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

• Published in: Glycoconjugate journal 2013-07, Vol.30 (5), p.463-472
• Main Authors: Yokouchi, Daisuke, Ono, Natsuko, Nakamura, Kosuke, Maeda, Megumi, Kimura, Yoshinobu
• Format: Article
• Language: English
• Subjects: Biochemistry; Biomedical and Life Sciences; Carbohydrate Sequence; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Fruit - chemistry; Fruit - enzymology; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Life Sciences; Lycopersicon esculentum - chemistry; Lycopersicon esculentum - enzymology; Molecular Sequence Data; Molecular Weight; Pathology; Polysaccharides - chemistry; Substrate Specificity; Temperature; Xylosidases - chemistry; Xylosidases - isolation & purification
• ISSN: 0282-0080; 1573-4986
• DOI: 10.1007/s10719-012-9441-y

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 Man 1 Xyl 1 GlcNAc 2 -PA, Man 1 Xyl 1 Fuc 1 GlcNAc 2 -PA, and Man 2 Xyl 1 Fuc 1 GlcNAc 2 -PA, but not that from Man 3 Xyl 1 GlcNAc 2 -PA or Man 3 Xyl 1 Fuc 1 GlcNAc 2 -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.