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Quantitative study of yeast Alg1 beta-1, 4 mannosyltransferase activity, a key enzyme involved in protein N-glycosylation.
- Source :
-
Biochimica et biophysica acta. General subjects [Biochim Biophys Acta Gen Subj] 2017 Jan; Vol. 1861 (1 Pt A), pp. 2934-2941. Date of Electronic Publication: 2016 Sep 23. - Publication Year :
- 2017
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Abstract
- Background: 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.<br />Methods: 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.<br />Results: Using purified Alg1, its biochemical characteristics were investigated, including the apparent K <subscript>m</subscript> and V <subscript>max</subscript> 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.<br />General Significance: This work provides an efficient method for production of Alg1 and a new MS-based quantitative assay of its activity.<br /> (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Blotting, Western
Chromatography, Liquid
Disaccharides chemistry
Disaccharides metabolism
Electrophoresis, Polyacrylamide Gel
Glycosylation
Mannosyltransferases chemistry
Mass Spectrometry
Mutant Proteins chemistry
Mutant Proteins isolation & purification
Mutant Proteins metabolism
Recombinant Proteins biosynthesis
Recombinant Proteins isolation & purification
Mannosyltransferases metabolism
Saccharomyces cerevisiae enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0304-4165
- Volume :
- 1861
- Issue :
- 1 Pt A
- Database :
- MEDLINE
- Journal :
- Biochimica et biophysica acta. General subjects
- Publication Type :
- Academic Journal
- Accession number :
- 27670784
- Full Text :
- https://doi.org/10.1016/j.bbagen.2016.09.023