LIPID-LINKED SACCHARIDES IN GLYCOPROTEIN SYNTHESIS

糖蛋白合成中的脂联糖

• 批准号: 3227140
• 负责人: Alan D Elbein
• 金额: $ 15.63万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3227140
• 关键词: Golgi apparatus; affinity chromatography; antibody; beta glucosidases; biological transport; carbohydrate biosynthesis; cell free system; density gradient ultracentrifugation; dolichol; gel filtration chromatography; glycosyltransferase; ion exchange chromatography; laboratory rabbit; lipids; mannose; mannosidase; mass spectrometry; oligosaccharides; plants; protein biosynthesis; tissue /cell culture; xylose; Golgi apparatus; affinity chromatography; antibody; beta glucosidases; biological transport; carbohydrate biosynthesis; cell free system; density gradient ultracentrifugation; dolichol; gel filtration chromatography; glycosyltransferase; ion exchange chromatography; laboratory rabbit; lipids; mannose; mannosidase; mass spectrometry; oligosaccharides; plants; protein biosynthesis; tissue /cell culture; xylose

Many plasma membrane receptors, such as those for insulin, for LDL, for acetylcholine, etc., are glycoproteins. All of these receptors contain N-linked oligosaccharides. Although many of the steps are known that lead to the assembly of the lipid-linked oligosaccharide donor, few of the enzymes have been purified or characterized, and little is known about how the pathway is regulated. Such information is of considerable importance since alterations in carbohydrate structure may be involved is such disease conditions as atherosclerosis, diabetes, cancer, etc. We propose to purify and study several enzymes that appear likely to be regulatory sites in the pathway. One of these enzymes is the GlcNAc-1-P transferase that catalyzes the first step in the pathway; another is the mannosyl transferase that adds the sixth mannose to the lipid- linked oligosaccharides. In this latter reaction, there is a change in mannosyl donor from GDP-mannose to dolichyl-P-mannose. We think we have some good assays for these enzymes and some reasonable affinity steps for purification. We propose to do these studies in several plant systems for a number of reasons including the fact that soybean or sycamore cells are very inexpensive to grow in culture and it is quite reasonable to grow large amounts of cells (i.e., up to a Kg of cells). In addition, the enzymes of the lipid-linked saccharide pathway in plants appear to be much more stable after solubilization than those of animal cells. Thus purification should be more feasible in the plant system. We also plan to purify several of the processing enzymes to homogeneity in order to prepare polyclonal antibodies. Such antibodies will be used for enzyme localization as well as for studies on biosynthesis and targeting of these enzymes. We now have antibody against glucosidase II, an early processing enzyme, and will finish the purification of mannosidase II, a mid stage enzyme, and GlcNAc transferase II, a late-stage processing enzyme. Among the questions to be addressed is whether plants have cis, medial and trans Golgi, and whether the various activities are in different areas of the Golgi. Other questions deal with the signals that direct these proteins to the specific sites. Since we have many of these processing enzymes purified, we will attempt to set up an in vitro processing system. As glycoproteins become cloned in bacteria, it will become important to be able to glycosylate and process these proteins in cell-free systems.

许多质膜受体，例如用于LDL的胰岛素受体， 对于乙酰胆碱等，是糖蛋白。 所有这些受体 含有N连接的寡糖。 虽然许多步骤是 已知导致脂质连接的寡糖组装 供体，很少有酶纯化或表征的酶，并且 关于路径的调节方式知之甚少。 这样的 信息非常重要，因为 碳水化合物结构可能涉及这种疾病状况 作为动脉粥样硬化，糖尿病，癌症等 并研究几种似乎是调节部位的酶 在路径中。 这些酶之一是GlcNAC-1-P转移酶 这催化了路径中的第一步；另一个是 甘露糖基转移酶，将第六个甘露糖添加到脂质中 连接的寡糖。 在后一个反应中，有一个 甘露糖基供体从GDP-甘露糖变为dolichyl-p-mannose的变化。 我们认为我们对这些酶有一些好的测定法和一些 合理的纯化步骤。 我们建议这样做 多种原因在几种工厂系统中进行研究，包括 大豆或无障碍细胞的事实非常便宜 在文化中成长，大量生长是很合理的 细胞（即，最多到千克细胞）。 另外， 植物中脂质连接的糖途径似乎很大 溶解后比动物细胞更稳定。 因此 纯化在工厂系统中应该更可行。 我们还计划将几种加工酶纯化为 同质性以制备多克隆抗体。 这样的 抗体将用于酶定位以及 这些酶的生物合成和靶向的研究。 我们现在 具有针对葡萄糖酶II的抗体，一种早期加工酶， 并将完成甘露糖苷酶II的纯化，一个中阶段 酶和GlcNAC转移酶II，一种晚期加工酶。 要解决的问题之一是植物是否有顺式 内侧和反式高尔基，以及各种活动是否在 高尔基的不同区域。 其他问题涉及 将这些蛋白质引导到特定位点的信号。 自从 我们有许多这样的加工酶纯化，我们将尝试 建立一个体外处理系统。 随着糖蛋白的变化 克隆在细菌中，能够很重要 糖基化并在无细胞系统中处理这些蛋白质。