CONTROLLED EXPRESSION OF NEOGLYCANS IN ANIMAL CELLS

动物细胞在其细胞表面合成一种复杂的寡糖模式 糖偶联物。在单个细胞中，糖蛋白可能显示出- 特异性糖基化，受控的微观异质性 每个位置的低聚糖，以及相对于以下位置的差异糖基化 鞘糖脂中发现的低聚糖。由于配置 细胞表面的低聚糖被推定为提供个性 细胞功能和行为，这些糖基化反应必须是 精心控制；然而，我们对许多元素的理解 参与调节动物细胞糖基化的研究非常有限。这个 这项研究的目的是为了更多地了解 糖基转移酶在糖共轭生物合成调控中的作用 动物细胞。这将通过监测结构 由单一基因引起的糖共轭化合物(新聚糖)的变化 事件，即一个“新的”糖基转移酶基因被引入到一个 “野性类型”背景。中国仓鼠卵巢(CHO)细胞将用作 获得稳定表达编码A基因的克隆的cDNA的“幼稚”接受者 小鼠(α1，3)半乳糖基转移酶和三种人岩藻糖基转移酶： H(α1，2)岩藻糖基转移酶血型，Lewis血型(Fuc 至GlcNAc-α1，3/4)岩藻糖基转移酶，以及(Fuc至 1，3)半乳糖基转移酶。的结构特点 这些细胞系合成的代谢性放射性标记糖偶联物 将被测定并与亲本细胞低聚糖进行比较。至 确定这些细胞系中的特定糖蛋白是否优先 用新聚糖糖基化，代谢放射性标记的糖蛋白将 通过十二烷基硫酸钠-PAGE凝胶分离，并进行部分结构 它们的新聚糖的特征。CHO细胞转铁蛋白受体 和溶酶体相关膜蛋白(LAMP)，已知展示 不同的低聚糖侧链，将被用作“记者” 以确定其N-连接是否以及在多大程度上连接 寡糖被新表达的糖基转移酶修饰。 成绩单之间有直接关系的程度 新聚糖的含量、特定的酶活性和相对含量 由转基因细胞合成的将通过分析 在如此构建的细胞系中代谢放射性标记的糖共轭化合物 糖基转移酶基因的剂量增加。这些研究 将提供重要的新信息，使我们了解 末端糖基化反应中的糖基转移酶，以及， 有助于确定协调复杂低聚糖的因子 动物细胞表达的模式。

Animal cells synthesize a complex pattern of oligosaccharides on their glycoconjugates. In a single cell, glycoproteins may demonstrate site- specific glycosylation, controlled microheterogeneity of the oligosaccharide at each site, and differential glycosylation relative to the oligosaccharides found on glycosphingolipids. Since the configuration of oligosaccharides at cell surfaces is presumed to provide individuality of cellular function and behavior, these glycosylation reactions must be exquisitely controlled; however, our understanding of the many elements involved in regulating animal cell glycosylation is severely limited. The objective of this research is to understand more about the precise role of glycosyltransferases in the regulation of glycoconjugate biosynthesis in animal cells. This will be accomplished by monitoring the structural changes in glycoconjugates (neoglycans) as a result of a single genetic event, namely, the introduction of a "new" glycosyltransferase gene into a "wild type" background. Chinese hamster ovary (CHO) cells will be used as "naive" recipients for the stable expression of cloned cDNAs encoding a murine (alpha1,3)galactosyltransferase and three human fucosyltransferases: the blood group H (alpha1,2)fucosyltransferase, the Lewis blood group (Fuc to GlcNAc-alpha1,3/4)fucosyltransferase, and the (Fuc to GlcNAcalpha1,3)galactosyltransferase. The structural features of metabolically radiolabeled glycoconjugates synthesized by these cell lines will be determined and compared to parental-cell oligosaccharides. To determine if specific glycoproteins in these cell lines are preferentially glycosylated with neoglycans, metabolically radiolabeled glycoproteins will be resolved by SDS-PAGE gels and subjected to partial structural characterization of their neoglycans. The CHO cell transferrin receptor and Lysosome Associated Membrane Proteins (LAMPs), known to exhibit different oligosaccharide side chains, will be used as "reporter glycoproteins" to determine if and to what degree their N-linked oligosaccharides are modified by the newly expressed glycosyltransferase. The degree to which there is a direct relationship between transcript levels, specific enzyme activities, and the relative amounts of neoglycan synthesized by transfected cells will be determined by analyses of the metabolically radiolabeled glycoconjugates in cell lines constructed so that the dosages of glycosyltransferase genes are increased. These studies will provide important new information on the precise role of glycosyltransferases in terminal glycosylation reactions, as well as, contribute to defining factors that orchestrate the complex oligosaccharide patterns expressed by animal cells.