Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes

项目负责人/主要研究者（末、首、中）：郭忠武 GPI偶联物的合成及其与细胞膜的结合研究 将糖基磷脂酰肌醇（GPIs）（一种复杂糖脂家族）连接到蛋白质C-末端是一个重要的过程。 常见的和重要的翻译后修饰，其用于将蛋白质锚到细胞外表面。 GPIs和GPI锚定蛋白/糖蛋白在各种生物和病理过程中起着关键作用。 然而，由于缺乏适当的方法，对GPIs和GPI锚定蛋白的深入研究是具有挑战性的。 方法来访问和工具来研究这些结构复杂，多样性和两亲分子。 该研究项目旨在通过建立新的合成方法来应对这一挑战， 结构上同质和确定的GPI连接蛋白/糖蛋白和相关类似物， 促进GPI锚定蛋白/糖蛋白的分离和研究的新工具和策略， GPI结合分子，从而填补GPI研究的空白。因此，未来的研究方向， 该项目将包括：（1）开发新的方法，用于合成天然GPI连接的蛋白质的基础上， GPI转酰胺酶催化的酶促GPI/蛋白质连接和无痕施陶丁格反应-和硫代酸/叠氮化物 胺化衍生的化学选择性GPI/蛋白质连接;（2）GPI在细胞上的组织和取向的研究 表面荧光共振能量转移（FRET）技术，采用发色团标记的GPIs作为 分子工具;（3）使用发现和研究与GPI结合/相互作用的细胞膜成分 携带可光活化的亲和探针以拉下GPI结合分子的GPI衍生物;（4）定性和定量分析GPI衍生物的性质。 通过GPI的代谢工程定量分析由各种细胞表达的GPI锚定蛋白 生物合成途径以促进GPI锚定蛋白的标记和下拉以用于其快速分离， 表征和分析。 拟议的研究是创新的，将产生重大和广泛的影响，因为它解决了一系列问题。 这是GPI研究中重要但尚未解决的问题，填补了对GPI锚定理解的空白。具体地说， GPI锚定蛋白和糖蛋白的实用合成方法将允许获得这些重要的 结构均匀和确定形式的分子及其官能化类似物，其可用于 各种生物学研究。系统深入地研究GPI锚定蛋白质组学， 以及GPI与细胞膜中其他分子的相互作用， 通过分子探针和本文提出的策略，将导致更好和更深入的理解 GPI的锚定。这不仅有助于更详细地揭示其功能和作用机制， GPIs还有助于发现新的疾病标志物，包括GPI锚定蛋白/糖蛋白和GPI- 结合分子，从而实现GPI应用于分子医学的承诺，如开发 新的诊断和治疗方法。

Program Director/Principal Investigator (Last, First, Middle): GUO, Zhongwu Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes Attaching glycosylphosphatidylinositols (GPIs), a family of complex glycolipids, to the protein C-terminus is a common and important posttranslational modification, which serves to anchor proteins to the extracellular surface. GPIs and GPI-anchored proteins/glycoproteins play a critical role in various biological and pathological processes. However, in-depth investigation of GPIs and GPI-anchored proteins is challenging because of the lack of proper methods to access and tools to study these structurally complex, diverse, and amphipathic molecules. This research project aims to address the challenge by establishing novel synthetic methods to enable access to structurally homogeneous and defined GPI-linked proteins/glycoproteins and related analogs and developing new tools and strategies to facilitate the isolation and investigation of GPI-anchored proteins/glycoproteins and GPI-binding molecules, so as to fill the gaps in GPI research. Accordingly, the future research directions of this project will include: (1) development of new methods for the synthesis of natural GPI-linked proteins based on GPI transamidase-catalyzed enzymatic GPI/protein ligation and traceless Staudinger reaction- and thio acid/azide amination-derived chemoselective GPI/protein ligations; (2) study of GPI organization and orientation on the cell surface by fluorescence resonance energy transfer (FRET) technology employing chromophore-labeled GPIs as molecular tools; (3) discovery and investigation of cell membrane components that bind/interact with GPIs using GPI derivatives that carry a photoactivatable affinity probe to pull down GPI-binding molecules; (4) qualitative and quantitative analysis of GPI-anchored proteins expressed by various cells through metabolic engineering of GPI biosynthetic pathways to facilitate the labeling and pull-down of GPI-anchored proteins for their rapid isolation, characterization, and analysis. The proposed research is innovative and will have a significant and broad impact, as it addresses a series of important but unsolved problems in GPI research and fills the gaps in understanding GPI anchorage. Specifically, a practical synthetic method for GPI-anchored proteins and glycoproteins will allow for access to these important molecules and their functionalized analogs in structurally homogeneous and defined forms, which are useful for various biological studies. Systematic and in-depth investigation of GPI-anchored proteomics, GPI organization and orientation on the cell surface, and GPI interaction with other molecules in the cell membrane, which will be enabled by the molecular probes and strategies proposed herein, will lead to a better and deeper understanding of GPI anchorage. This will not only help reveal more details about the functions and functional mechanisms of GPIs but also help discover new disease markers, including both GPI-anchored proteins/glycoproteins and GPI- binding molecules, thereby to fulfill the promise of GPI application to molecular medicine, such as development of new diagnostic and therapeutic methods.