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 Man₂Xyl₁Fuc₁GlcNAc₂-PA, but not that from Man₃Xyl₁GlcNAc₂-PA or Man₃Xyl₁Fuc₁GlcNAc₂-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.

The N-Glycan Cluster from Xanthomonas campestris pv. campestris

“…NixE and NixH are assumed to act at a later stage, by removing the ␣-1,3-fucose and the ␤-1,4-mannose, respectively. This sequential mode of action corroborates literature data reporting that, in the context of fruit ripening, the removal of ␣-1,3-mannose from N-glycan is a prerequisite for hydrolysis of the ␤-1,2-xylose by a tomato xylosidase (42). In addition, in the human N-glycan degrading Firmicutes, a sequential model is proposed, with the removal of the ␣-1,3-mannose by a GH38 as a first step necessary for the activity of a GH125 subsequently involved in the removal of the ␣-1,6-mannose (19).…”

Rice α-fucosidase active against plant complex type N-glycans containing Lewis a epitope: purification and characterization

“…7) When the pyridylaminated lacto-N-fucopentaose III (LNFP III, Takara Bio Inc., Japan) was used as a substrate, the products obtained were analyzed with a Cosmosil 5C18-AR column (6.0 × 250 mm) at a flow rate of 1.0 mL/min using two-solvent system (0.02% TFA/water and 20% acetonitrile/water) described in our previous report. 8) Rice α-fucosidase (α-fucosidase Os, α-fucosidase from Oriza sativa) was purified about 100-fold to homogeneity (total units, 0.21 mU; specific activity, 16.9 mU/mg), as shown in Fig. 2-I.…”

Purification and Substrate Specificity of AGinkgo bilobaGlycosidase Active in β-1,2-Xylosidic Linkage in Plant Complex TypeN-Glycans

“…In a previous study, 1) we purified and characterized a tomato -xylosidase (-Xyl'ase Le1) that released a xylosyl residue from truncated type plant N-glycans.…”

“…Through all the purification steps, the activity of -xylosidase was measured using pNP--xylopyranoside (pNP--Xyl) as synthetic substrate and Xyl1-2Man1-4GlcNAc1-4(Fuc1-3)GlcNAc-PA (MFX). 1) The enzyme solution (20 mL) was added to 40 mL of pNP--Xyl (final concentration 5 mM) in 0.1 M Na-acetate buffer (pH 4.0), and the reaction mixture was incubated at 37 C for 3 h. The amount of p-nitrophenol released was quantified by measuring the absorbance at 420 nm. One unit of enzyme activity was defined as the amount of enzyme releasing 1 mmol of p-nitrophenol per min at 37 C. When a fluorescence-labeled oligosaccharide was used as pseudo-natural substrate, the reaction mixture (30 mL), containing about 27 pmol of M3FX in 0.15 M sodium acetate buffer (pH 4.0), was incubated at 37 C for 14 h. The products obtained were analyzed with a Jasco 880-PU HPLC apparatus with a Jasco Intelligent spectrofluorometer (Jasco, Tokyo) and a Cosmosil 5C18-AR column (0:6 Â 25 cm, Nacalai Tesque, Kyoto, Japan).…”

“…1) Ginkgo -xylosidase was purified by a combination of ion-exchange chromatography (Q-Sepharose and Shodex QAE column (Showa Denko, Tokyo)), hydrophobic interaction chromatography (Butyl-Toyopearl and Shodex Phenyl column (Showa Denko, Tokyo, Japan), and gel filtration by the purification procedure used in our previous study. 1) Ginkgo -xylosidase (-Xyl'ase Gb) was purified about 160-fold to homogeneity (total units, 2.2 mU; specific activity, 37.9 mU/mg), as shown in Fig. 1A.…”