Purification and characterization of [beta]-xylosidase that is active for plant complex type N-glycans from tomato (Solanum lycopersicum): removal of core [alpha]1-3 mannosyl residue is prerequisite for hydrolysis of [beta]1-2 xylosyl residue

• Published in: Glycoconjugate journal 2013-07, Vol.30 (5), p.463
• Main Authors: Yokouchi, Daisuke, Ono, Natsuko, Nakamura, Kosuke, Maeda, Megumi, Kimura, Yoshinobu
• Format: Article
• Language: English
• Subjects: Agricultural biotechnology; Enzymes; Plant biology; Tomatoes
• ISSN: 0282-0080; 1573-4986
• DOI: 10.1007/s10719-012-9441-y

In this study, we purified and characterized the [beta]-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified [beta]-xylosidase ([beta]-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 [beta]-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, [beta]-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. [beta]-Xyl'ase Le-1 hydrolyzed the [beta]1-2 xylosyl residue from Man^sub 1^Xyl^sub 1^GlcNAc^sub 2^-PA, Man^sub 1^Xyl^sub 1^Fuc^sub 1^GlcNAc^sub 2^-PA, and MaN2Xyl^sub 1^Fuc^sub 1^GlcNAc^sub 2^-PA, but not that from Man^sub 3^Xyl^sub 1^GlcNAc^sub 2^-PA or Man^sub 3^Xyl^sub 1^Fuc^sub 1^GlcNAc^sub 2^-PA, indicating that the [alpha]1-3 arm mannosyl residue exerts significant steric hindrance for the access of [beta]-Xyl'ase Le-1 to the xylosyl residue, whereas the [alpha]1-3 fucosyl residue exerts little effect. These results suggest that the release of the [beta]1-2 xylosyl residue by [beta]-Xyl'ase Le-1 occurs at least after the removal the [alpha]-1,3-mannosyl residue in the core trimannosyl unit.[PUBLICATION ABSTRACT]