Quantitative study of yeast Alg1 beta-1, 4 mannosyltransferase activity, a key enzyme involved in protein N-glycosylation.
Asparagine (N)-linked glycosylation begins with a stepwise synthesis of the dolichol-linked oligosaccharide (DLO) precursor, Glc3Man9GlcNAc2-PP-Dol, which is catalyzed by a series of endoplasmic reticulum membrane-associated glycosyltransferases. Yeast ALG1 (asparagine-linked glycosylation 1) encodes a β-1, 4 mannosyltransferase that adds the first mannose onto GlcNAc2-PP-Dol to produce a core trisaccharide Man1GlcNAc2-PP-Dol. ALG1 is essential for yeast viability, and in humans mutations in the ALG1 cause congenital disorders of glycosylation known as ALG1-CDG. Alg1 is difficult to purify because of its low expression level and as a consequence, has not been well studied biochemically. Here we report a new method to purify recombinant Alg1 in high yield, and a mass spectral approach for accurately measuring its β-1, 4 mannosyltransferase activity.



N-terminally truncated yeast His-tagged Alg1 protein was expressed in Escherichia coli and purified by HisTrap HP affinity chromatography. In combination with LC-MS technology, we established a novel assay to accurately measure Alg1 enzyme activity. In this assay, a chemically synthesized dolichol-linked oligosaccharide analogue, phytanyl-pyrophosphoryl-α-N, N'-diacetylchitobioside (PPGn2), was used as the acceptor for the β-1, 4 mannosyl transfer reaction.



Using purified Alg1, its biochemical characteristics were investigated, including the apparent Kmand Vmaxvalues for acceptor, optimal conditions of activity, and the specificity of its nucleotide sugar donor. Furthermore, the effect of ALG1-CDG mutations on enzyme activity was also measured.



This work provides an efficient method for production of Alg1 and a new MS-based quantitative assay of its activity.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: lishe520@126.com.
Sheng-Tao
ST
Li
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: wangning@jiangnan.edu.cn.
Ning
N
Wang
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: xusha1984@jiangnan.edu.cn.
Sha
S
Xu
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: jianyin@jiangnan.edu.cn.
Jian
J
Yin
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: hideki@jiangnan.edu.cn.
Hideki
H
Nakanishi
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, United States. Electronic address: neta.dean@stonybrook.edu.
Neta
N
Dean
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: xdgao@jiangnan.edu.cn.
Xiao-Dong
XD
Gao
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008364:Mannosyltransferases / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004187:Disaccharides; D050505:Mutant Proteins; D011994:Recombinant Proteins; C086865:phytanylpyrophosphoryl-N,N'-diacetylchitobioside; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase
ALG1-CDG; Alg1; N-glycosylation; Phytanyl-pyrophosphate-GlcNAc2