LIPID-LINKED SACCHARIDES IN GLYCOPROTEIN SYNTHESIS

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，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 已知导致脂质连接寡糖组装 供体，很少有酶被纯化或表征，并且 关于该途径是如何调节的知之甚少。 等 信息是相当重要的，因为改变 碳水化合物结构可能涉及这样的疾病条件 如动脉粥样硬化，糖尿病，癌症等。我们建议净化 并研究几种可能是调节位点的酶 在通道中。 其中一种酶是GlcNAc-1-P转移酶 催化途径中的第一步;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。 在后一种反应中， 甘露糖基供体从GDP-甘露糖变为长壁醇-β-甘露糖。 我们认为我们对这些酶有一些很好的检测方法， 用于纯化的合理亲和步骤。 我们建议这样做 在几个植物系统中进行的研究， 大豆或西卡莫尔细胞对于 在文化中生长，大量生长是相当合理的 细胞（即，高达1 Kg的细胞）。 此外， 植物中脂连接糖途径似乎 溶解后比动物细胞更稳定。 因此 净化在工厂系统中应该更可行。 我们还计划纯化几种加工酶， 以制备多克隆抗体。 等 抗体将用于酶定位以及用于 这些酶的生物合成和靶向研究。 我们现在 具有抗葡萄糖苷酶II（一种早期加工酶）的抗体， 并将完成甘露糖苷酶II的纯化， 酶和GlcNAc转移酶II，一种后期加工酶。 其中一个需要解决的问题是植物是否有顺式， 内侧和transGolgi，以及各种活动是否在 高尔基体的不同区域。 其他问题涉及 将这些蛋白质导向特定位点的信号。 以来 我们有许多纯化的加工酶，我们将尝试 建立一个体外处理系统 当糖蛋白变成 克隆到细菌中， 糖基化并在无细胞系统中加工这些蛋白质。

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.