title: Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline.
abstract: BACKGROUND
  An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-ß-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.
  
  
  METHODS
  We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.
  
  
  RESULTS
  Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.
  
  
  CONCLUSIONS
  Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.
  
  
  CONCLUSIONS
  Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kyoto, Japan. mumekawa@umich.edu
authors.forename: Midori
authors.initials: M
authors.lastname: Umekawa
authors.forename: Takayuki
authors.initials: T
authors.lastname: Higashiyama
authors.forename: Yurie
authors.initials: Y
authors.lastname: Koga
authors.forename: Tomonari
authors.initials: T
authors.lastname: Tanaka
authors.forename: Masato
authors.initials: M
authors.lastname: Noguchi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kobayashi
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Shoda
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
authors.forename: Hisashi
authors.initials: H
authors.lastname: Ashida
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006020:Glycopeptides / Q000378:metabolism; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology* / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009844:Oligosaccharides / Q000378:metabolism*; D010080:Oxazoles / Q000302:isolation & purification / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism*
chemical_list: D006020:Glycopeptides; D009844:Oligosaccharides; D010080:Oxazoles; D012794:Sialic Acids; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase