采用新颖的照片识别和主动站点-SH 在P.I.实验室中开发的标记方法。一个具体目标 将确定在活跃位点的潜在“甘露糖基主题” 酶。概述了实验策略以获得多克隆 即使对单个酶的抗体抗体抗性 同质性。标准和基于PCR的技术将用于 获得甘露糖基转移酶的cDNA探针。这些的可用性 试剂应为未来对法规的调查打开大门 乳腺和其他组织中蛋白质N-糖基化的蛋白质。这 乳腺为研究人员提供了独特的模型 生物化学和分子生物学的糖蛋白生物合成 等级。它的生长受到多种激素的强烈调节 以及在女性生殖生活中的分化。它可以 有可能在转基因动物中充当出色的生物反应器 收获大量生物医学意义的糖蛋白 分泌，即牛奶。 α-的分泌取得了初步成功 抗胰蛋白酶，组织纤溶酶原激活剂和血液凝结因子 看起来很有希望。