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

Research output: Contribution to journalArticle

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 1 2013

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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