Purification and substrate specificity of a Ginkgo biloba glycosidase active in ß-1,2-xylosidic linkage in plant complex type N-glycans.

The ß-xylosidase, which is active against plant complex type N-glycans, was purified to homogeneity from Ginkgo biloba seeds. The N-terminal amino acid sequence, G-S-A-A-G-N-R-, of the Ginkgo ß-xylosidase (ß-Xyl'ase Gb) was consistent with the deduced internal amino acid sequence of an Arabidopsis ß-xylosidase (AtBXL1). ß-Xyl'ase Gb hydrolyzed the ß1-2 xylosyl residue from Xylß1-2Manß1-4GlcNAcß1-4GlcNAc-PA and Xylß1-2Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA, but not that from Manα1-6(Manα1-3)(Xylß1-2)Manß1-4GlcNAcß1-4(Fucα1-3)GlcNAc-PA.

Purification and characterization of ß-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 ß1-2 xylosyl residue.

In this study, we purified and characterized the ß-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified ß-xylosidase (ß-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 ß-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, ß-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. ß-Xyl'ase Le-1 hydrolyzed the ß1-2 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 ß-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 ß1-2 xylosyl residue by ß-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.