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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 1; pp. 2934 - 2941
• Main Authors: Li, Sheng-Tao, Wang, Ning, Xu, Sha, Yin, Jian, Nakanishi, Hideki, Dean, Neta, Gao, Xiao-Dong
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2017
• Subjects: affinity chromatography; Alg1; ALG1-CDG; Amino Acid Sequence; asparagine; Blotting, Western; Chromatography, Liquid; Disaccharides - chemistry; Disaccharides - metabolism; Electrophoresis, Polyacrylamide Gel; endoplasmic reticulum; enzyme activity; Escherichia coli; Glycosylation; glycosyltransferases; humans; mannose; Mannosyltransferases - chemistry; Mannosyltransferases - metabolism; Mass Spectrometry; Mutant Proteins - chemistry; Mutant Proteins - isolation & purification; Mutant Proteins - metabolism; mutation; N-glycosylation; Phytanyl-pyrophosphate-GlcNAc2; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Saccharomyces cerevisiae - enzymology; trisaccharides; viability; yeasts; DLO; PPGn2; ESI-MS; Phytanyl-pyrophosphate-GlcNAc2; TMD; Alg1; EDTA; ER; N-glycosylation; UPLC; ALG1-CDG; GlcNAc; Man; DPGn2; Alg; PCR; Glc
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.09.023

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 Km and Vmax values 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. •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.