Jack bean  -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).

Mechanistic Insight into the Binding of Multivalent Pyrrolidines to α‐Mannosidases

“…To support our experimental data and to propose a binding mode of the ligands into the active site of the α‐mannosidase, a molecular modeling approach was performed. The X‐ray crystal structure of JBMan is not available yet; nevertheless, the complete primary sequence, recently reported by Kumar et al, was used to check the homology between JBMan and other α‐mannosidases. In particular, the sequence alignment was performed with two α‐mannosidases with two reported X‐ray crystal structures, the lysosomal α‐mannosidase from Bos taurus (bovine, bLAM) and the Golgi α‐mannosidase II from Drosophila melanogaster (fruit fly, GMII) .…”

“…Binding studies with bLAM gave a 93 % identity and 100 % of similarity (see the Supporting Information for the sequence alignment). Moreover, by comparing these three different mannosidase sequences, it is noteworthy that the active site aminoacids, His23, Asp25, Trp28, Tyr35, Asp145 (the catalytic residue), Arg170, Tyr210, Asp268, Phe269, Tyr325, Trp333, His385, His386, Asp387, Thr392, and Tyr625, are highly conserved in all the mannosidases. In addition, by superimposing the crystal structures of GMII and bLAM, the same 3D orientation of the active site residues was maintained (see the Supporting Information).…”

“…To obtain insights into the binding modes that explain the large multivalent effect observed, the JBMan–ligand complexes were also analyzed by transmission electron microscopy with negative staining. JBMan is a high molecular weight metalloenzyme of about 220 KDa, which comprises two pairs of subunits that have molecular weights of 66 and 44 KDa, respectively, and is visible by TEM. For the mannosidase alone, the image clearly showed particles of approximately 10×20 nm (Figure a), which matches the size of one dimer of the JBMan heterodimeric subunits, as previously reported .…”

Luffa acutangulaagglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry

“…The peptides were prepared for de novo sequencing by subjecting the lectin to in‐gel as well as in‐solution proteolytic digestions using trypsin, chymotrypsin, and thermolysin as described in ref. .…”

“…The amino acids isoleucine (I) and leucine (L) are isobaric and are mostly assigned to as I or L based on appropriate database homology comparisons. Similarly, the assignment for glutamine (Q) and lysine (K) were made based on amino acids exhibiting an increment mass of 128 to Q based on appropriate database homology comparisons or K unless proven by a tryptic cleavage site . The protein sequence data reported in the current study will appear in the UniProt Knowledgebase under the accession number C0HJV2.…”