Novel Approaches to Access GPIs and GPI-Anchored Proteins for the Study of GPI An

获取 GPI 和 GPI 锚定蛋白用于 GPI An 研究的新方法

• 批准号: 8136594
• 负责人: Zhongwu Guo
• 金额: $ 29.38万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136594
• 关键词: Abbreviations; Acetylcholinesterase; Acetylglucosamine; Acyl Coenzyme A; Address; Alkenes; Alkynes; Anabolism; Binding; Biological; Biological Process; Biotin; Biotinylation; CD55 Antigens; Cell membrane; Cell surface; Cell-Free System; Cells; Chemicals; Complement; Coupled; Coupling; Development; Diagnostic Procedure; Dicyclohexylcarbodiimide; Disease; Endoplasmic Reticulum; Enzymes; Esters; Fluorides; GPI Membrane Anchors; Galactose; Glucose; Glycolipids; Glycopeptides; Glycoproteins; Glycosylphosphatidylinositols; Goals; Hydroxylamine; Investigation; Keto Acids; Label; Ligation; Link; Mannose; Membrane; Membrane Glycoproteins; Membrane Proteins; Methods; Molecular Target; Peptides; Phosphate Buffer; Phosphatidylinositols; Phospholipase C; Play; Polysaccharides; Positioning Attribute; Preparation; Process; Proteins; Proteomics; Pyrrolidinones; Reaction; Research; Role; Saline; Silver; Source; Streptavidin; Structure; Sulfhydryl Compounds; Sulfides; Surface; Trifluoroacetic Acid; Ursidae Family; Variant; acyl group; alkyl group; benzotriazole; chemical synthesis; design; functional group; inorganic phosphate; link protein; man; methoxyamine; mutant; novel strategies; phosphoethanolamine; public health relevance; rapid technique; transamidases; trifluoromethanesulfonic acid

DESCRIPTION (provided by applicant): Many surface proteins and glycoproteins are anchored to cell membranes by glycosylphosphatidylinositols (GPIs), and GPI-anchored proteins and glycoproteins play an important role in various biological processes. To investigate these processes and to explore GPI anchorage, it is necessary to have access to homogenous and structurally defined GPIs, GPI derivatives, and GPI-anchored proteins and glycoproteins. This proposal aims to develop a practical method for the synthesis of these important molecules and study GPI-anchored proteomics by means of synthetic GPIs. The proposal has three specific aims. Specific Aim 1 is to develop a general strategy for the synthesis of natural GPIs and various important GPI derivatives bearing functionalities such as alkene, alkyne, ester, thiol, and sulfide. This research plans to use the p-methoxybenzyl (PMB) group as a global and permanent protecting group in the synthesis to assure that the final deprotection can be achieved selectively in the presence of a variety of functionalities as mentioned. Specific Aim 2 is to develop a practical method for the chemical synthesis of natively linked GPI-anchored peptides/glycopeptides or proteins/glycoproteins. The fundamental synthetic design is to prepare the GPI and peptide/glycopeptide segments separately and then link them together via a mild and chemoselective reaction. For this purpose, synthetic GPIs will be modified to bear a hydroxylamine or methoxylamine group at the GPI glycan non-reducing end, and peptides will be modified to bear an 1-ketoacid functionality at the C-terminus. Subsequently, the two segments will be coupled by Bode's ligation method under mild conditions. Specific Aim 3 is to develop a feasible method for rapid isolation and identification of GPI-anchored proteins and glycoproteins and use it to study surface GPI anchorage. For this purpose, the endoplasmic reticulum (ER) membranes of mutant cells lacking GPI biosynthesis functionality will be isolated and used to react with synthetic biotinylated GPIs. Since these ER membranes contain both GPI transamidases, the enzymes that catalyze GPI addition to proteins, and all nascent proteins destined for GPI anchorage, their reaction with biotinylated GPIs will result in the biotinylation of all GPI-anchored proteins. This will create a cell-free system for obtaining the entire complement of GPI-anchored proteins in the biotinylated form. Thereafter, GPI-anchored proteins will be easily isolated with a streptavidin column and analyzed by MS and other methods established for proteomic studies. The broad impact of this project: An effective and widely useful method for the synthesis of homogenous and structurally defined GPIs and GPI-anchored peptides/glycopeptides/proteins is essential for the study and understanding of GPI anchorage and GPI-anchored molecules. Systematic analysis of GPI-anchored proteins on cells will provide important information about the profiles of cell GPI anchorage. This information can help identify GPI-anchored protein markers associated with certain diseases, which can be used as new molecular targets for the design and development of more effective diagnostic techniques and disease treatments. PUBLIC HEALTH RELEVANCE: This proposal aims to develop a practical synthetic method for glycosylphosphatidylinositols (GPIs) and GPI- anchored peptides/glycopeptides or proteins/glycoproteins that play pivotal roles in many biological processes and to develop a practical method for identifying and analyzing GPI-anchored proteins/glycoproteins expressed by cells. This research is important for the study and understanding of the functions of GPI anchors and GPI- anchored molecules. Systematic analysis of GPI-anchored proteins on cells will provide important information that can help identify disease-associated GPI-anchored protein markers that are useful molecular targets for the design and development of more effective diagnostic techniques and disease treatments.

