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

描述（由申请人提供）：许多表面蛋白和糖蛋白通过糖基磷脂酰肌醇（gpi）锚定在细胞膜上，gpi锚定的蛋白和糖蛋白在各种生物过程中发挥重要作用。为了研究这些过程并探索GPI锚定，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。本课题旨在开发一种实用的合成这些重要分子的方法，并通过合成gpi来研究gpi锚定的蛋白质组学。该提案有三个具体目标。具体目标1是为天然GPI和各种重要的GPI衍生物（如烯烃、炔、酯、硫醇和硫化物）的合成制定一个总体策略。本研究计划在合成中使用对甲氧基（PMB）基团作为全局和永久的保护基团，以确保在上述各种功能存在的情况下可以选择性地实现最终的去保护。具体目标2是开发一种化学合成天然连接gpi锚定肽/糖肽或蛋白质/糖蛋白的实用方法。基本的合成设计是分别制备GPI和肽/糖肽段，然后通过温和的化学选择性反应将它们连接在一起。为此，合成的GPI将被修饰为在GPI聚糖非还原端具有羟胺或甲氧基，而肽将被修饰为在c端具有1-酮酸功能。随后，在温和的条件下，用Bode结扎法将两段进行耦合。具体目标3是建立一种可行的快速分离和鉴定GPI锚定蛋白和糖蛋白的方法，并将其用于研究表面GPI锚定。为此，缺乏GPI生物合成功能的突变细胞的内质网（ER）膜将被分离并用于合成生物素化的GPI。由于这些内质网膜既含有GPI转氨酶（催化GPI加入蛋白质的酶），也含有所有用于GPI锚定的新生蛋白，它们与生物素化的GPI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系统，以获得生物素化形式的gpi锚定蛋白的整个补体。此后，gpi锚定蛋白将很容易地与链亲和素柱分离，并通过质谱和其他为蛋白质组学研究建立的方法进行分析。该项目的广泛影响：一种有效和广泛使用的方法来合成均质和结构明确的GPI和GPI锚定肽/糖肽/蛋白质，对于GPI锚定和GPI锚定分子的研究和理解至关重要。对细胞上GPI锚定蛋白的系统分析将为了解细胞GPI锚定的概况提供重要信息。这些信息有助于确定与某些疾病相关的gpi锚定蛋白标记物，可作为设计和开发更有效的诊断技术和疾病治疗的新分子靶标。

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.