TY - JOUR
T1 - 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
AU - Umekawa, Midori
AU - Higashiyama, Takayuki
AU - Koga, Yurie
AU - Tanaka, Tomonari
AU - Noguchi, Masato
AU - Kobayashi, Atsushi
AU - Shoda, Shin ichiro
AU - Huang, Wei
AU - Wang, Lai Xi
AU - Ashida, Hisashi
AU - Yamamoto, Kenji
N1 - Funding Information:
We thank Mr. Jun Watanabe of Burker Daltonics for analyzing ESI-TOF MS of sialo-complex-type glycoform of RNaseB. This work was partially supported by the Japan Society for the Promotion of Science (Grant-in-Aid for JSPS Research Fellowships for Young Scientists no. 214774 to MU and for Scientific Research (B) no. 20380052 to KY) and JST-CREST.
PY - 2010/11
Y1 - 2010/11
N2 - 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-β- 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. General significance: Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.
AB - 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-β- 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. General significance: Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.
KW - Chemo-enzymatic synthesis of glycoprotein
KW - Glycosynthase-like mutant
KW - Sialo-complex-type glycan
KW - Sugar oxazoline
KW - Transglycosylation
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U2 - 10.1016/j.bbagen.2010.07.003
DO - 10.1016/j.bbagen.2010.07.003
M3 - Article
C2 - 20647032
AN - SCOPUS:77956267399
VL - 1800
SP - 1203
EP - 1209
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0006-3002
IS - 11
ER -