Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea 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.