STRUCTURE AND FUNCTION OF GLYCOINOSITOL

糖肌醇的结构和功能

• 批准号: 6105352
• 负责人: TERRONE L ROSENBERRY
• 金额: $ 14.93万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6105352
• 关键词: N acetylglucosamine; X ray crystallography; acetylcholinesterase; chemical structure function; chemical synthesis; glucosamine; glycolipids; guanosine diphosphate; inositol; insulin; ion exchange chromatography; lipid structure; mannose; methylation; nitrous acid; paroxysmal nocturnal hemoglobinuria; phospholipase C; phospholipase D; tissue /cell culture; tritium; uridine diphosphate

Our focus will remain on the structure and function of glycoinositol phospholipids (GPIs) which include glycolipids based on a GlcN-(1 -> 6)- inositol phospholipid core. The first aim is to identify, isolate and characterize free glycolipids with GPI structures from mammalian cells. Three approaches to their identification will be pursued: a) biosynthetic labeling of intact cells with GPI components like [3H]glucosamine or [3H]inositol; b) exogenous radiomethylation of glucosamine-containing lipids in cell extracts; and c) in vitro labeling of microsomal lipids with UDP-[3H]GlcNAc or GDP-[3H]mannose. GPIs will be identified by their susceptibility to several enzymatic and chemical fragmentation agents including bacterial phosphatidylinositol-specific phospholipase C (PIPLC); trypanosome GPI-specific PLC (GPI-PLC); serum GPI-specific PLD (GPI-PLD); and nitrous acid deamination. The glycan cleavage products will be isolated and analyzed by ion exchange chromatography to identify characteristic GPI components and to deduce GPI structural variants. The second aim is to compare the actions of glycans derived from GPIs with those of insulin on intact cells and cell extracts to test the hypothesis that a glycan product formed by PIPLC cleavage of a GPI mediates many of the metabolic effects of insulin. We will continue to pursue this glycan among the GPIs isolated in aim 1. In an alternative approach, the major glycan phosphate derived from the human erythrocyte (Ehu) acetylcholinesterase (AChE) GPI anchor will be compared to insulin in several intact cell and in vitro assays of metabolic enzymes. Preliminary observations that show similar effects with this glycan and insulin will be extended to examine the effects of other glycan fragments derived from the Ehu AChE GPI anchor. In the third aim we will identify and characterize GPI-specific phospholipases. The phospholipid cofactor requirements of PIPLC for optimal activity will be determined as a model for other phospholipases. We will initiate protocols to obtain x-ray crystallographic structural information about the PIPLC active site. The phospholipid requirements for trypanosome GPI-PLC and serum GPI-PLD also will be studied and applied to a search for other endogenous GPI phospholipases in mammalian cells, including those activated by insulin. We propose the chemical synthesis of a simple GPI in the fourth aim. We will undertake the synthesis of radiolabeled GlcN-(1 -> 6)-inositol phospholipids in which the glycerolipid contains short C7 - C10 acyl or alkyl groups. These short lipid groups will insure rapid diffusion into the cell membrane. These GPIs may be useful as 1) substrates for identifying endogenous GPI-specific phospholipases; 2) precursors for glycan extension either in vivo or in vitro; 3) substrates for identifying GPI inositol acylating and/or deacylating enzymes; and 4) labeled ligands to search for receptors on the cell surface.

我们的重点将保留在糖称醇的结构和功能上 磷脂（GPI）包括基于GLCN-（1-> 6） - 肌醇磷脂核。 第一个目的是识别，隔离和 用哺乳动物细胞的GPI结构表征游离糖脂。 将采用三种识别方法：a）生物合成 用[3H]葡萄糖胺或GPI成分标记完整细胞 [3H]肌醇； b）含葡萄糖的外源性放射甲基化 细胞提取物中的脂质； c）与微粒体脂质的体外标记 UDP- [3H] GlcNAC或GDP- [3H]甘露糖。 GPI将由他们的 对几种酶和化学碎片化剂的敏感性 包括细菌性磷脂酰肌醇特异性磷脂酶C（PIPLC）； 锥虫特异性PLC（GPI-PLC）;血清GPI特异性PLD（GPI-PLD）; 和亚硝酸脱氨酸。 聚糖裂解产品将是 通过离子交换色谱分离和分析，以识别 特征性GPI组件并推断GPI结构变体。 这 第二个目的是将源自GPI的聚糖的行为与 完整细胞和细胞提取物上胰岛素的胰岛素以检验假设 GPI的PIPLC裂解形成的聚糖产品介导了许多 胰岛素的代谢作用。 我们将继续追求这个聚糖 在AIM 1中孤立的GPI中。在另一种方法中，主要是 源自人红细胞（EHU）的聚糖磷酸盐 将将乙酰胆碱酯酶（ACHE）GPI锚与胰岛素进行比较 几个完整的细胞和代谢酶的体外测定。 初步的 与该聚糖和胰岛素相似的观察结果将是 扩展以检查从源自 Ehu Ache GPI锚。 在第三个目标中，我们将确定和表征 GPI特异性磷脂酶。 磷脂辅因子的要求 最佳活动的PIPLC将被确定为其他 磷脂酶。 我们将启动协议以获得X射线 有关PIPLC活动位点的晶体学结构信息。 这 锥虫GPI-PLC和血清GPI-PLD的磷脂要求也 将研究并应用于其他内源GPI 哺乳动物细胞中的磷脂酶，包括胰岛素激活的磷脂酶。 我们提出了第四个目标中简单GPI的化学合成。 我们 将承担放射标记的GLCN-（1-> 6） - 肌醇的合成 甘油中含有短C7 -C10酰基或的磷脂 烷基。 这些短脂质组将确保快速扩散到 细胞膜。 这些GPI可能是有用的1） 鉴定内源性GPI特异性磷脂酶； 2）前体 聚糖在体内或体外延伸； 3）用于识别的基板 GPI肌醇酰化和/或脱酰基酶； 4）标记配体 在细胞表面搜索受体。