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.

拟议研究计划的最终目标是描绘 动物生长过程中基因表达的分子基础， 发展调查的重点是提纯和定性 中的多萜醇循环的需要β-甘露糖的甘露糖基转移酶 大鼠乳腺，并开发分子试剂，即单特异性 针对纯化酶的多克隆抗体及其cDNA。 这些调查是在早期研究的背景下提出的 从私家侦探那里的实验室表明，一些ER定位的酶， 蛋白质N-糖基化在生殖周期中被调节， 乳腺的生长和分化直至达到高峰 哺乳中期的水平。胰岛素、催乳素和 氢化可的松似乎将它们的表达调节到高于基础水平。 一个工作假设已经制定，根据该长叶醇- 连接的组装和多肽连接的成熟， 用于N-糖基化的寡糖前体协同上调 通过胰岛素、催乳素和氢化可的松的协同作用 乳腺的催乳分化。 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， 是生物识别的基础糖蛋白的改变 代谢与多种病理学有关，例如，恶性肿瘤， 动脉粥样硬化，许多遗传疾病和最初的宿主-寄生虫 互动导致艾滋病。十七种糖基转移酶的协同作用 和两种糖苷酶，即，葡萄糖苷酶I和II是最低限度需要的， 所有N-连接糖蛋白的组装。其中，5个GDP-- 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性，极不稳定， 由于缺乏明确的受体底物，这些酶 一直无视许多实验室净化它们的努力。在 在拟议的研究中，这些酶将被纯化， 其特征在于，采用新的光识别和活性位点-SH P.I.开发的标记方法的实验室。一个具体的目标 将是确定一个潜在的"甘露糖基序"的活性位点内的 酶。概述了一种实验策略， 针对单个酶的抗体，即使它们抵抗纯化， 同质性将采用标准和基于PCR的技术， 获得甘露糖基转移酶的cDNA探针。网站或资源的可用性 试剂应该为未来的法规调查打开大门 蛋白质N-糖基化在乳腺和其他组织。的 乳腺为研究者提供了一个独特的研究模型 糖蛋白生物合成的生物化学和分子生物学 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。