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.

计划主任/首席研究员（最后，第一，中间）： GPI共轭物和GPI锚定在细胞膜的合成和生物学研究 将复杂糖脂家族（GPIS）连接到蛋白质C末端是A是A 常见和重要的翻译后修饰，将蛋白质锚定在细胞外表面。 GPI和GPI锚定的蛋白/糖蛋白在各种生物学和病理过程中起着至关重要的作用。 但是，对GPI和GPI锚定蛋白的深入研究很具有挑战性，因为缺乏适当的 访问和研究这些结构复杂，多样和两亲性分子的方法。 该研究项目旨在通过建立新颖的合成方法来解决挑战 到结构均匀且定义的GPI连接蛋白/糖蛋白和相关类似物以及发展 新工具和策略，以促进GPI锚定蛋白/糖蛋白的隔离和调查 GPI结合分子，以填补GPI研究中的空白。因此，未来的研究指示 项目将包括：（1）开发基于自然GPI连接蛋白质的新方法 GPI跨成绩酶催化的酶促GPI/蛋白质连接以及无可透性的Staudinger反应和硫代酸/叠氮化物 胺化的化学选择性GPI/蛋白质连接； （2）研究细胞的GPI组织和方向 通过荧光共振能量传递（FRET）技术，采用发色团标记的GPI作为技术 分子工具； （3）发现和研究使用使用GPI结合/相互作用的细胞膜成分 携带光活化亲和力探针以下拉GPI结合分子的GPI衍生物； （4）定性和 通过GPI代谢工程表达的各种细胞表达的GPI锚定蛋白的定量分析 生物合成途径，以促进GPI锚定蛋白的标签和下拉，以快速隔离， 表征和分析。 拟议的研究具有创新性，将产生重大和广泛的影响，因为它解决了一系列 GPI研究中的重要但未解决的问题，并填补了理解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使用这些重要的 分子及其在结构均匀和定义的形式中的官能化类似物，这对 各种生物学研究。 GPI蛋白质组学的系统和深入研究GPI组织 和细胞表面的方向，以及与细胞膜中其他分子的相互作用，这将是 由本文提出的分子探针和策略启用，将带来更好，更深入的理解 GPI锚固。这不仅将有助于揭示有关功能和功能机制的更多细节 GPI，但也有助于发现新的疾病标志物，包括GPI锚定的蛋白/糖蛋白和GPI- 结合分子，从而实现GPI对分子医学的诺言，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