Purification and characterization of β-xylosidase that is active for plant complex type N-glycans from tomato (Solanum lycopersicum)

Removal of core α1-3 mannosyl residue is prerequisite for hydrolysis of β1-2 xylosyl residue

Daisuke Yokouchi, Natsuko Ono, Kosuke Nakamura, Megumi Maeda, Yoshinobu Kimura

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9 Citations (Scopus)

Abstract

In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl'ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl'ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, β-Xyl'ase Le-1 showed optimum activity at about pH 5 at 40 C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl'ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man1Xyl1GlcNAc2-PA, Man1Xyl1Fuc1GlcNAc2-PA, and Man 2Xyl1Fuc1GlcNAc2-PA, but not that from Man3Xyl1GlcNAc2-PA or Man 3Xyl1Fuc1GlcNAc2-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl'ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.

Original languageEnglish
Pages (from-to)463-472
Number of pages10
JournalGlycoconjugate Journal
Volume30
Issue number5
DOIs
Publication statusPublished - Jul 2013

Fingerprint

Xylosidases
Lycopersicon esculentum
Purification
Polysaccharides
Hydrolysis
Chemical modification
Plant Cells
Molecular mass
Vacuoles
Cell Wall
Polyacrylamide Gel Electrophoresis
Cells
Amino Acids
Substrates
Enzymes

Keywords

  • β-xylosidase
  • Fruit ripening
  • Plant N-glycan
  • Solanum lycopersicum

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

@article{cae7905dfb1b4b0bb0f7cac66bcfb38f,
title = "Purification and characterization of β-xylosidase that is active for plant complex type N-glycans from tomato (Solanum lycopersicum): Removal of core α1-3 mannosyl residue is prerequisite for hydrolysis of β1-2 xylosyl residue",
abstract = "In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl'ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl'ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, β-Xyl'ase Le-1 showed optimum activity at about pH 5 at 40 C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl'ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man1Xyl1GlcNAc2-PA, Man1Xyl1Fuc1GlcNAc2-PA, and Man 2Xyl1Fuc1GlcNAc2-PA, but not that from Man3Xyl1GlcNAc2-PA or Man 3Xyl1Fuc1GlcNAc2-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl'ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.",
keywords = "β-xylosidase, Fruit ripening, Plant N-glycan, Solanum lycopersicum",
author = "Daisuke Yokouchi and Natsuko Ono and Kosuke Nakamura and Megumi Maeda and Yoshinobu Kimura",
year = "2013",
month = "7",
doi = "10.1007/s10719-012-9441-y",
language = "English",
volume = "30",
pages = "463--472",
journal = "Glycoconjugate Journal",
issn = "0282-0080",
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number = "5",

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T1 - Purification and characterization of β-xylosidase that is active for plant complex type N-glycans from tomato (Solanum lycopersicum)

T2 - Removal of core α1-3 mannosyl residue is prerequisite for hydrolysis of β1-2 xylosyl residue

AU - Yokouchi, Daisuke

AU - Ono, Natsuko

AU - Nakamura, Kosuke

AU - Maeda, Megumi

AU - Kimura, Yoshinobu

PY - 2013/7

Y1 - 2013/7

N2 - In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl'ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl'ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, β-Xyl'ase Le-1 showed optimum activity at about pH 5 at 40 C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl'ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man1Xyl1GlcNAc2-PA, Man1Xyl1Fuc1GlcNAc2-PA, and Man 2Xyl1Fuc1GlcNAc2-PA, but not that from Man3Xyl1GlcNAc2-PA or Man 3Xyl1Fuc1GlcNAc2-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl'ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.

AB - In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl'ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl'ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, β-Xyl'ase Le-1 showed optimum activity at about pH 5 at 40 C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl'ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man1Xyl1GlcNAc2-PA, Man1Xyl1Fuc1GlcNAc2-PA, and Man 2Xyl1Fuc1GlcNAc2-PA, but not that from Man3Xyl1GlcNAc2-PA or Man 3Xyl1Fuc1GlcNAc2-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl'ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.

KW - β-xylosidase

KW - Fruit ripening

KW - Plant N-glycan

KW - Solanum lycopersicum

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U2 - 10.1007/s10719-012-9441-y

DO - 10.1007/s10719-012-9441-y

M3 - Article

VL - 30

SP - 463

EP - 472

JO - Glycoconjugate Journal

JF - Glycoconjugate Journal

SN - 0282-0080

IS - 5

ER -