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

蛋白和糖蛋白c端附着的糖基磷脂酰肌醇（GPI）是真核生物物种中普遍存在的重要的翻译后修饰，它有助于将蛋白和糖蛋白锚定在胞外膜上。gpi锚定蛋白和糖蛋白在各种生物和病理过程中起着关键作用。然而，目前对这些分子及其功能的详细研究是有限的，主要原因是难以获得纯净的形式和足够的数量，并且缺乏适当的工具来分析这些多样、复杂和两亲性的分子。因此，非常希望有策略可以促进访问和研究gpi锚定蛋白和糖蛋白。本研究项目的最终目标是开发能够获得均匀且结构明确的天然gpi锚定蛋白和糖蛋白的策略，以及能够快速，有效地分离和分析gpi锚定蛋白和糖蛋白的策略。因此，这项建议有三个具体目标。目的1是制备GPI转氨酶（GPI- t）的重组催化亚基GPI8（真核细胞用于将GPI附着在蛋白质上的天然酶）和来源于细胞内质网（ER）的膜相关完整GPI- t，并利用它们创建一种可能用于酶合成天然GPI锚定蛋白和糖蛋白的通用方法。目标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锚定蛋白开发一种实用的策略，即为细胞或分离的内质网提供一种标记的合成GPI类似物，GPI- t可以使用该类似物将其添加到结合GPI的蛋白上。这将导致gpi锚定蛋白的特异性标记，使其能够快速分离，然后进行基于ms的蛋白质组学分析。目的3是开发一种实用的策略，通过使用与GPI锚点具有高亲和力结合的细菌毒素CAPM因子来研究细胞表面GPI锚定蛋白质组学，以促进磷脂酰肌醇特异性磷脂酶C酶处理后细胞释放的GPI连接蛋白和糖蛋白的分离，并随后进行GPI锚定蛋白质组学分析。无论是天然GPI锚定蛋白和糖蛋白合成策略，还是GPI锚定蛋白质组学研究的两种策略，都具有独创性和创新性，因为目前还没有真正天然的GPI锚定蛋白/糖蛋白合成方法，也没有合适的方法对GPI锚定蛋白质组学进行系统的研究。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允许以纯净和明确的形式获得这些重要分子及其功能化类似物，用于各种生物和生物物理研究。允许系统研究gpi锚定蛋白质组学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物。这一结果将为开发具有调节活性、靶向能力等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.