GLYCOPROTEIN MANNOSYLTRANSFERASES

糖蛋白甘露糖基转移酶

基本信息

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
项目类别：
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

项目摘要

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.

这里提出的研究的最终目标是描述调节基因表达在蛋白质N-糖基化期间的基因表达乳腺激素调节的生长和分化。这调查的重点是净化和表征GDP甘露糖 - 需要Dolichol循环的甘露糖基转移酶，腺体并开发分子试剂，抗体，抗体和cDNA 相同的。这些研究是在早期研究的背景下提出的从p.i.s实验室表明，蛋白质N-的许多酶在乳腺的个体发育过程中调节糖基化；这胰岛素，催乳素和氢化可的松的联合作用似乎调节其在基础水平上的表达。我们的工作假设假设Dolichol连接的组件和多肽连接 N-糖基化的寡糖前体的成熟是上述激素的协同作用协调上调在大鼠乳腺的乳脂分化过程中。天冬酰胺连接的糖蛋白包括最大类的糖蛋白并参与了无数现象生物识别。糖蛋白代谢的改变是与许多病理和宿主相互作用相关。一个十七个糖基转移酶和两个糖苷酶的音乐会很常见所有N连接糖蛋白的组装。在这五个GDP-Mannose中需要甘露糖基转移酶构成一个酶家族。使用新颖的三方策略，这些酶之一已经被纯化并在P.I的实验室中进行了特征。拟议的研究将扩展采购其他酶的总体方法。一个具体目标是确定这些潜在的“甘露糖基主题” 酶。概述了一个实验策略，类似于活动在P.I.的实验室中开发的STIT-SH标记方法以获取酶产生酶的催化多肽，如果是难以获得同质酶。基于PCR的标准技术将被用来获得酶的cDNA。可用性这些试剂应为将来的调查打开大门调节动物系统中蛋白质N-糖基化的调节。乳腺为研究人员提供独特的模型，用于研究糖蛋白生物合成和生化和分子生物学的调节等级。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。