植烷醇磷酸甘露糖的酶法合成及应用  

Enzymatic Synthesis and Application of Phytanol-Phosphate Mannose

作　　者：李盛陶 王宁[1,2] 高晓冬 LI Shengtao;WANG Ning;GAO Xiaodong(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学糖化学与生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2020年第3期49-55,共7页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(21778023);江苏省自然科学基金项目(BK20170174)。

摘　　要：内质网(ER)腔内的甘露糖基化修饰共同存在于N-糖基化、糖基磷脂酰肌醇(GPI)锚定化、O-和C-甘露糖基化中,对于真核生物来说具有重要的作用。ER腔内的甘露糖基化的底物供体为多萜醇磷酸甘露糖(Dol-P-Man),因此Dol-P-Man的合成对于理解不同种类的糖基化修饰具有重要的意义。本研究原核表达纯化了酵母Dol-P-Man的合成酶(Dpm1),并鉴定了重要的DXD motif。利用纯化的Dpm1蛋白,及简单的植烷醇(Phytanol)代替复杂的多萜醇(Dolichol),体外酶法合成了Dol-P-Man的替代结构Phy-P-Man。在N-糖基化途径中Alg3催化Dol-PP-GlcNAc2-Man5和Dol-P-Man生成Dol-PP-GlcNAc2-Man6。通过体外酶反应,本研究证明Phy-P-Man同样能够作为Alg3的底物供体用于N-糖基化途径的研究,为以后合成高甘露糖型的N糖结构及GPI、O-和C-甘露糖基化的研究奠定了基础。Mannosylation in the endoplasmic reticulum(ER)lumen is common to four glycosylation pathways,and involves N-linked glycosylation,glycosylphosphatidylinositol(GPI)-anchor and protein O-and C-mannosylation.Thus,mannosylation in the ER lumen is vital for eukaryotes.The donor for mannosylation in the ER lumen is dolichol-phosphate-mannose(Dol-P-Man),thus the synthesis of Dol-P-Man is important for understanding different kinds of glycosylation modification.Dol-P-Man synthase catalyzes the transfer of mannose from GDP-mannose(GDP-Man)to dolichol-phosphate(Dol-P)to produce Dol-P-Man.This study expressed yeast Dol-P-Man synthase(Dpm1)in Escherichia coli,and purified it.The conserved DXD motif in Dpm1 playsed crucial roles for these activities.We used phytanol with a short lipid chain to substitute for its dolichol homologue with a long lipid chain,and synthesized phytanol-phosphate mannose(Phy-P-Man)using purified Dpm1.Furthermore,Phy-P-Man could be used as a substrate for Alg3 for N-glycosylation research.

关 键 词：甘露糖基化 Dpm1 植烷醇 Alg3 

分 类 号：Q81[生物学—生物工程]