GPI转酰胺酶合成GPI锚定蛋白的研究

GPI anchored proteins, which are widely present in the eukaryotic cell membrane, play a key role in many physiological and pathological processes. Due to the complexity of GPI anchors, it is difficult to obtain defined, homogeneous GPI-linked proteins from biological resources, thus greatly restricting the basic research of GPI proteins and their application in the treatment of important diseases. Consequently, in vitro synthesis of GPI protein analogues become one of the hotspots of this field and has made some progress, but the artificial synthesis of natural GPI anchored proteins has not yet been realized. In vivo, GPI anchored proteins are formed through the GPI transamidase catalyzed cleavage of precursor proteins and subsequent attachment with GPI anchors. Therefore, GPI transamidase is an ideal enzyme source for the synthesis of natural GPI proteins in vitro. In this project, the GPI transamidase is prepared from natural resource and the artificial GPI protein precursor bearing natural GPI signal is designed and tested by the enzyme. The transpeptidation activity is subsequently determined in the presence of the precursor and hydrazide-biotin. Afterwards, GPI anchor analogs are synthesized and the transpeptidation reaction is performed to obtain GPI protein analogs. This work would explore the in vitro catalytic property of the GPI transamidase and also establish a new method for the synthesis of natural GPI proteins bearing natural GPI signal, which would lay a foundation for the synthesis and functional analysis of various natural GPI proteins.

糖基磷脂酰肌醇(GPI)锚定蛋白普遍存在于真核细胞膜上，在多种生理和病理过程中发挥关键作用，但GPI锚结构复杂，难以纯化获得均一的GPI锚定蛋白，极大地限制了该类蛋白的基础研究及其在重大疾病治疗方面的应用。GPI锚定蛋白的体外高效合成因此成为该领域的研究热点之一，已取得进展，但天然GPI锚定蛋白的人工合成仍未实现。GPI转酰胺酶是生物体内通过转肽作用将GPI蛋白前体和GPI锚偶联成GPI锚定蛋白的关键酶，因而是体外合成天然GPI蛋白的理想酶源。本项目拟从天然来源制备GPI转酰胺酶，设计制备融合天然GPI信号序列的蛋白前体底物，研究该酶对蛋白底物的识别能力；以蛋白底物和肼酰生物素研究该酶的转肽活性；进而设计合成GPI锚类似物并建立酶的转肽反应，合成带天然GPI信号序列的GPI锚定蛋白类似物。该项目将建立GPI锚定蛋白合成的新方法，为各类天然GPI锚定蛋白的合成及生物学功能研究奠定基础。

糖基磷脂酰肌醇(GPI)锚定蛋白普遍存在于真核细胞膜上，在多种生理和病理过程中发挥关键作用。本项目研究了GPI转酰胺酶的制备、对蛋白底物的识别及GPI锚定蛋白类似物的合成。天然提取了酵母GPI转酰胺酶粗酶，并探索了GPI转酰胺酶可溶性重组表达体系，利用pET系列质粒通过融合麦芽糖结合蛋白在大肠杆菌成功表达了酿酒酵母、布氏锥虫、墨西哥利什曼原虫GPI转酰胺酶的GPI8亚基，重组酶分子量大小与理论分子量基本一致；通过分子对接设计并合成了多种多肽底物，筛选到带荧光标记的多肽底物ATKD-AMC，能用于测定不同来源GPI转酰胺酶的酶活，首次建立了GPI转酰胺酶的快速测定方法；以绿色荧光蛋白为模式蛋白，C末端融合酵母天然GPI锚定蛋白GPI信号序列，构建GPI转酰胺酶蛋白底物，并将信号序列截短，构建一系列蛋白底物分子，证实GPI转酰胺酶对人工蛋白底物的识别依赖于GPI信号序列；同时采用酶法和化学法合成GPI锚类似物，采用GPI转酰胺酶以蛋白底物和GPI锚类似物为底物进行反应，检测到GPI锚定蛋白类似物的生成。