Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline.

ABSTRACT

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-ß-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. CONCLUSIONS: This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.