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Alg2 甘露糖基转移酶的拓扑和酶学分析揭示了其在脂连接寡糖生物合成途径中的作用。

Topological and enzymatic analysis of human Alg2 mannosyltransferase reveals its role in lipid-linked oligosaccharide biosynthetic pathway.

机构信息

Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

Zaozhuang Jienuo enzyme co., Ltd, Zaozhuang, China.

出版信息

Commun Biol. 2022 Feb 8;5(1):117. doi: 10.1038/s42003-022-03066-9.

DOI:10.1038/s42003-022-03066-9
PMID:35136180
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8827073/
Abstract

N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER). Alg2 mannosyltransferase adds both the α1,3- and α1,6-mannose (Man) onto ManGlcNAc-pyrophosphate-dolichol (MGn-PDol) in either order to generate the branched MGn-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop a liquid chromatography-mass spectrometry quantitative kinetics assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man onto MGn before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of MGn substrate, both of which trigger production of the MGn(α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of MGn (α-1,6) intermediate.

摘要

N-糖基化始于内质网 (ER) 上脂质连接寡糖 (LLO) 的生物合成。Alg2 甘露糖基转移酶以任意顺序在 ManGlcNAc-焦磷酸-dolichol (MGn-PDol) 上添加α1,3-和α1,6-甘露糖 (Man)，从而生成支化的 MGn-PDol 产物。经过充分研究的酵母 Alg2 通过四个疏水区与 ER 膜相互作用。出乎意料的是，我们发现酵母和人类之间的 Alg2 结构已经发生了分歧。人类 Alg2（hAlg2）通过一个单一的膜结合结构域与 ER 结合，在体外显著更稳定。这些特性被利用来开发一种用于研究纯化的 hAlg2 的液相色谱-质谱定量动力学测定法。在生理条件下，hAlg2 更倾向于在添加α1,6-Man 之前将α1,3-Man 转移到 MGn 上。然而，这种偏好会被 GDP-Man 供体过量或 MGn 底物水平增加所改变，这两者都会触发 MGn(α-1,6)-PDol 的产生。这些结果表明，Alg2 可能通过控制 MGn（α-1,6）中间产物的积累来调节 LLO 生物合成途径。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/18d24140bad5/42003_2022_3066_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/150b787ab12e/42003_2022_3066_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/2bcb8658654a/42003_2022_3066_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/fe1457386132/42003_2022_3066_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/8ef6df4e94ad/42003_2022_3066_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/d18f1ffd19d4/42003_2022_3066_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/18d24140bad5/42003_2022_3066_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/150b787ab12e/42003_2022_3066_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/2bcb8658654a/42003_2022_3066_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/fe1457386132/42003_2022_3066_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/8ef6df4e94ad/42003_2022_3066_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/d18f1ffd19d4/42003_2022_3066_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2404/8827073/18d24140bad5/42003_2022_3066_Fig6_HTML.jpg

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