天冬酰胺(N)-连接的糖基化开始于逐步合成的dolichol连接的寡糖(DLO)前体,Glc3Man9GlcNAc2-PP-Dol,由一系列内质网膜相关糖基转移酶催化。酵母ALG1(天冬酰胺连接的糖基化1)编码β-1,4甘露糖基转移酶,该酶将第一个甘露糖添加到GlcNAc2-PP-Dol上以产生核心三糖Man1GlcNAc2-PP-Dol。ALG1对酵母活力至关重要,在人类中,ALG1的突变会导致先天性糖基化障碍,称为ALG1-CDG。Alg1由于其表达水平低而难以纯化,因此,没有得到很好的生物化学研究。在这里,我们报道了一种高产率纯化重组Alg1的新方法,和用于精确测量其β-1,4甘露糖基转移酶活性的质谱方法。
N末端截短的酵母His标记的Alg1蛋白在大肠杆菌中表达,并通过HisTrapHP亲和层析纯化。结合LC-MS技术,我们建立了一种新的测定法来准确测量Alg1酶活性。在这个试验中,一种化学合成的多力康联寡糖类似物,植烷基-焦磷酰基-α-N,N'-二乙酰几丁糖苷(PPGn2),用作β-1,4甘露糖基转移反应的受体。
使用纯化的Alg1,研究其生化特性,包括受体的表观Km和Vmax值,最佳的活动条件,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.
这项工作提供了一种生产Alg1的有效方法以及一种新的基于MS的定量测定其活性的方法。
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.