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 糖基磷脂酰肌醇(GPI)附着在蛋白质和糖蛋白C末端是一种重要的 真核生物中普遍存在的翻译后修饰，它有助于锚定蛋白质和糖蛋白 胞外膜。GPI锚定蛋白和糖蛋白在多种生物和 病理过程。然而，目前对这些分子及其功能的详细研究还很有限， 主要是因为很难以纯形式和足够的数量获取它们，而且缺乏适当的工具来 分析这些不同的、复杂的和两亲性分子。因此，制定战略是非常必要的 这有助于获得和研究GPI锚定蛋白和糖蛋白。 这项研究项目的最终目标是制定战略，使人们能够获得同质和 结构上定义的天然GPI锚定蛋白和糖蛋白以及能够快速、有效地实现的策略 GPI锚定蛋白和糖蛋白的分离与分析。因此，这项建议有三个具体的 目标。目的1制备重组GPI转氨酶催化亚基GPI8(GPI-T) 真核细胞用来将GPI连接到蛋白质上的酶，以及来源于 细胞内质网(ER)，并用它们来创造一种潜在的通用的酶合成方法 天然GPI锚定蛋白和糖蛋白。目标2是为GPI的研究制定一个实用的策略-- 通过GPI-锚定蛋白生物合成途径的代谢工程由细胞表达锚定蛋白， 也就是说，给细胞或分离的内质网一个标记的合成GPI类似物，GPI-T可以使用它来添加到蛋白质中 发往GPI附件。这将导致GPI锚定蛋白的特定标记，以使其能够快速 分离并进行基于MS的蛋白质组学分析。目标3是为细胞的研究开发一种实用的策略 表面GPI锚定的蛋白质组学-CAPM因子，一种与GPI高亲和力结合的细菌毒素 锚定，以便于分离GPI连接的蛋白和细胞在处理后释放的糖蛋白 磷脂酰肌醇特异性磷脂酶C酶和随后的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的两种合成策略。 锚定蛋白质组学的研究具有原创性和创新性，因为目前还没有合成的方法 缺乏真正天然的GPI锚定蛋白/糖蛋白，没有合适的方法对GPI锚定蛋白进行系统研究 蛋白质组学。拟议的研究将产生广泛而重大的影响。一种实用的合成方法 GPI锚定的蛋白质和糖蛋白将允许访问这些重要的分子和它们的功能化 用于各种生物学和生物物理研究的纯形式和定义形式的类似物。允许系统化的策略 GPI锚定蛋白质组学的研究将有助于揭示GPI锚定蛋白与疾病的关系， 以及其他重要信息，并帮助识别新的蛋白质标记物。这一结果将广泛适用于 开发新的诊断和治疗策略，具有可调节的活动和靶向能力等。

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 use 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.