New Methods to Access GPI-Anchored Proteins and Study GPI-Anchored Proteomics

获取 GPI 锚定蛋白和研究 GPI 锚定蛋白质组学的新方法

• 批准号: 8989113
• 负责人: Zhongwu Guo
• 金额: $ 17.88万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2016-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8989113
• 关键词: Address; Affinity; Affinity Chromatography; Anabolism; Bacterial Toxins; Binding; Binding Proteins; Biocompatible Materials; Biological; Biological Process; Biosynthetic Proteins; Biotin; Catalytic Domain; Cell membrane; Cell surface; Cells; Chemicals; Chemistry; Cleaved cell; Complex; Coupled; Coupling; Development; Disease; Endoplasmic Reticulum; Enzymes; Eukaryotic Cell; Event; GPAA1 gene; GPI Membrane Anchors; Glycolipids; Glycopeptides; Glycoproteins; Glycosylphosphatidylinositols; Goals; HSV glycoprotein C; Health; Human; In Vitro; Investigation; Label; Lead; Ligation; Link; Literature; Membrane; Membrane Glycoproteins; Membrane Proteins; Methods; Molecular; Nature; PLCG2 gene; Pathologic Processes; Pathway interactions; Peptides; Pharmaceutical Preparations; Play; Post-Translational Protein Processing; Property; Protein Biosynthesis; Protein C; Proteins; Proteomics; Recombinant Proteins; Recombinants; Research; Research Project Grants; Role; Sepharose; Signal Transduction; Structure-Activity Relationship; Techniques; Tissues; Trypanosoma brucei brucei; analog; base; biophysical analysis; chemical synthesis; design; disease diagnosis; extracellular; genetically modified cells; innovation; link protein; metabolic engineering; molecular marker; novel; novel diagnostics; novel strategies; novel therapeutic intervention; protein biomarkers; research study; sortase; synthetic peptide; tool; transamidases

DESCRIPTION (provided by applicant): New Methods to Access GPI-Anchored Proteins and Study GPI-Anchored Proteomics Glycosylphosphatidylinositol (GPI) attachment to the protein and glycoprotein C-terminus is an important and ubiquitous posttranslational modification in eukaryotic species, which helps anchor proteins and glycoproteins to the extracellular membrane. GPI-anchored proteins and glycoproteins play a pivotal role in various biological and pathological processes. However, currently, detailed studies on these molecules and their functions are limited, mainly because of the difficulty to access them in pure form and sufficient quantity and the lack of proper tools to analyze these diverse, complex, and amphipathic molecules. Therefore, it is highly desirable to have strategies that can facilitate access to and investigation of GPI-anchored proteins and glycoproteins. The ultimate goals of this research project are to develop strategies that enable access to homogeneous and structurally defined natural GPI-anchored proteins and glycoproteins and strategies that enable rapid, effective isolation and analysis of GPI-anchored proteins and glycoproteins. Accordingly, this proposal has three specific aims. Aim 1 is to prepare both the recombinant catalytic subunit GPI8 of GPI transamidase (GPI-T), the natural enzyme used by eukaryotic cells to attach GPIs to proteins, and membrane-associated intact GPI-T derived from the cell endoplasmic reticulum (ER) and uses them to create a potentially general method for enzymatic synthesis of natural GPI-anchored proteins and glycoproteins. Aim 2 is to develop a practical strategy for the study of GPI- anchored proteins expressed by cells via metabolic engineering of GPI-anchored protein biosynthetic pathways, namely, to give cells or isolated ERs a tagged synthetic GPI analog that can be used by GPI-T to add to proteins bound for GPI attachment. This will result in the specific labeling of GPI-anchored proteins to enable their rapid isolation and then MS-based proteomics analysis. Aim 3 is to develop a practical strategy for the study of cell surface GPI-anchored proteomics by using CAPM factor, a bacterial toxin that has a high-affinity binding to GPI anchors, to facilitate the isolation of GPI-linked proteins and glycoproteins released from cells upon treatment with phosphatidylinositol-specific phospholipase C enzyme and subsequent GPI-anchored proteomics analysis. Both the strategy for natural GPI-anchored protein and glycoprotein synthesis and the two strategies for GPI- anchored proteomics study are original and innovative, because currently there is no method for the synthesis of truly natural GPI-anchored proteins/glycoproteins and no proper method for systematic study of GPI-anchored proteomics. The proposed research will have a broad and significant impact. A practical synthetic method for GPI-anchored proteins and glycoproteins will allow access to these important molecules and their functionalized analogs in pure and defined forms for various biological and biophysical studies. Strategies allowing systematic study of GPI-anchored proteomics will help reveal the relationships between GPI-anchored proteins and diseases, as well as other important information, and help identify new protein markers. The results will be widely useful for the development of new diagnostic and therapeutic strategies with modulated activity, targeting ability, etc.