描述（由申请人提供）：许多表面蛋白和糖蛋白被糖基磷脂酰肌醇（GPIS）锚定在细胞膜上，而GPI锚定的蛋白质和糖蛋白在各种生物学过程中起着重要作用。为了研究这些过程并探索GPI锚固，有必要获得同质和结构定义的GPI，GPI衍生物以及GPI锚定的蛋白质和糖蛋白。该建议旨在开发一种实用方法来合成这些重要分子，并通过合成GPI研究GPI锚定的蛋白质组学。该提案具有三个具体目标。 具体目的1是制定一般策略来合成天然GPI和各种重要的GPI衍生物，具有碱，碱，酯，酯，硫醇和硫化物等功能。这项研究计划将P-甲氧基苯甲酰基（PMB）组作为综合中的全球和永久保护组，以确保在提到的各种功能的存在下可以选择性地实现最终脱位。 具体目的2是开发一种实用方法来化学合成本质上连接的GPI锚定肽/糖肽或蛋白质/糖蛋白。基本的合成设计是分别准备GPI和肽/糖肽片段，然后通过轻度和化学的反应将它们连接在一起。为此，将修改合成GPI，以在GPI GLYCAN非还原端携带羟胺或甲氧基胺基，并将修饰肽在C-末端具有1-酮酸功能。随后，这两个部分将在轻度条件下通过Bode的连接方法耦合。 具体目标3是开发一种可行的方法，用于快速分离和鉴定GPI锚定的蛋白质和糖蛋白，并使用它来研究表面GPI锚固。为此，将分离缺乏GPI生物合成功能的突变细胞的内质网（ER）膜，并用于与合成生物素化的GPI反应。由于这些ER膜均包含GPI转酰胺酶，因此催化GPI添加到蛋白质的酶，以及所有针对GPI锚固的新生蛋白质，它们与生物素化GPI的反应将导致所有GPI锚定蛋白的生物素化。这将创建一个无细胞的系统，用于以生物素化形式获得GPI锚定蛋白的整个补体。此后，将GPI锚定的蛋白很容易用链霉亲和素柱分离，并通过MS和其他用于蛋白质组学研究的方法进行分析。 该项目的广泛影响：一种有效且广泛有用的方法，可用于合成同质和结构定义的GPI和GPI锚定肽/糖肽/蛋白质，对于研究和理解GPI锚定和GPI恒河分子是必不可少的。对细胞上GPI锚定的蛋白质的系统分析将提供有关细胞GPI锚固概况的重要信息。该信息可以帮助识别与某些疾病相关的GPI锚定蛋白标记，这些蛋白质标志物可以用作设计和开发更有效的诊断技术和疾病治疗的新分子靶标。 公共卫生相关性：该建议旨在开发一种实用的合成方法，用于糖基磷脂酰肌醇（GPIS）和GPI-锚定肽/糖肽或蛋白质/糖蛋白，这些肽或蛋白/糖蛋白在许多生物学过程中起着关键作用在许多生物学过程中发挥关键作用，并通过识别和分析的Gpi-protect和contectectect和contectect和contectect和分析的蛋白质。这项研究对于研究和理解GPI锚和GPI锚定分子的功能很重要。对细胞上GPI锚定蛋白的系统分析将提供重要信息，以帮助鉴定与疾病相关的GPI锚定蛋白标记物，这些标志物是设计和开发更有效的诊断技术和疾病治疗的有用分子靶标。