Quantitative study of yeast Alg1 beta-1, 4 mannosyltransferase activity, a key enzyme involved in protein N-glycosylation

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• 作者：Sheng-Tao Li^a ; ^{lishe520@126.com} ; Ning Wang^a ; ^{wangning@jiangnan.edu.cn} ; Sha Xu^a ; ^{xusha1984@jiangnan.edu.cn} ; Jian Yin^a ; ^{jianyin@jiangnan.edu.cn} ; Hideki Nakanishi^a ; ^{hideki@jiangnan.edu.cn} ; Neta Dean^b ; ^{neta.dean@stonybrook.edu} ; Xiao-Dong Gao^a ; ^{xdgao@jiangnan.edu.cn}
• 关键词：Alg ; asparagine-linked glycosylation ; DLO ; dolichol-linked oligosaccharide ; DPGn2 ; dolichol pyrophosphate-GlcNAc2 ; EDTA ; ethylenediaminetetraacetic acid ; ESI-MS ; electrospray ionization mass spectrometry ; ER ; endoplasmic reticulum ; Glc ; glucose ; GlcNAc ; N-acetylglucosamine ; Man ; mannose ; PCR ; polymerase chain reaction ; PPGn2 ; phytanyl-pyrophosphate-GlcNAc2 ; TMD ; transmembrane-spanning domain ; UPLC ; ultra-performance liquid chromatography
• 刊名：Biochimica et Biophysica Acta (BBA) - General Subjects
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：1861
• 期：1
• 页码：2934-2941
• 全文大小：1049 K
• 卷排序：1861

文摘

Recombinant Alg1 glycosyltransferase was purified in high yield. A novel quantitative assay was established to study Alg1 β-1, 4 mannosyltransferase activity. These methods were applied to measure the effect on Alg1 activity of mutations identified in human ALG1-CDG patients. These new methods open the possibility to scale up DLO product PPGn2-Man for further downstream enzymatic reactions.