Alg蛋白相关脂肪醇寡糖的合成及其分子识别机制的研究

A majority of newly synthesized polypeptides are modified by N-linked glycan in a co- or a post-translational manner, which plays important roles in maintaining the structural stability and functional diversity of proteins. In the endoplasmic reticulum (ER), the assembly of N-glycoproteins starts from the biosynthesis of lipid-linked oligosaccharide (LLO) on the ER membrane, catalyzing by a series of glycosyltransferases named Alg (asparagine linked glycosylation) proteins. It has recently been revealed that the abnormality of human Alg protein will result in the congenital disorder of glycosylation (CDG). Recent study also indicated that the activity of Alg proteins is closely related to the structure of its LLO substrate, especially the lipid chain. However, the detailed mechanism of protein-substrate recognition is still unclear, due to the diversity of LLO structures in the ER and the difficulties to obtain pure Alg proteins and LLO substrates. In this study, we plan to build up an LLO library with various lengths of lipid chains, which can serve as the mimic substrates to study the relationship between the Alg protein activity and the structure of lipid chain in LLO. To this end, three Alg proteins (Alg1, Alg2 and Alg11) located on the cytoplasmic side of ER will be prepared, and applied to the in vitro activity assay to give an insight into the LLO biosynthesis pathway. Our study will systematically investigate the substrate selectivity of Alg proteins from different species, and explore potential LLO structures with simple lipid chains, providing alternative substrates for investigating the molecular mechanism of ER glycosyltransferases in vitro.

糖基化是最为普遍的蛋白质修饰之一，对于维持蛋白质的结构稳定性及功能多样性至关重要。蛋白质N-糖基化起始于内质网膜上脂肪醇寡糖（LLO）的合成，该过程由多种糖基转移酶Alg蛋白催化完成，而人体Alg异常会导致先天性糖基化障碍。近期研究结果表明Alg蛋白的活性可能与LLO的脂肪链结构相关，但因内质网天然LLO结构多样且分离困难，使其分子机制至今尚未完全阐明。本课题拟化学合成一系列含不同长度脂肪链的LLO底物，同时大量表达不同物种来源的3个内质网胞浆侧Alg蛋白：Alg1，Alg2和Alg11，进而系统地研究各种Alg蛋白活性与底物中脂肪链结构间相互关系，深入解析LLO生物途径的分子机制。本研究将揭示不同物种来源的Alg蛋白对底物LLO中脂肪链长度选择性的差异，并开发结构简化的LLO类似物，为进行后续的体外内质网糖基转移酶活性测试打下基础。

蛋白质N-糖基化起始于内质网膜上脂肪醇寡糖（LLO）的合成，该过程由多种糖基转移酶Alg蛋白催化完成，而人体Alg异常会导致先天性糖基化障碍。前期研究结果表明Alg蛋白的活性可能与LLO的脂肪链结构相关，但因内质网天然LLO结构多样且分离困难，使其分子机制至今尚未完全阐明。本研究首次成功半合成含不同长度脂肪链的乙酰壳二糖与乙酰壳糖作为后续研究的底物，并利用大肠杆菌体系大量表达酵母和人源的各种Alg蛋白，使用液质联用方法对这些蛋白的底物特异性进行定量分析。结果表明，随着LLO中脂肪链长度的增加，Alg蛋白识别效率增加，且脂肪醇末端异戊烯结构也影响其识别效率，同时人源Alg蛋白对不同长度与结构的底物特异性高于酵母来源Alg蛋白。研究首次确认了不同物种来源的Alg蛋白对底物LLO中脂肪链长度选择性具有差异，揭示了LLO生物合成途径中的分子识别机制，为开发结构简化的LLO类似物用于体外内质网糖基转移酶活性测试打下了基础。相关研究成果发表论文8篇，申请专利2项，培养研究生6名。项目组成员参加多次国内、国际学术会议并进行口头报告。