title: Characterization of α-mannosidase from Dolichos lablab seeds: partial amino acid sequencing and N-glycan analysis.
abstract: 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 IC(50) 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.
authors: Protein Biochemistry and Glycobiology Laboratory, Department of Biochemistry, University of Hyderabad, Hyderabad 500046, India.
authors: B S
authors: BS
authors: Gnanesh Kumar
authors: G
authors: G
authors: Pohlentz
authors: M
authors: M
authors: Mormann
authors: N
authors: N
authors: Siva Kumar
mesh_terms: D031294:Dolichos / Q000201:enzymology*; D006020:Glycopeptides / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D011134:Polysaccharides / Q000737:chemistry*; D012639:Seeds / Q000201:enzymology; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D043323:alpha-Mannosidase / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D006020:Glycopeptides; D011134:Polysaccharides; D043323:alpha-Mannosidase