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

项目总监/首席调查员(末位、第一位、中位)：郭忠武 GPI偶联物及其对细胞膜的锚定作用的合成和生物学研究 糖基磷脂酰肌醇(GPIs)是一种复杂的糖脂家族，它与蛋白C末端的连接是一种 常见且重要的翻译后修饰，用于将蛋白质锚定在细胞外表面。 GPI和GPI锚定蛋白/糖蛋白在多种生物和病理过程中发挥着重要作用。 然而，深入研究GPI和GPI锚定蛋白是具有挑战性的，因为缺乏适当的 研究这些结构复杂、多样和两亲性分子的方法和工具。 该研究项目旨在通过建立新的合成方法来实现访问，以应对这一挑战 结构均一和明确的GPI连接的蛋白质/糖蛋白和相关类似物 促进分离和研究GPI锚定蛋白/糖蛋白和 GPI结合分子，从而填补GPI研究的空白。据此，本研究的未来研究方向 该项目将包括：(1)开发合成天然GPI连接蛋白的新方法 GPI转氨酶催化的GPI/蛋白质连接和无迹Staudinger反应--以及硫代酸/叠氮 氨基衍生的GPI/蛋白质化学选择性连接；(2)GPI在细胞上的组织和定位研究 表面荧光共振能量转移(FRET)技术利用发色团标记的GPI作为 分子工具；(3)发现和研究与GPI结合/相互作用的细胞膜成分 携带可光激活亲和探针的GPI衍生物，以拉下GPI结合分子；(4)定性和 利用GPI代谢工程定量分析不同细胞表达的GPI锚定蛋白 便于快速分离GPI锚定蛋白的标记和下拉的生物合成途径， 刻画和分析。 拟议的研究是创新的，将产生重大和广泛的影响，因为它涉及一系列 GPI研究中重要但尚未解决的问题，并填补了理解GPI锚定的空白。具体来说， 一种实用的GPI锚定蛋白和糖蛋白的合成方法将允许获得这些重要的 分子及其官能化类似物的结构均一和定义的形式，这是有用的 各种生物学研究。系统深入地研究GPI锚定蛋白质组学、GPI组织 和细胞表面的取向，以及GPI与细胞膜上的其他分子相互作用，这将是 通过这里提出的分子探针和策略，将导致更好和更深入的理解 在GPI安克雷奇。这不仅有助于揭示更多关于BMP的功能和作用机制的细节 GPI还有助于发现新的疾病标记物，包括GPI锚定的蛋白/糖蛋白和GPI- 从而实现了GPI在分子医学等方面的应用前景 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.