COMPLEX CARBOHYDRATES--STRUCTURE, FUNCTION, SYNTHESIS

N-连接糖基化是膜蛋白的主要修饰 低聚糖前体生物合成中的大部分反应 并将其加工成成熟的形式，都是众所周知的。然而，我们知道相对 关于N-连接的糖基化在细胞中是如何控制的，以及 只有几种动物糖基转移酶被纯化到 同质性的或具有同质性的用蛋白质处理培养的动物细胞 合成抑制剂或能量微扰剂，产生截短的脂质连接 低聚糖，表明对一个或多个 脂联低聚糖途径的糖基转移酶。 然而，监管的机制尚不清楚。我们现在的处境很好 提纯关键酶的位置(即DOL-P-MAN和DOL-P-GLC合成酶， LLO途径的GlcNAc-1-P转移酶、β-甘露糖转移酶) 因为：1.我们之前已经从主动脉中溶解了这些酶 并对其中几个进行了广泛纯化，2.我们有底物 光亲和探针(如N3-GDP[32P]-甘露糖)用于识别 SDS-PAGE上的催化亚基和3.我们现在有更多的经验 以及更好的纯化方法来分离这些蛋白质。 一旦我们提纯了蛋白质，我们就会准备针对每种蛋白质的抗体 并检测体内酶活性和酶蛋白的含量 以干扰LLO途径的方式(如用蛋白质)处理的细胞 合成抑制剂或在葡萄糖匮乏的细胞中)。我们还将检查 正常和治疗前后各酶的合成率和转化率 细胞。此外，我们将确定这些酶中是否有任何一种 受制于项目结束限制或其他反馈规定，或受 共价键修饰。 N-连接的糖基化的一个功能是影响一些 蛋白质。这一过程似乎是通过识别和结合来调节的 从伴侣到糖基化的高甘露糖结构在未折叠或 变性蛋白质。因为我们已经合成了各种放射性标记 糖化蛋白质和糖化低聚糖(如GLC[3-1]- Man9GlcNAc)，并使纯化的葡萄糖苷酶I和II可用于 研究中，我们将确定伴侣蛋白与这些分子的相互作用 酶，以及伴侣蛋白与标记底物的结合。我们 还将寻找一种专门阻止移除 最后用葡萄糖苷酶II测定葡萄糖。

N-linked glycosylation is the major modification of membrane proteins and most of the reactions in the biosynthesis of the oligosaccharide precursor and its processing to mature forms, are known. Yet, we know relatively little about how N-linked glycosylation is controlled in the cell, and only a few of the animal glycosyltransferases have been purified to homogeneity or characterized. Cultured animal cells treated with protein synthesis inhibitors or energy perturbants, produce truncated lipid-linked oligosaccharides, indicating regulation of one or more of the glycosyltransferases of the lipid-linked oligosaccharide pathway. However, the mechanism of regulation is unknown. We are in a good position to purify key enzymes (i.e., dol-P-man and dol-P-Glc synthases, GlcNAc-1-P transferase, beta-mannosyl-transferase) of the LLO pathway since: 1. We have previously solubilized these enzymes from aorta microsomes and purified several of them extensively, 2. We have substrate photoaffinity probes (such as N3-GDP[32P]-mannose) to identify the catalytic subunits on SDS-PAGE, and 3. We now have much more experience and better purification methods to isolate these proteins. Once we have the proteins purified, we will prepare antibody against each of thee and examine the amounts of enzyme activity, and enzyme protein in cells treated in ways that disturb the LLO pathway (such as with protein synthesis inhibitors or in glucose-starved cells). We will also examine the rate of synthesis and turnover of each enzyme in normal and treated cells. In addition, we will determine whether any of these enzymes are subjected to end-project inhibition or other feedback regulations, or to covalent modification. One function of N-linked glycosylation is to affect folding of some proteins. This process appears to be mediated by recognition and binding of chaperonins to glucosylated-high-mannose structures on unfolded or denatured proteins. Since we have synthesized various radiolabeled glucosylated-proteins and glucosylated-oligosaccharides (such as Glc[3-1]- Man9GlcNAc), and have the purified glucosidase I and II available to study, we will determine the interactions of chaperonins with these enzymes, and the binding of chaperonins to the labeled substrates. We will also look for an inhibitor that specifically blocks the removal of the last glucose by glucosidase II.