GLYCOPROTEIN MANNOSYLTRANSFERASES

糖蛋白甘露糖基转移酶

• 批准号: 2701623
• 负责人: INDER K VIJAY
• 金额: $ 27万
• 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 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701623
• 关键词: active sites; affinity chromatography; affinity labeling; carbohydrate biosynthesis; carbohydrate structure; dolichol; enzyme activity; gene expression; genetic regulation; glycoproteins; glycosylation; hexosyltransferase; high performance liquid chromatography; immunoprecipitation; laboratory rabbit; mammary gland; mannose; microsomes; molecular cloning; nucleic acid probes; polymerase chain reaction; protein purification; protein sequence; thiols; western blottings

The ultimate objective of the research proposed here is to delineate the regulation of gene expression for protein N-glycosylation during the hormonally-modulated growth and differentiation of the mammary gland. The focus of the investigation is to purify and characterize the GDP-Mannose- requiring mannosyltransferases of the dolichol cycle int he rat mammary gland and develop molecular reagents, viz, antibodies and cDNAs for the same. These studies are being proposed in the context of earlier studies from the P.I.s lab showing that a number of enzymes of protein N- glycosylation are modulated during the ontogeny of the mammary gland; the combined action of insulin, prolactin and hydrocortisone appears to regulate their expression over basal levels. Our working hypothesis postulates that the dolichol-linked assembly and polypeptide-linked maturation of the oligosaccharide precursor for N-glycosylation are coordinately upregulated by the synergistic action of the above hormones during the lactogenic differentiation of the rat 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 numerous pathologies and host-parasite interaction. A concert of seventeen glycosyltransferases and two glycosidases is common to the assembly of all N-linked glycoproteins. Among these five GDP-Mannose requiring mannosyltransferases constitute a family of enzymes. Using a novel, tripartite strategy, one of these enzymes has already been purified and characterized in the P.I's lab. The proposed study will extend the same overall approach to procure the other enzymes. A specific goal is to identify a potential "mannosyl motif" within the active site of these enzymes. An experimental strategy is outlined, analogous to the active site-SH tagging methodology developed in the P.I.'s laboratory to obtain catalytic polypeptides of the enzymes for antibody production, should it be difficult to obtain homogenous enzymes. Standard PCR-based technologies will be employed to obtain the cDNAs for the enzymes. The availability of these reagents should open the door for future investigations on the regulation of protein N-glycosylation in animal systems. The mammary gland offers a unique model to the investigator for studying glycoprotein biosynthesis and regulation 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.

本研究的最终目的是描述 蛋白质N-糖基化过程中基因表达的调节 乳腺的经尿道调节的生长和分化。 的 研究的重点是纯化和表征GDP-甘露糖， 需要大鼠乳腺中长醇循环的甘露糖基转移酶 腺和开发分子试剂，即，抗体和cDNA的 一样的 这些研究是在早期研究的背景下提出的 实验室的数据显示，一些蛋白质N- 糖基化在乳腺的个体发育过程中受到调节; 胰岛素、催乳素和氢化可的松的联合作用似乎 在基础水平上调节它们的表达。 我们的工作假设 假设长轴醇连接的装配和多肽连接的 用于N-糖基化的寡糖前体的成熟是 通过上述激素的协同作用协同上调 在大鼠乳腺的催乳分化过程中。 天冬酰胺连接的糖蛋白包括最大的一类糖蛋白 并参与了无数的现象， 生物识别。 糖蛋白代谢的改变是 与许多病理和宿主-寄生虫相互作用有关。 一 十七种糖基转移酶和两种糖苷酶的音乐会是常见的， 所有N-连接糖蛋白的组装。 在这五个GDP-甘露糖 需要甘露糖基转移酶构成酶家族。 使用 一种新的三方策略，其中一种酶已经被纯化 并在私家侦探的实验室进行了鉴定 拟议的研究将扩大 同样的方法来获得其他酶。 具体目标是 在这些化合物的活性位点内鉴定潜在的“甘露糖基基序”， 内切酶 一个实验策略概述，类似于积极的 在P.I.的实验室获得 用于抗体产生的酶的催化多肽， 很难获得均匀的酶。 标准PCR技术 将用于获得酶的cDNA。 的可用性 这些试剂应该为未来的研究打开大门。 调节动物系统中的蛋白质N-糖基化。 乳腺 为研究糖蛋白的研究者提供了一个独特的模型 生物化学和分子生物学的生物合成和调控 水平 它的生长受到多种激素的强烈调节 在女性的整个生殖过程中， 它可以 可能在转基因动物中用作优良的生物反应器， 收获大量具有生物医学意义的糖蛋白， 分泌，即，牛奶 初步成功地分泌了阿尔法- 抗胰蛋白酶、组织纤溶酶原激活剂和凝血因子 看起来很有希望。