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

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，如胰岛素受体、低密度脂蛋白受体、 对于乙酰胆碱等，都是糖蛋白。所有这些受体 含有N-连接的低聚糖。尽管许多步骤都是 已知导致脂质连接的寡糖的组装 供体，很少有酶被提纯或鉴定，而且 人们对该途径是如何调控的知之甚少。是这样的 信息是相当重要的，因为 碳水化合物结构可能参与了这种疾病的情况 如动脉粥样硬化、糖尿病、癌症等。我们建议提纯 并研究几种似乎是调节位点的酶 在这条路上。其中一种酶是GlcNAc-1-P转移酶 它催化了该途径的第一步；另一步是 甘露糖基转移酶，将第六种甘露糖添加到脂质中- 连接的低聚糖。在后一种反应中，有一个 甘露糖供体由GDP-甘露糖转变为Dolichyl-P-甘露糖。 我们认为我们有一些很好的检测这些酶的方法 合理的亲和力步骤进行纯化。我们建议这样做 对几个植物系统的研究，原因有很多，包括 事实上，大豆或梧桐树细胞对 在文化中生长，大量种植是很合理的 细胞的数量(即最多一公斤细胞)。此外，黄曲霉的酵素 植物中的脂联糖途径似乎有很多 溶解后比动物细胞更稳定。因此， 净化在植物系统中应该更可行。 我们还计划提纯几种加工酶来 均一，以制备多克隆抗体。是这样的 抗体将用于酶的定位，以及用于 这些酶的生物合成和靶向性研究。我们现在 有对葡萄糖苷酶II的抗体，葡萄糖苷酶II是一种早期加工酶， 并将完成甘露糖苷酶II的提纯，这是一个中期 酶和后期加工酶GlcNAc转移酶II。 需要解决的问题之一是植物是否有CI， 内侧和跨高尔基体，以及各种活动是否在 高尔基山脉的不同地区。其他问题涉及 将这些蛋白质引导到特定位置的信号。自.以来 我们已经提纯了许多这样的处理酶，我们将尝试 建立体外处理系统。当糖蛋白变成 在细菌中克隆，它将成为重要的能够 糖基化并在无细胞系统中处理这些蛋白质。