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.
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.
Umekawa
Midori
M
Graduate School of Biostudies, Kyoto University, Kyoto, Japan. mumekawa@umich.edu
Higashiyama
Takayuki
T
Koga
Yurie
Y
Tanaka
Tomonari
T
Noguchi
Masato
M
Kobayashi
Atsushi
A
Shoda
Shin-Ichiro
S
Huang
Wei
W
Wang
Lai-Xi
LX
Ashida
Hisashi
H
Yamamoto
Kenji
K
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*
D006020:Glycopeptides; D009844:Oligosaccharides; D010080:Oxazoles; D012794:Sialic Acids; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase