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

描述（由申请人提供）：获取GPI锚定蛋白质和研究GPI锚定蛋白质组学的新方法糖基磷脂酰肌醇（GPI）与蛋白质和糖蛋白C-末端的连接是真核生物中重要且普遍存在的翻译后修饰，其有助于将蛋白质和糖蛋白锚至细胞外膜。GPI锚定蛋白和糖蛋白在各种生物学和病理学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，主要是因为难以获得它们的纯形式和足够的数量，以及缺乏适当的工具来分析这些多样的，复杂的，两亲性的分子。因此，非常需要具有可以促进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转酰胺酶（GPI transamidase，GPI-T）的重组催化亚基GPI 8和来自细胞内质网的膜结合完整GPI-T，并利用它们建立一种潜在的通用的酶促合成天然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开发用于研究由细胞表达的GPI锚定蛋白的实用策略，即，给予细胞或分离的ER标记的合成GPI类似物，其可被GPI-T用于添加到结合用于GPI附着的蛋白。这将导致GPI锚定蛋白的特异性标记，以使其能够快速分离，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CAPM因子，具有高亲和力结合GPI锚的细菌毒素，以促进分离GPI连接的蛋白质和糖蛋白后释放的磷脂酰肌醇特异性磷脂酶C酶处理细胞和随后的GPI锚定蛋白质组学分析的细胞表面GPI锚定蛋白质组学的研究。 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性的，因为目前还没有真正天然GPI锚定蛋白/糖蛋白的合成方法，也没有合适的方法来系统地研究GPI锚定蛋白质组学。拟议的研究将产生广泛而重大的影响。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分子及其功能化类似物，其以纯净和确定的形式用于各种生物和生物物理研究。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。这些结果对于开发具有调节活性、靶向能力等的新的诊断和治疗策略具有广泛的意义。

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.