PPGn2-Man

• 分子结构分类: 有机化合物 - 有机氧化合物
• 英文名称: PPGn2-Man
• 英文别名: [(2R,3R,4R,5S,6R)-3-acetamido-5-[(2S,3R,4R,5S,6R)-3-acetamido-4-hydroxy-6-(hydroxymethyl)-5-[(2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)oxan-2-yl] [hydroxy(3,7,11,15-tetramethylhexadecoxy)phosphoryl] hydrogen phosphate
• 化学式: C₄₂H₈₀N₂O₂₂P₂
• 分子量: 1027.04
• InChiKey: AGWSVFLKGIRVRN-XRXKXNHBSA-N

计算性质

• 辛醇/水分配系数(LogP)：
  0.4
• 重原子数：
  68
• 可旋转键数：
  29
• 环数：
  3.0
• sp3杂化的碳原子比例：
  0.95
• 拓扑面积：
  369
• 氢给体数：
  12
• 氢受体数：
  22

反应信息

• 作为反应物：
  描述：

  PPGn2-Man 在 盐酸 、 Helix pomatia β-mannosidase 作用下， 以 甲醇 为溶剂， 反应 17.0h， 生成 N,N'-Diacetylchitobiose
  参考文献：
  名称：

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

  摘要：

  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 beta-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 beta-1, 4 mannosyltransferase activity.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-alpha-N,N'-diacetylchitobioside (PPGn2), was used as the acceptor for the beta-1, 4 mannosyl transfer reaction.Results: Using purified Alg1, its biochemical characteristics were investigated, including the apparent K-m and V-max 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.General significance: This work provides an efficient method for production of Alg1 and a new MS-based quantitative assay of its activity. (C) 2016 Elsevier B.V. All rights reserved.

  DOI：

  10.1016/j.bbagen.2016.09.023
• 作为产物：
  描述：

  phytanyl-pyrophosphoryl-α-N,N′-diacetylchitobioside 、 guanosine 5'-diphospho-D-mannose 在 乙二胺四乙酸 、 dithiothreitol 、 recombinant His6-tagged S. cerevisiae Alg1 Δtransmembrane-spanning domain 、 magnesium chloride 、 NP-40 作用下， 以 aq. buffer 为溶剂， 反应 0.5h， 生成 PPGn2-Man
  参考文献：
  名称：

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

  摘要：

  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 beta-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 beta-1, 4 mannosyltransferase activity.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-alpha-N,N'-diacetylchitobioside (PPGn2), was used as the acceptor for the beta-1, 4 mannosyl transfer reaction.Results: Using purified Alg1, its biochemical characteristics were investigated, including the apparent K-m and V-max 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.General significance: This work provides an efficient method for production of Alg1 and a new MS-based quantitative assay of its activity. (C) 2016 Elsevier B.V. All rights reserved.

  DOI：

  10.1016/j.bbagen.2016.09.023

文献信息

• Efficient Enzymatic Synthesis of the Core Trisaccharide ofN-Glycans with a Recombinantβ-Mannosyltransferase
  作者：Gregory M. Watt、Leigh Revers、Matthew C. Webberley、Iain B. H. Wilson、Sabine L. Flitsch

  DOI：10.1002/anie.199723541

  日期：1997.11.14
• Quantitative study of yeast Alg1 beta-1, 4 mannosyltransferase activity, a key enzyme involved in protein N-glycosylation
  作者：Sheng-Tao Li、Ning Wang、Sha Xu、Jian Yin、Hideki Nakanishi、Neta Dean、Xiao-Dong Gao

  DOI：10.1016/j.bbagen.2016.09.023

  日期：2017.1

  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 beta-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 beta-1, 4 mannosyltransferase activity.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-alpha-N,N'-diacetylchitobioside (PPGn2), was used as the acceptor for the beta-1, 4 mannosyl transfer reaction.Results: Using purified Alg1, its biochemical characteristics were investigated, including the apparent K-m and V-max 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.General significance: This work provides an efficient method for production of Alg1 and a new MS-based quantitative assay of its activity. (C) 2016 Elsevier B.V. All rights reserved.