title: Jack bean α-mannosidase: amino acid sequencing and N-glycosylation analysis of a valuable glycomics tool.
abstract: Jack bean (Canavalia ensiformis) seeds contain several biologically important proteins among which α-mannosidase (EC 3.2.1.24) has been purified, its biochemical properties studied and widely used in glycan analysis. In the present study, we have used the purified enzyme and derived its amino acid sequence covering both the known subunits (molecular mass of ∼66,000 and ∼44,000 Da) hitherto not known in its entirety. Peptide de novo sequencing and structural elucidation of N-glycopeptides obtained either directly from proteolytic digestion or after zwitterionic hydrophilic interaction liquid chromatography solid phase extraction-based separation were performed by use of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry and low-energy collision-induced dissociation experiments. De novo sequencing provided new insights into the disulfide linkage organization, intersection of subunits and complete N-glycan structures along with site specificities. The primary sequence suggests that the enzyme belongs to glycosyl hydrolase family 38 and the N-glycan sequence analysis revealed high-mannose oligosaccharides, which were found to be heterogeneous with varying number of hexoses viz, Man8-9GlcNAc2 and Glc1Man9GlcNAc2 in an evolutionarily conserved N-glycosylation site. This site with two proximal cysteines is present in all the acidic α-mannosidases reported so far in eukaryotes. Further, a truncated paucimannose type was identified to be lacking terminal two mannose, Man1(Xyl)GlcNAc2 (Fuc).
authors.affiliation: Protein Biochemistry and Glycobiology Laboratory, Department of Biochemistry, University of Hyderabad, Hyderabad 500046, India.
authors.forename: B S
authors.initials: BS
authors.lastname: Gnanesh Kumar
authors.forename: Gottfried
authors.initials: G
authors.lastname: Pohlentz
authors.forename: Mona
authors.initials: M
authors.lastname: Schulte
authors.forename: Michael
authors.initials: M
authors.lastname: Mormann
authors.forename: Nadimpalli
authors.initials: N
authors.lastname: Siva Kumar
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D032449:Canavalia / Q000201:enzymology*; D054794:Glycomics / Q000379:methods; D006031:Glycosylation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D043323:alpha-Mannosidase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D043323:alpha-Mannosidase
keywords: N-glycans; de novo sequencing; jack bean; mass spectrometry; α-mannosidase