Yujiro Higuchi1, Yasunari Eshima1, Yibo Huang1, Takashi Kinoshita2, Wataru Sumiyoshi2, Shin-Ichi Nakakita3, Kaoru Takegawa4. 1. Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan. 2. Fushimi Pharmaceutical Co. Ltd., Marugame, Kagawa, 763-8605, Japan. 3. Department of Functional Glycomics, Life Science Research Center, Kagawa University, Miki-Cho, Kagawa, 761-0793, Japan. 4. Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan. takegawa@agr.kyushu-u.ac.jp.
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-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. CONCLUSIONS: This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.
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-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.
Authors: Thomas C Donahue; Guanghui Zong; Nicholas A O'Brien; Chong Ou; Jeffrey C Gildersleeve; Lai-Xi Wang Journal: Bioconjug Chem Date: 2022-06-10 Impact factor: 6.069