Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline.
Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline.
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DOI：
10.1016/j.bbagen.2010.07.003
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发表时间：
2010-11
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通讯作者：
M. Umekawa;T. Higashiyama;Yurie Koga;Tomonari Tanaka;M. Noguchi;A. Kobayashi;S. Shoda;Wei Huang;Lai-Xi Wang;H. Ashida;Kenji Yamamoto
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作者：
M. Umekawa;T. Higashiyama;Yurie Koga;Tomonari Tanaka;M. Noguchi;A. Kobayashi;S. Shoda;Wei Huang;Lai-Xi Wang;H. Ashida;Kenji Yamamoto
背景一种有效的合成同源糖蛋白的方法对于阐明糖蛋白聚糖的结构和功能作用是必不可少的。我们重点研究了来自Mucor hiemalis的内切-β-N-乙酰氨基葡萄糖苷酶（Endo-M）作为糖缀合物合成工具的转糖基活性，因为它不仅可以将高甘露糖型的寡糖，而且可以将复合型的N-聚糖整体转移到具有N-乙酰氨基葡萄糖残基的各种受体上。然而，其实际应用存在两大瓶颈：转糖基化产物的产率低，且难以获得活化的糖恶唑啉底物，尤其是唾液酸复合物型底物。方法我们利用Endo-M的糖基化酶样N175 Q突变体进行转糖基化，发现其具有增强的以糖恶唑啉作为供体底物的转糖基化活性，结合从蛋黄中的天然唾液酸糖肽容易地制备唾液酸复合物型糖恶唑啉，TSEndo-M-N175 Q显示出向生物活性肽和牛核糖核酸酶B上的唾液酸复合物型糖恶唑啉的有效转糖基化，并且每种唾液酸化的化合物以显著高的产率获得。通过唾液酸复合物型糖唑啉的简单合成和Endo-M-N175 Q催化的转糖基化的组合，能够酶促合成各种唾液酸化的化合物。一般意义我们的方法对于生物学上重要的糖肽和糖蛋白的实际合成是非常有用的。
BACKGROUNDAn efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.METHODSWe carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.RESULTSEndo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.CONCLUSIONSHighly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.GENERAL SIGNIFICANCEOur method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.