植烷醇磷酸甘露糖的酶法合成及应用

Enzymatic Synthesis and Application of Phytanol-Phosphate Mannose

DOI：10.3969/j.issn.1673-1689.2020.03.007

中文关键词: 甘露糖基化 Dpm1 植烷醇 Alg3

英文关键词: mannosylation,Dpm1,phytanol,Alg3

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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，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。

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. 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）inEscherichia 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.

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