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

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

研究成果: ジャーナルへの寄稿記事

108 引用 (Scopus)

抄録

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.

元の言語英語
ページ(範囲)3094-3099
ページ数6
ジャーナルJournal of Biological Chemistry
270
発行部数7
DOI
出版物ステータス出版済み - 1 1 1995
外部発表Yes

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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

これを引用

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

:: Journal of Biological Chemistry, 巻 270, 番号 7, 01.01.1995, p. 3094-3099.

研究成果: ジャーナルへの寄稿記事

Takegawa, K. ; Tabuchi, M. ; Yamaguchi, S. ; Kondo, A. ; Kato, I. ; Iwahara, S. / Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-β-N-acetylglucosaminidase. :: Journal of Biological Chemistry. 1995 ; 巻 270, 番号 7. pp. 3094-3099.
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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.",
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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.

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