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文章摘要
植烷醇磷酸甘露糖的酶法合成及应用*
Enzymatic Synthesis and Application of Phytanol-phosphate Mannose
投稿时间:2018-03-29  修订日期:2018-04-09
DOI:
中文关键词: 甘露糖基化  Dpm1  植烷醇
英文关键词: mannosylation  Dpm1  phytanol
基金项目:国家自然科学基金 (21778023);江苏省自然科学基金(BK20170174)
作者单位E-mail
李盛陶 江南大学生物工程学院 糖化学与生物技术教育部重点实验室 lishe520@126.com 
王宁 江南大学生物工程学院 糖化学与生物技术教育部重点实验室  
高晓冬 江南大学生物工程学院 糖化学与生物技术教育部重点实验室 xdgao@jiangnan.edu.cn 
摘要点击次数: 67
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中文摘要:
      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,因此对于真核生物来说具有重要的作用。由于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。
英文摘要:
      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. Due to 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. Dolichol-phosphate mannose synthase catalyzes the transfer of mannose from GDP-mannose (GDP-Man) to dolichol-phosphate (Dol-P) to produce dolichol-phosphate mannose (Dol-P-Man). This study expressed yeast dolichol-phosphate mannose synthase (Dpm1) in Escherichia coli, and purified. The conserved DXD motif in Dpm1 plays 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 was proved that it could be used as a substrate for Alg3 for N-glycosylation research.
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