α-Mannosidase involved in turnover of plant complex type N-glycans in tomato (Lycopersicum esculentum) fruits

In this study, we purified and characterized an α-mannosidase to homogeneity from mature red tomato fruits. Purified α-mannosidase (α-Man LE-1) gave two separate bands, of molecular masses of 70 kDa (L-subunit) and 47 kDa (S-subunit), on SDS-PAGE under non-reducing and reducing conditions. On the other hand, the molecular weight was estimated to be 230 kDa by gel filtration, indicating that α-Man LE-1 functions in a tetrameric structure in plant cells. The N-terminal sequence of the L-subunit and the S-subunit were determined to be L-Y-M-V-Y-M-T-K-Q-G- and X-X-L-E-Q/K-S-F-S-Y-Y respectively. When pyridylaminated N-glycans were used as substrates, α-Man LE-1 showed optimum activity at about pH 6 and at 40°C, and the activity was completely inhibited by both swainsonine and 1-deoxy- mannojirimycin. α-Man LE-1 hydrolyzed the α-mannosidic linkages from both high-mannose type and plant complex type N-glycan, but preferred a truncated plant complex type structure to high-mannose type N-glycans bearing α1-2 mannosyl residues.