title: Site specific N-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-binding bark Sambucus nigra agglutinin using LC-MSnrevealed differential glycosylation.
abstract: The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(α2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(α2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MSnmode. MS2of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS3of Y1/Y1αions (peptide + HexNAc)+nof corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, 560012, India.
authors.forename: B S
authors.initials: BS
authors.lastname: Gnanesh Kumar
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, 560012, India. surolia@mbu.iisc.ernet.in.
authors.forename: Avadhesha
authors.initials: A
authors.lastname: Surolia
mesh_terms: D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D009844:Oligosaccharides / Q000737:chemistry*; D037121:Plant Lectins / Q000737:chemistry*; D054788:Ribosome Inactivating Proteins / Q000737:chemistry*; D027924:Sambucus nigra / Q000737:chemistry*
chemical_list: D006020:Glycopeptides; D009844:Oligosaccharides; D037121:Plant Lectins; C050600:Sambucus nigra lectins; D054788:Ribosome Inactivating Proteins
keywords: Complex; High mannose; N-glycans; Paucimannose; Site specificity; Type II RIPs