内质网胞质侧DLO动态平衡机制的研究

糖为细胞提供能量，还能与其他生命物质形成糖聚合物（如糖蛋白、糖脂以及多糖等）。糖类物质是维持细胞乃至机体生命活动的关键, 其代谢调控机制在糖生物学领域受到关注。最近，越来越多的研究证明蛋白质N-糖基化修饰被控制在一个平衡的状态，由内质网（ER）膜上多萜醇寡糖（DLO）的合成和降解途径共同调控。尽管DLO合成途径的基因大多已被鉴定，其调控机制尚不明确；而降解途径的研究更加滞后，导致DLO动态平衡的调控机制未得到解析。本课题拟利用分子生物学和化学生物学的交叉方法，以ER胞质侧DLO途径为目标，首先研究合成关键糖基转移酶Alg2的性质及立体结构，阐明其在DLO合成中的调节机制；同时初次分离并鉴定降解DLO中间体的焦磷酸酶（DLODP），分析其对DLO平衡的调控作用。该研究从合成和降解两方面解析ER胞质侧DLO动态平衡调节机制，对探索N-糖基化稳态的生理原理，揭示相关疾病的病理机制具有重要的意义。

Carbohydrates, which are also generically referred in molecular biology as “glycans”, play important biological roles in all aspects of life. They not only provide energy or serve as structural materials in cells, but also modify other biological macromolecules to generate glycoconjugates such as glycoproteins and glycolipids etc. Studies on regulating the metabolism of glycans are always prominent matters of interest in glycobiology field. Recently, a growing number of evidences suggest that several key enzymes in the early stage of dolichol-linked oligosaccharide (DLO) synthetic pathway on the endoplasmic reticulum (ER) membrane involved in the regulation of N-glycan homeostasis. Despite most genes participating in DLO pathway have been identified, molecular mechanisms regulating the synthetic and degradation pathways of DLO are still ambiguous. In this study, we propose to investigate the mechanisms of DLO homeostasis on cytoplasmic face of ER using the interdisciplinary methods of molecular biology and chemical biology. In our strategy, the key enzyme Alg2 in the DLO synthetic pathway will be studied by the kinetics analysis and 3D structure modeling; then DLO-diphosphatase (DLODP) protein will be firstly separated from mice liver and applied to the verification of its function in DLO degradation pathway; finally the interactions between the synthetic and degradation of DLO will be investigated through the above mentioned two enzymes. Our work will elucidate the mechanisms of the DLO homeostasis in the ER, leading to the further studies of N-glycan regulation and the reveal of related diseases.