Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-beta-N-acetylglucosaminidase.
We describe a novel method for the enzymatic synthesis of neoglycoproteins. Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) had high levels of transglycosylation activity. The enzyme activity of Endo-A was markedly increased by adding 4-L-aspartyl-glycosylamine (GlcNAc-Asn) to the reaction mixture. Digesting (Man)6(GlcNAc)2 with the enzyme in the presence of GlcNAc-Asn gave a mixture of hydrolytic ((Man)6GlcNAc) and transglycosylic ((Man)6(GlcNAc)2-Asn) products. By means of transglycosylation, (Man)6GlcNAc was transferred en bloc to the partially deglycosylated ovalbumin glycopeptide (EEKYN(GlcNAc)LTSVL) concomitant with the hydrolysis of (Man)6-GlcNAc)2Asn. The structure of the transglycosylation product was designated as (Man)6(GlcNAc)2-peptide by amino acid composition and sequence analysis as well as ion mass spectrometry. The enzyme also transferred oligosaccharide to partially deglycosylated ribonuclease B (GlcNAc-protein) during the hydrolysis of (Man)6-(GlcNAc)2Asn. Native ribonuclease B had (Man)5-9 (GlcNAc)2 as its heterogeneous N-linked sugar chains. High performance liquid chromatography showed that all of the N-linked sugar chains of the synthetic neoribonuclease of the pyridylamino derivatives were modified to (Man)6(GlcNAc)2.
Takegawa
K
K
Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Japan.
Tabuchi
M
M
Yamaguchi
S
S
Kondo
A
A
Kato
I
I
Iwahara
S
S
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006020:Glycopeptides / Q000138:chemical synthesis* / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000302:isolation & purification; D007202:Indicators and Reagents; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D010047:Ovalbumin; D012260:Ribonucleases / Q000138:chemical synthesis / Q000302:isolation & purification
D006020:Glycopeptides; D006023:Glycoproteins; D007202:Indicators and Reagents; D009844:Oligosaccharides; D010047:Ovalbumin; D012260:Ribonucleases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase