COMPLEX, CARBOHYDRATES, STRUCTURE, FUNCTION, SYNTHESIS

复杂、碳水化合物、结构、功能、合成

• 批准号: 6363481
• 负责人: Alan D Elbein
• 金额: $ 29.2万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1975
• 资助国家: 美国
• 起止时间: 1975-02-01 至 2004-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363481
• 关键词: N glycosidase; SDS polyacrylamide gel electrophoresis; carbohydrate metabolism; carbohydrate structure; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; glycolipids; glycoprotein structure; glycosylation; glycosyltransferase; immunologic assay /test; intermolecular interaction; membrane proteins; molecular chaperones; oligosaccharides; protein folding; protein purification; protein structure function; tissue /cell culture

DESCRIPTION (Adapted from applicant's abstract): N-linked glycosylation is the major non-protein modification that occurs on membrane and secretory glycoproteins and therefore involves a major and important series of reactions. In spite of the widespread distribution of this pathway, very little is known about the regulation of these reactions, nor on how the entire pathway is controlled. In addition, very few of the enzymes involved in the pathway have been purified or characterized. The PI has evidence that the pyrophosphorylases that synthesize GDP-mannose (GDPMPP) and UDP-G1cNAc (UDPHexNAccPP), two key precursors of N- and O-glycosylation, have unusual substrate specificities and other properties that suggest that they play a regulatory role in carbohydrate metabolism and/or biosynthesis. In addition, the UDPHexNAcPP, which is membrane-bound, may reside in a complex linked to other ER or Golgi proteins and this complex may regulate G1cNAc transfer. In this study, the PI will purify GDPMPP and determine whether one subunit synthesizes GDP-glucose and the other GDP-mannose, and what regulates these activities and how. The PI will clone and express GDPMPP and then delete or alter the activity to determine its role in N-glycosylation. Similar experiments will be done with membrane-associated UDPHexNAcPP. The PI will use these pyrophosphorylases to prepare photoprobes, such as N3-UDP [32P]-G1cNAc, to isolate the transferases involved in N-linked glycosylation so that their properties and roles in regulation of glycosylation can be assessed. These enzymes are likely to be important in metabolic disease such as diabetes, atherosclerosis and glycoprotein deficiency syndromes.

描述（改编自申请人摘要）：N-连接糖基化是 发生在细胞膜上的主要非蛋白质修饰， 糖蛋白，因此涉及主要和重要的一系列反应。 尽管这一途径广泛分布， 关于这些反应的调节，也不知道整个途径是如何 控制。此外，很少有参与该途径的酶具有 被纯化或表征。PI有证据表明焦磷酸化酶 其合成GDP-甘露糖（GDPMPP）和UDP-G1 cNAc（UDPHexNAccPP），这两个关键 N-和O-糖基化的前体，具有不寻常的底物特异性， 其他特性表明它们在碳水化合物中起着调节作用， 代谢和/或生物合成。此外，UDPHexNAcPP， 膜结合，可能存在于与其他ER或高尔基体蛋白连接的复合物中 该复合物可调节G1 cNAc的转运。在本研究中，PI将 纯化GDPMPP，并确定是否一个亚基合成GDP-葡萄糖， 其他国内生产总值甘露糖，以及什么调节这些活动，以及如何。PI将 克隆并表达GDPMPP，然后删除或改变其活性以确定其 N-糖基化的作用。类似的实验将在 膜相关UDPHexNAcPP。PI将使用这些焦磷酸化酶， 制备光探针，如N3-UDP [32 P]-G1 cNAc，以分离转移酶 参与N-连接的糖基化，因此它们的性质和作用， 可以评估糖基化的调节。这些酶很可能是 在代谢性疾病如糖尿病、动脉粥样硬化 糖蛋白缺乏综合征