GLYCOPROTEIN MANNOSYLTRANSFERASES

这里提出的研究的最终目标是勾勒出 蛋白质N-糖基化过程中基因表达的调控 荷尔蒙调节的乳腺的生长和分化。这个 调查的重点是提纯和表征GDP-甘露糖- 大鼠乳房需要甘露糖基转移酶的二羟苯二酚循环 腺体和开发分子试剂，即抗体和cDNA 一样的。这些研究是在早期研究的背景下提出的 来自P.I.s实验室的研究表明，蛋白质N的一些酶- 糖基化是在乳腺的个体发育过程中调节的； 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。我们的工作假说 假设多醇连接的组装和多肽连接 N-糖基化低聚糖前体的成熟度是 通过上述激素的协同作用而协同上调 在大鼠乳腺的乳源性分化过程中。 天冬酰胺连接的糖蛋白是糖蛋白中最大的一类。 并参与了无数的现象，这些现象是 生物识别。糖蛋白代谢的改变是 与多种病理和宿主-寄生虫相互作用有关。一个 17种糖基转移酶和两种糖苷酶共同作用于 所有N-连接的糖蛋白的组装。这五大GDP中--甘露 需要甘露糖基转移酶构成一个酶家族。使用 新的三方策略，其中一种酶已经被提纯 在私家侦探的实验室里。拟议的研究将延长 获得其他酶的总体方法相同。一个具体的目标是 在这些基因的活性部位中确定一个潜在的“甘露糖基序” 酵素。概述了一种类似于主动策略的实验策略 P.I.S实验室开发的Site-SH标记方法 催化多肽产生抗体的酶，应该是 很难获得同质酶。基于标准的聚合酶链式反应技术 将被用来获得酶的cDNA。是否可以使用 这些试剂应该会为未来对 动物系统中蛋白质N-糖基化的调节。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中的生物合成与调控 水平。它的生长受到多种激素的强烈调节。 以及在雌性生殖生命的整个过程中的分化。它可以 在转基因动物中潜在地作为一个很好的生物反应器 在ITS中收获大量具有生物医学意义的糖蛋白 分泌物，即牛奶。分泌α-干扰素的初步成功 抗胰蛋白酶、组织型纤溶酶原激活剂和凝血因子 看起来很有前途。

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.