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.

• Umekawa, Midori

• Higashiyama, Takayuki

• Koga, Yurie

• Tanaka, Tomonari

• Noguchi, Masato

• Kobayashi, Atsushi

• Shoda, Shin-Ichiro

• Huang, Wei

• Wang, Lai-Xi

• Ashida, Hisashi

• Yamamoto, Kenji

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. GENERAL SIGNIFICANCE: Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.

Journal

• Biochimica et biophysica acta

  Biochimica et biophysica acta 1800 (11), 1203-1209, 2010-11

Keywords

• Chemo-enzymatic synthesis of glycoprotein
• Glycosynthase-like mutant
• Sialo-complex-type glycan
• Sugar oxazoline
• Transglycosylation
• Carbohydrate Conformation
• Carbohydrate Sequence
• Glycopeptides/metabolism
• Glycosylation
• Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/genetics
• Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase/metabolism
• Molecular Sequence Data
• Mucor/enzymology
• Mucor/genetics
• Mutation/genetics
• Oligosaccharides/metabolism
• Oxazoles/isolation & purification
• Oxazoles/metabolism
• Sialic Acids/metabolism

Details

• CRID: 1050282676917102592
• NII Article ID: 120002647341
• NII Book ID: AA00564635
• ISSN: 00063002
• HANDLE: 2433/131837
• Text Lang: en
• Article Type: journal article
• Data Source:
  • IRDB
  • CiNii Articles