Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-β-N-acetylglucosaminidase

Kaoru Takegawa, M. Tabuchi, S. Yamaguchi, A. Kondo, I. Kato, S. Iwahara

Research output: Contribution to journalArticle

108 Citations (Scopus)

Abstract

We describe a novel method for the enzymatic synthesis of neoglycoproteins. Endo-β-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)6(GlcNAc) and transglycosylic ((Man)6(GlcNAc)2-Ash) 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.

Original languageEnglish
Pages (from-to)3094-3099
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number7
DOIs
Publication statusPublished - Jan 1 1995

Fingerprint

Acetylglucosaminidase
Oligosaccharides
Glycoproteins
Ashes
Sugars
Hydrolysis
Enzymes
Arthrobacter
Glycopeptides
Protein Sequence Analysis
Ovalbumin
High performance liquid chromatography
Enzyme activity
Mass spectrometry
Mass Spectrometry
High Pressure Liquid Chromatography
Ions
Derivatives
Amino Acids
Peptides

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-β-N-acetylglucosaminidase. / Takegawa, Kaoru; Tabuchi, M.; Yamaguchi, S.; Kondo, A.; Kato, I.; Iwahara, S.

In: Journal of Biological Chemistry, Vol. 270, No. 7, 01.01.1995, p. 3094-3099.

Research output: Contribution to journalArticle

Takegawa, Kaoru ; Tabuchi, M. ; Yamaguchi, S. ; Kondo, A. ; Kato, I. ; Iwahara, S. / Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-β-N-acetylglucosaminidase. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 7. pp. 3094-3099.
@article{0d2e77b9ae4b4486a5d409eee343432a,
title = "Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-β-N-acetylglucosaminidase",
abstract = "We describe a novel method for the enzymatic synthesis of neoglycoproteins. Endo-β-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)6(GlcNAc) and transglycosylic ((Man)6(GlcNAc)2-Ash) 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.",
author = "Kaoru Takegawa and M. Tabuchi and S. Yamaguchi and A. Kondo and I. Kato and S. Iwahara",
year = "1995",
month = "1",
day = "1",
doi = "10.1074/jbc.270.7.3094",
language = "English",
volume = "270",
pages = "3094--3099",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "7",

}

TY - JOUR

T1 - Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-β-N-acetylglucosaminidase

AU - Takegawa, Kaoru

AU - Tabuchi, M.

AU - Yamaguchi, S.

AU - Kondo, A.

AU - Kato, I.

AU - Iwahara, S.

PY - 1995/1/1

Y1 - 1995/1/1

N2 - We describe a novel method for the enzymatic synthesis of neoglycoproteins. Endo-β-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)6(GlcNAc) and transglycosylic ((Man)6(GlcNAc)2-Ash) 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.

AB - We describe a novel method for the enzymatic synthesis of neoglycoproteins. Endo-β-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)6(GlcNAc) and transglycosylic ((Man)6(GlcNAc)2-Ash) 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.

UR - http://www.scopus.com/inward/record.url?scp=0028843229&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028843229&partnerID=8YFLogxK

U2 - 10.1074/jbc.270.7.3094

DO - 10.1074/jbc.270.7.3094

M3 - Article

C2 - 7852391

AN - SCOPUS:0028843229

VL - 270

SP - 3094

EP - 3099

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 7

ER -