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

GPI 缀合物和 GPI 细胞膜锚定的合成和生物学研究

• 批准号: 10584557
• 负责人: Zhongwu Guo
• 金额: $ 33.48万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584557
• 关键词: Acids; Address; Affinity; Amination; Azides; Binding; Biological; Biological Process; Cell membrane; Cell surface; Cells; Complex; Development; Disease; Disease Marker; Family; Fluorescence Resonance Energy Transfer; GPI Membrane Anchors; Glycolipids; Glycoproteins; Glycosylphosphatidylinositols; Investigation; Label; Ligation; Membrane Glycoproteins; Membrane Proteins; Methods; Molecular; Molecular Medicine; Molecular Probes; Pathologic Processes; Pathway interactions; Play; Post-Translational Protein Processing; Principal Investigator; Process; Protein C; Proteins; Proteomics; Reaction; Research; Research Project Grants; Role; Series; Surface; Technology Transfer; analog; chromophore; extracellular; innovation; link protein; metabolic engineering; molecular marker; novel; novel diagnostics; novel therapeutic intervention; novel therapeutics; programs; tool; transamidases

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.

项目主任/首席研究员（后、一、中）：郭忠武 GPI 缀合物和 GPI 细胞膜锚定的合成和生物学研究 将糖基磷脂酰肌醇 (GPI)（一类复杂糖脂）附着到蛋白质 C 末端是一种 常见且重要的翻译后修饰，用于将蛋白质锚定到细胞外表面。 GPI 和 GPI 锚定蛋白/糖蛋白在各种生物和病理过程中发挥着关键作用。 然而，由于缺乏适当的方法，对 GPI 和 GPI 锚定蛋白的深入研究具有挑战性。 获取这些结构复杂、多样化和两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应对这一挑战 结构均质且明确的 GPI 连接蛋白/糖蛋白和相关类似物，并正在开发 促进 GPI 锚定蛋白/糖蛋白分离和研究的新工具和策略 GPI结合分子，从而填补了GPI研究的空白。据此，本课题未来的研究方向 项目将包括：（1）开发合成天然GPI连接蛋白的新方法 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 氨基化衍生的化学选择性 GPI/蛋白质连接； (2)GPI在细胞上的组织和定向研究 通过荧光共振能量转移 (FRET) 技术，采用发色团标记的 GPI 作为表面 分子工具； (3) 使用 GPI 发现和研究与 GPI 结合/相互作用的细胞膜成分 GPI 衍生物携带光激活亲和探针以拉下 GPI 结合分子； (4) 定性和 通过 GPI 代谢工程定量分析各种细胞表达的 GPI 锚定蛋白 促进 GPI 锚定蛋白的标记和下拉以实现快速分离的生物合成途径， 表征和分析。 拟议的研究具有创新性，将产生重大而广泛的影响，因为它解决了一系列问题 GPI 研究中重要但尚未解决的问题，填补了理解 GPI 锚固的空白。具体来说， 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 分子及其功能化类似物具有结构均匀和确定的形式，可用于 各种生物学研究。系统深入研究GPI锚定蛋白质组学、GPI组织 和细胞表面的方向，以及 GPI 与细胞膜中其他分子的相互作用，这将是 通过本文提出的分子探针和策略，将带来更好、更深入的理解 GPI 锚地。这不仅有助于揭示有关功能和作用机制的更多细节 GPI 还有助于发现新的疾病标记物，包括 GPI 锚定蛋白/糖蛋白和 GPI- 结合分子，从而实现 GPI 应用到分子医学的承诺，例如开发 新的诊断和治疗方法。

项目成果

Ganglioside GM1 and the Central Nervous System.

• DOI: 10.3390/ijms24119558
• 发表时间: 2023-05-31
• 期刊: International journal of molecular sciences
• 影响因子: 5.6

Labeling cell surface glycosylphosphatidylinositol-anchored proteins through metabolic engineering using an azide-modified phosphatidylinositol.

• DOI: 10.1016/j.bbrc.2023.01.029
• 发表时间: 2023-02-19
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Kundu, Sayan;Jaiswal, Mohit;Craig, Kendall C.;Guo, Jiatong;Guo, Zhongwu
• 通讯作者: Guo, Zhongwu

Spin-Labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells

• DOI: 10.1007/s00723-023-01624-w
• 发表时间: 2023-10-09
• 期刊: APPLIED MAGNETIC RESONANCE
• 影响因子: 1
• 作者: Jaiswal，Mohit;Tran，Trang T.;Fanucci，Gail E.
• 通讯作者: Fanucci，Gail E.

Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

自旋标记洞察化学固定如何影响细胞上的聚糖组织。

• DOI: 10.21203/rs.3.rs-3039983/v1
• 发表时间: 2023
• 期刊: Research square
• 作者: Jaiswal,Mohit;Tran,TrangT;Guo,Jiatong;Zhou,Mingwei;Kunda,Sayan;Guo,Zhongwu;Fanucci,Gail
• 通讯作者: Fanucci,Gail