Transfer of Man 9 GlcNAc to L-fucose by endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae

Jian Qiang Fan, Long H. Huynh, Bruce B. Reinhold, Vernon N. Reinhold, Kaoru Takegawa, Shojiro Iwahara, Akihiro Kondo, Ikunoshin Kato, Yuan C. Lee

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

We have reported that transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995) J Biol Chem 270: 17723-29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man 9 GlcNAc, to L-Fuc using Man 9 GlcNAc 2 Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2 M L-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence of L-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man 9 GlcNAc-L-Fuc-PA, revealed that a β-anomeric configuration linkage was formed between GlcNAc and L-Fuc. The GlcNAc was found to be 1,2-linked to L-Fuc by two methods: i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man 9 GlcNAc-L-Fuc; and ii) preparation of Man 9 GlcNAc-L-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man 9 GlcNAcβ(1,2)-L-Fuc. Methyl β-L-fucopyranoside, L-Gal are also accepters for the enzymic transglycosylation. However, transglycosylation failed when methyl α-L-fucopyranoside, D-Fuc and D-Gal were used These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a 4 C 1 -conformation or equivalent conformation of the acceptor to perform transglycosylation.

Original languageEnglish
Pages (from-to)643-652
Number of pages10
JournalGlycoconjugate Journal
Volume13
Issue number4
DOIs
Publication statusPublished - Jan 1 1996
Externally publishedYes

Fingerprint

Arthrobacter
Acetylglucosaminidase
Fucose
Acetone
Oligosaccharides
Oxidation-Reduction
Mass Spectrometry
High Pressure Liquid Chromatography
Mass spectrometry
Conformations
Glycoside Hydrolases
High performance liquid chromatography
Digestion
Hydrolysis
Carbon
Sugars
Fans
endo-alpha-sialidase
mannosyl(9)-N-acetylglucosamine
Decomposition

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Transfer of Man 9 GlcNAc to L-fucose by endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae . / Fan, Jian Qiang; Huynh, Long H.; Reinhold, Bruce B.; Reinhold, Vernon N.; Takegawa, Kaoru; Iwahara, Shojiro; Kondo, Akihiro; Kato, Ikunoshin; Lee, Yuan C.

In: Glycoconjugate Journal, Vol. 13, No. 4, 01.01.1996, p. 643-652.

Research output: Contribution to journalArticle

Fan, JQ, Huynh, LH, Reinhold, BB, Reinhold, VN, Takegawa, K, Iwahara, S, Kondo, A, Kato, I & Lee, YC 1996, ' Transfer of Man 9 GlcNAc to L-fucose by endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae ', Glycoconjugate Journal, vol. 13, no. 4, pp. 643-652. https://doi.org/10.1007/BF00731453
Fan, Jian Qiang ; Huynh, Long H. ; Reinhold, Bruce B. ; Reinhold, Vernon N. ; Takegawa, Kaoru ; Iwahara, Shojiro ; Kondo, Akihiro ; Kato, Ikunoshin ; Lee, Yuan C. / Transfer of Man 9 GlcNAc to L-fucose by endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae In: Glycoconjugate Journal. 1996 ; Vol. 13, No. 4. pp. 643-652.
@article{21535663522a460b8d5d09dd0dbff473,
title = "Transfer of Man 9 GlcNAc to L-fucose by endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae",
abstract = "We have reported that transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30{\%} acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995) J Biol Chem 270: 17723-29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man 9 GlcNAc, to L-Fuc using Man 9 GlcNAc 2 Asn as donor substrate in a medium containing 35{\%} acetone. The transglycosylation yield was greater than 25{\%} when 0.2 M L-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence of L-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man 9 GlcNAc-L-Fuc-PA, revealed that a β-anomeric configuration linkage was formed between GlcNAc and L-Fuc. The GlcNAc was found to be 1,2-linked to L-Fuc by two methods: i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man 9 GlcNAc-L-Fuc; and ii) preparation of Man 9 GlcNAc-L-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man 9 GlcNAcβ(1,2)-L-Fuc. Methyl β-L-fucopyranoside, L-Gal are also accepters for the enzymic transglycosylation. However, transglycosylation failed when methyl α-L-fucopyranoside, D-Fuc and D-Gal were used These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a 4 C 1 -conformation or equivalent conformation of the acceptor to perform transglycosylation.",
author = "Fan, {Jian Qiang} and Huynh, {Long H.} and Reinhold, {Bruce B.} and Reinhold, {Vernon N.} and Kaoru Takegawa and Shojiro Iwahara and Akihiro Kondo and Ikunoshin Kato and Lee, {Yuan C.}",
year = "1996",
month = "1",
day = "1",
doi = "10.1007/BF00731453",
language = "English",
volume = "13",
pages = "643--652",
journal = "Glycoconjugate Journal",
issn = "0282-0080",
publisher = "Springer Netherlands",
number = "4",

}

TY - JOUR

T1 - Transfer of Man 9 GlcNAc to L-fucose by endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae

AU - Fan, Jian Qiang

AU - Huynh, Long H.

AU - Reinhold, Bruce B.

AU - Reinhold, Vernon N.

AU - Takegawa, Kaoru

AU - Iwahara, Shojiro

AU - Kondo, Akihiro

AU - Kato, Ikunoshin

AU - Lee, Yuan C.

PY - 1996/1/1

Y1 - 1996/1/1

N2 - We have reported that transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995) J Biol Chem 270: 17723-29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man 9 GlcNAc, to L-Fuc using Man 9 GlcNAc 2 Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2 M L-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence of L-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man 9 GlcNAc-L-Fuc-PA, revealed that a β-anomeric configuration linkage was formed between GlcNAc and L-Fuc. The GlcNAc was found to be 1,2-linked to L-Fuc by two methods: i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man 9 GlcNAc-L-Fuc; and ii) preparation of Man 9 GlcNAc-L-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man 9 GlcNAcβ(1,2)-L-Fuc. Methyl β-L-fucopyranoside, L-Gal are also accepters for the enzymic transglycosylation. However, transglycosylation failed when methyl α-L-fucopyranoside, D-Fuc and D-Gal were used These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a 4 C 1 -conformation or equivalent conformation of the acceptor to perform transglycosylation.

AB - We have reported that transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995) J Biol Chem 270: 17723-29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man 9 GlcNAc, to L-Fuc using Man 9 GlcNAc 2 Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2 M L-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence of L-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man 9 GlcNAc-L-Fuc-PA, revealed that a β-anomeric configuration linkage was formed between GlcNAc and L-Fuc. The GlcNAc was found to be 1,2-linked to L-Fuc by two methods: i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man 9 GlcNAc-L-Fuc; and ii) preparation of Man 9 GlcNAc-L-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man 9 GlcNAcβ(1,2)-L-Fuc. Methyl β-L-fucopyranoside, L-Gal are also accepters for the enzymic transglycosylation. However, transglycosylation failed when methyl α-L-fucopyranoside, D-Fuc and D-Gal were used These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a 4 C 1 -conformation or equivalent conformation of the acceptor to perform transglycosylation.

UR - http://www.scopus.com/inward/record.url?scp=0029658569&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029658569&partnerID=8YFLogxK

U2 - 10.1007/BF00731453

DO - 10.1007/BF00731453

M3 - Article

C2 - 8872122

AN - SCOPUS:0029658569

VL - 13

SP - 643

EP - 652

JO - Glycoconjugate Journal

JF - Glycoconjugate Journal

SN - 0282-0080

IS - 4

ER -