Highly efficient transglycosylation of sialo-complex-type oligosaccharide using <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> endoglycosidase and sugar oxazoline

Objectives

To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities.

Results

Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase (Endo-CC^N180H) 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-CC^N180H in 20 μl 20 mM Tris/HCl pH 7.5 at 30 °C for 30–60 min.

Conclusions

This transglycosylation method using SG-oxazoline and Endo-CC^N180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.