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

描述（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锚定蛋白和糖蛋白在各种生物过程中发挥着重要作用。为了研究这些过程并探索 GPI 锚定，有必要获得同质且结构明确的 GPI、GPI 衍生物以及 GPI 锚定蛋白和糖蛋白。该提案旨在开发一种合成这些重要分子的实用方法，并通过合成 GPI 来研究 GPI 锚定蛋白质组学。该提案有三个具体目标。 具体目标 1 是开发合成天然 GPI 和具有烯烃、炔烃、酯、硫醇和硫化物等功能的各种重要 GPI 衍生物的总体策略。本研究计划在合成中使用对甲氧基苄基（PMB）基团作为全局且永久的保护基团，以确保在上述各种官能团存在的情况下可以选择性地实现最终的脱保护。 具体目标 2 是开发一种化学合成天然连接的 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/糖肽片段，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 聚糖非还原端带有羟胺或甲氧基胺基团，并且肽将被修饰为在 C 末端带有 1-酮酸官能团。随后，这两个片段将在温和条件下通过博德连接法偶联。 具体目标 3 是开发一种快速分离和鉴定 GPI 锚定蛋白和糖蛋白的可行方法，并用它来研究表面 GPI 锚定。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，并用于与合成的生物素化 GPI 反应。由于这些 ER 膜含有 GPI 转酰胺酶（催化 GPI 添加到蛋白质上的酶）以及所有用于 GPI 锚定的新生蛋白质，它们与生物素化 GPI 的反应将导致所有 GPI 锚定蛋白质的生物素化。这将创建一个无细胞系统，用于获得生物素化形式的 GPI 锚定蛋白的全部补充。此后，GPI 锚定蛋白将很容易用链霉亲和素柱分离，并通过 MS 和为蛋白质组研究建立的其他方法进行分析。 该项目的广泛影响：一种有效且广泛使用的合成均质且结构明确的 GPI 和 GPI 锚定肽/糖肽/蛋白质的方法对于研究和理解 GPI 锚定和 GPI 锚定分子至关重要。对细胞上 GPI 锚定蛋白的系统分析将提供有关细胞 GPI 锚定概况的重要信息。这些信息可以帮助识别与某些疾病相关的 GPI 锚定蛋白标记，这些标记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。 公共健康相关性：本提案旨在开发一种实用的合成方法，用于合成在许多生物过程中发挥关键作用的糖基磷脂酰肌醇 (GPI) 和 GPI 锚定肽/糖肽或蛋白质/糖蛋白，并开发一种用于识别和分析细胞表达的 GPI 锚定蛋白/糖蛋白的实用方法。该研究对于研究和理解GPI锚和GPI锚定分子的功能具有重要意义。对细胞上 GPI 锚定蛋白的系统分析将提供重要信息，帮助识别与疾病相关的 GPI 锚定蛋白标记物，这些标记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点。

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.