描述（由申请人提供）：获取 GPI 锚定蛋白和研究 GPI 锚定蛋白质组学的新方法 糖基磷脂酰肌醇 (GPI) 附着在蛋白质和糖蛋白 C 末端，是真核物种中重要且普遍存在的翻译后修饰，有助于锚定蛋白质和糖蛋白附着在细胞外膜上。 GPI 锚定蛋白和糖蛋白在各种生物和病理过程中发挥着关键作用。然而，目前对这些分子及其功能的详细研究是有限的，主要是因为难以以纯净的形式和足够的数量获得它们，并且缺乏适当的工具来分析这些多样化、复杂的两亲性分子。因此，非常需要有能够促进 GPI 锚定蛋白和糖蛋白的获取和研究的策略。 该研究项目的最终目标是开发能够获得均质且结构明确的天然 GPI 锚定蛋白和糖蛋白的策略，以及能够快速、有效分离和分析 GPI 锚定蛋白和糖蛋白的策略。因此，该提案具有三个具体目标。目标 1 是制备 GPI 转酰胺酶 (GPI-T) 的重组催化亚基 GPI8（真核细胞用于将 GPI 附着到蛋白质上的天然酶）以及源自细胞内质网 (ER) 的膜相关完整 GPI-T并利用它们创建了一种潜在的通用方法，用于酶促合成天然 GPI 锚定蛋白和糖蛋白。目标 2 是通过 GPI 锚定蛋白生物合成途径的代谢工程研究细胞表达的 GPI 锚定蛋白的实用策略，即为细胞或分离的 ER 提供可被 GPI 使用的标记的合成 GPI 类似物。 T 添加到与 GPI 连接结合的蛋白质中。这将导致 GPI 锚定蛋白的特异性标记，从而能够快速分离它们，然后进行基于 MS 的蛋白质组学分析。目标 3 是利用 CAPM 因子（一种与 GPI 锚定点具有高亲和力的细菌毒素）研究细胞表面 GPI 锚定蛋白质组学的实用策略，以促进从细胞中释放的 GPI 连接蛋白和糖蛋白的分离。使用磷脂酰肌醇特异性磷脂酶 C 酶处理细胞并随后进行 GPI 锚定蛋白质组学分析。 天然GPI锚定蛋白和糖蛋白的合成策略以及GPI锚定蛋白质组学研究的两种策略都是原创和创新的，因为目前还没有真正天然的GPI锚定蛋白/糖蛋白的合成方法，也没有合适的方法来合成GPI锚定蛋白/糖蛋白。 GPI 锚定蛋白质组学的系统研究。拟议的研究将产生广泛而重大的影响。 GPI 锚定蛋白和糖蛋白的实用合成方法将允许以纯净和确定的形式获得这些重要分子及其功能化类似物，用于各种生物学和生物物理研究。对 GPI 锚定蛋白质组学进行系统研究的策略将有助于揭示 GPI 锚定蛋白质与疾病之间的关系以及其他重要信息，并有助于识别新的蛋白质标记。研究结果将广泛用于开发具有调节活性、靶向能力等的新诊断和治疗策略。