Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline
To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities.
Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase (Endo-CCN180H) were employed. pH 7.5 was ideal for both SG-oxazoline’s stability and Endo-CC’s transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 μg GlcNAc-RNase B, 200 μg SG-oxazoline and 3 μg Endo-CCN180H in 20 μl 20 mM Tris/HCl pH 7.5 at 30 °C for 30–60 min.
This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.
KeywordsEndo-β-N-Acetylglucosaminidase Complex-type oligosaccharide Coprinopsis N-Linked glycan Sialylglyco-oxazoline Sugar oxazoline Transglycosylation
We would like to thank Dr. Jun Hirabayashi (AIST Japan) and Masahito Dozaki (Fushimi Pharm. Co. Ltd) for numerous discussions. This study was supported in part by JSPS KAKENHI Grant Number 26292054 and Mizutani Foundation for Glycoscience Grant Number 160190 (K.T.).
Supplementary Fig. 1—Transglycosylation of deglycosylated RNase B under several reaction conditions.Supplementary Fig. 2—Images of original SDS-PAGE gels used in this study to be used in conjunction with Figs. 2, 3 and 4 and also Supplementary Fig. 1.
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