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

描述(申请人提供)：获得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末端是真核生物中一种重要的和普遍存在的翻译后修饰，它有助于将蛋白质和糖蛋白锚定到细胞膜上。GPI锚定蛋白和糖蛋白在多种生物和病理过程中发挥着重要作用。然而，目前对这些分子及其功能的详细研究是有限的，主要是因为很难以纯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的、复杂的两亲性分子。因此，非常希望有一种策略可以促进对GPI锚定蛋白和糖蛋白的获取和研究。这项研究项目的最终目标是开发能够获得同质和结构定义的天然GPI锚定蛋白和糖蛋白的策略，以及能够快速、有效地分离和分析GPI锚定蛋白和糖蛋白的策略。因此，这项提议有三个具体目标。目的1制备GPI转氨酶的重组催化亚基GPI-T(GPI-T)和膜结合完整的GPI-T，GPI-T是真核细胞用于将GPI连接到蛋白质上的天然酶，并利用它们创建一种潜在的通用的酶合成天然GPI锚定蛋白和糖蛋白的方法。目的2是通过GPI锚定蛋白生物合成途径的代谢工程，为研究细胞表达的GPI锚定蛋白提供一种实用的策略，即给细胞或分离的ERs一个标记的合成GPI类似物，GPI-T可以用它来添加到结合到GPI结合的蛋白质中。这将导致对GPI锚定蛋白的特定标记，以便快速分离它们，然后进行基于MS的蛋白质组学分析。目的3是建立一种实用的研究细胞表面GPI锚定蛋白质组学的策略，利用CAPM因子，一种与GPI锚定具有高亲和力的细菌毒素，以便于分离磷脂酰肌醇特异性磷脂酶C酶处理后细胞释放的GPI连接蛋白和糖蛋白，以及随后的GPI锚定蛋白质组学分析。无论是天然GPI锚定蛋白和糖蛋白的合成策略，还是GPI锚定蛋白质组学研究的两种策略都是原创性和创新性的，因为目前还没有合成真正天然的GPI锚定蛋白质/糖蛋白的方法，也没有合适的方法来系统研究GPI锚定蛋白质组学。拟议的研究将产生广泛而重大的影响。一种实用的GPI锚定蛋白和糖蛋白的合成方法将使人们能够获得这些重要的分子及其功能类似物，这些分子和它们的功能类似物以纯的和确定的形式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。这些结果将为开发具有调节活性、靶向性等的新的诊断和治疗策略提供广泛的帮助。

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.