CONTROLLED EXPRESSION OF NEOGLYCANS IN ANIMAL CELLS

动物细胞中新聚糖的受控表达

• 批准号: 3305402
• 负责人: DAVID F SMITH
• 金额: $ 19.38万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305402
• 关键词: CHO cells; SDS polyacrylamide gel electrophoresis; affinity chromatography; carbohydrate structure; gene expression; glycolipids; glycoprotein biosynthesis; glycoprotein structure; glycoproteins; glycosylation; glycosyltransferase; high performance liquid chromatography; human genetic material tag; lipid structure; membrane proteins; oligosaccharides; radiotracer; tissue /cell culture; transfection; transferrin receptor

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.

动物细胞合成一种复杂的寡糖， 糖缀合物 在单个细胞中，糖蛋白可以显示位点- 特异性糖基化， 每个位点的寡糖，以及相对于 在鞘糖脂上发现的寡糖。 由于配置 据推测，细胞表面的低聚糖 细胞功能和行为的，这些糖基化反应必须 然而，我们对许多元素的理解 参与调节动物细胞糖基化的基因受到严重限制。 的 这项研究的目的是了解更多关于确切的作用， 糖基转移酶在调节糖缀合物生物合成中的作用 动物细胞 这将通过监测结构来实现 糖缀合物（新聚糖）的变化是由于单一的遗传 事件，即，将“新的”糖基转移酶基因引入到 “野生型”背景。 中国仓鼠卵巢（CHO）细胞将用作 “幼稚”受体用于稳定表达克隆的编码 鼠（α 1，3）半乳糖基转移酶和三种人岩藻糖基转移酶： 血型H（α 1，2）岩藻糖基转移酶、刘易斯血型（Fuc 至GlcNAc-α 1，3/4）岩藻糖基转移酶，以及（Fuc至 GlcNA α 1，3）半乳糖基转移酶。 的结构特点 由这些细胞系合成的代谢放射性标记的糖缀合物 将被测定并与亲本细胞寡糖进行比较。 到 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SDS-PAGE凝胶分离，并进行部分结构分析。 其新聚糖的表征。 CHO细胞转铁蛋白受体 和溶酶体相关膜蛋白（LAMP），已知表现出 不同的寡糖侧链，将被用作“报告基因 糖蛋白”来确定它们的N-连接 寡糖被新表达的糖基转移酶修饰。 成绩单与学生成绩之间的直接关系 水平、特定酶活性和新聚糖的相对量 将通过分析转染细胞合成的蛋白质来确定。 代谢放射性标记的糖缀合物在细胞系中的构建， 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， 在末端糖基化反应中的糖基转移酶，以及， 有助于定义协调复杂寡糖的因素 动物细胞表达的模式。