Characterization of α-mannosidase from Dolichos lablab seeds: Partial amino acid sequencing and N-glycan analysis

α-Mannosidase is a key enzyme in processing and degradation of N-glycans in plants and animals. In the present study α-mannosidase from crude extracts of Dolichos lablab (Indian beans) has been purified by ammonium sulfate precipitation, anion exchange, galactose Sepharose, phenyl Sepharose, gel permeation and Con A Sepharose chromatography. The purified protein migrated as a single band corresponding to 116 kDa on SDS–PAGE under reducing conditions. The pH and temperature optima of α-mannosidase activity determined by use of p-nitrophenyl-α-D-mannopyranoside as substrate were found to be 5.0 and 60–65 °C, respectively. The KM was 1.48 mM and swainsonine was a potent inhibitor of the enzyme with IC50 value 50–80 nM. Additionally, the de novo amino acid sequencing showed active site regions highly conserved among other plant acidic α-mannosidases and yielded sequence coverage of approximately 32.5%. N-glycopeptide analysis revealed the presence of paucimannosidic type structure in a conserved N-glycosylation site as well as at least one oligo mannosidic glycan at an undetermined site after ZIC-HILIC enrichment of proteolytic glycopeptides. The partial biochemical and molecular characterization of this enzyme reveals that it is a class II α-mannosidase from the glycosyl hydrolase family 38.

► We purified α-mannosidase from Dolichos lablab seeds.
► The partial de novo sequencing of peptides revealed the highly conserved regions.
► N-glycosylation analysis showed the presence of paucimannosidic type structure.
► Results suggest the enzyme belongs to class II α-mannosidase in GH family 38.