CONTROL OF PROTEIN TERMINAL GLYCOSYLATION

描述：唾液酸寡糖在多种 重要的生物学过程，包括炎症、B细胞成熟 以及激活、病毒感染、胚胎和癌细胞的迁移， 和维持循环中的糖蛋白。这个项目的总体目标是 研究计划是为了了解控制 唾液酸寡糖的生物合成。在这项研究提案中， 申请者将研究控制阿尔法活动的机制。 天冬氨酸连接糖基化2，6-唾液酸基转移酶(ST6Gal I，ST)。这些 包括特定高尔基体池中的{1}酶区隔，{2} 两种催化活性不同的酶亚型的差异表达 活性、细胞定位和周转，以及不活跃的{3}形成 酶二聚体。三个假说将被检验。假设一： ST的特定高尔基分隔不仅涉及其 跨膜区，以及它的管腔干和催化区，以及 这些区域介导了导致高尔基体的寡聚事件。 留存。为了检验这一假设，申请者将确定哪个茎 高尔基体保留所需的序列，研究ST 两种酶的EM定位在高尔基体保留中的催化结构域 在该区域存在单一氨基酸差异的异构体，并决定 高尔基体滞留是否与ST寡聚有关。假设二： 两种ST亚型的差异表达深刻地影响了 特定细胞和组织对糖蛋白进行唾液酸化的能力。为了测试 这一假设，申请人将决定这两个分布 在不同组织和细胞类型中的ST亚型，并研究 体外和体内异构体活性的质和量差异 活着。假设III：高水平的ST表达导致了这种情况的形成 不活跃的ST同源二聚体是由二硫键形成的 半胱氨酸残基位于酶的供体结合部位。为了测试这一点 假设申请人将确定ST二聚体在哪里形成，如果 二聚反应提高了半胱氨酸残基的周转率 在二聚反应中，以及哪些条件有利于二聚反应。这些信息 从这次调查中获得的信息不仅将加深我们对 唾液酸寡糖的生物合成及其过程 在癌症和疾病期间发生改变，还允许申请者申请 所学到的增强和开发各种碳水化合物为主的 治疗策略。

DESCRIPTION: Sialylated oligosaccharides play pivotal roles in a variety of important biological processes, including inflammation, B cell maturation and activation, virus infection, migration of embryonic and cancer cells, and maintenance of glycoproteins in circulation. The overall goal of this research program is to understand the mechanisms that control the biosynthesis of sialylated oligosaccharides. In this research proposal the applicant will investigate mechanisms that control the activity of the alpha 2,6-sialyltransferase of Asn-linked glycosylation (ST6Gal I, ST). These include {1} enzyme compartmentation in specific Golgi cisternae, {2} differential expression of two enzyme isoforms that differ in catalytic activity, cellular localization and turnover, and {3} formation of inactive enzyme dimers. Three hypotheses will be tested. Hypothesis I: The specific Golgi compartmentation of the ST involves not only its transmembrane region, but also its lumenal stem and catalytic domains, and these regions mediate an oligomerization event that leads to Golgi retention. To test this hypothesis the applicant will determine which stem sequences are required for Golgi retention, investigate the role of the ST catalytic domain in Golgi retention by EM localization of two enzyme isoforms that differ by a single amino acid in this region, and determine whether Golgi retention correlates with ST oligomerization. Hypothesis II: The differential expression of two ST isoforms profoundly impacts the ability of specific cells and tissues to sialylate glycoproteins. To test this hypothesis the applicant will determine the distribution of these two ST isoforms in different tissues and cell types and investigate the qualitative and quantitative differences in isoform activity in vitro and in vivo. Hypothesis III: High levels of ST expression lead to the formation of inactive ST homodimers which are formed by disulfide bonds between cysteine residues in the enzyme's donor binding site. To test this hypothesis the applicant will determine where the ST dimer is formed, if dimerization increases turnover rate, which cysteine residues are involved in dimerization, and what conditions favor dimerization. The information gained from this investigation will not only further our understanding of the biosynthesis of sialylated oligosaccharides and how this process can be altered during cancer and disease, but also allow the applicant to apply what is learned to enhancing and developing a variety of carbohydrate-based therapeutic strategies.