title: Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline.
abstract: OBJECTIVE
  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-CCN180Hin 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-CCN180His beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.
authors.forename: Yujiro
authors.initials: Y
authors.lastname: Higuchi
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.
authors.forename: Yasunari
authors.initials: Y
authors.lastname: Eshima
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan.
authors.forename: Yibo
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Fushimi Pharmaceutical Co. Ltd., Marugame, Kagawa, 763-8605, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kinoshita
authors.affiliation: Fushimi Pharmaceutical Co. Ltd., Marugame, Kagawa, 763-8605, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Sumiyoshi
authors.affiliation: Department of Functional Glycomics, Life Science Research Center, Kagawa University, Miki-Cho, Kagawa, 761-0793, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Nakakita
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan. takegawa@agr.kyushu-u.ac.jp.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takegawa
mesh_terms: D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
keywords: Complex-type oligosaccharide; Coprinopsis; Endo-β-N-Acetylglucosaminidase; N-Linked glycan; Sialylglyco-oxazoline; Sugar oxazoline; Transglycosylation