Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, United States.
Biochim Biophys Acta Gen Subj. 2017 Jan;1861(1 Pt A):2934-2941. doi: 10.1016/j.bbagen.2016.09.023. Epub 2016 Sep 23.
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 K and V 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.
天冬酰胺（N）-连接糖基化始于多萜醇连接寡糖（DLO）前体的逐步合成，该前体由一系列内质网膜结合糖基转移酶催化。酵母 ALG1（天冬酰胺连接糖基化 1）编码一种β-1,4 甘露糖基转移酶，它将第一个甘露糖添加到 GlcNAc2-PP-Dol 上，生成核心三糖 Man1GlcNAc2-PP-Dol。ALG1 对酵母的生存至关重要，而人类中 ALG1 中的突变会导致先天性糖基化障碍，称为 ALG1-CDG。由于其低表达水平，Alg1 难以纯化，因此其生化特性尚未得到很好的研究。在这里，我们报告了一种新的方法，可以高效地纯化重组 Alg1，并采用质谱方法准确测量其β-1,4 甘露糖基转移酶活性。
在大肠杆菌中表达 N 端截断的酵母 His 标记 Alg1 蛋白，并通过 HisTrap HP 亲和层析进行纯化。结合 LC-MS 技术，我们建立了一种新的测定法，可准确测量 Alg1 酶活性。在该测定法中，使用化学合成的多萜醇连接寡糖类似物，植烷基焦磷酸-α-N,N'-二乙酰壳二糖苷（PPGn2）作为β-1,4 甘露糖基转移反应的受体。
使用纯化的 Alg1，研究了其生化特性，包括对受体的表观 K 和 V 值、最佳活性条件以及其核苷酸糖供体的特异性。此外，还测量了 ALG1-CDG 突变对酶活性的影响。
这项工作提供了生产 Alg1 的有效方法和基于 MS 的其活性定量测定法。
