title: N-Glycosylation analysis of yeast Carboxypeptidase Y reveals the ultimate removal of phosphate from glycans at Asn368.
abstract: Carboxypeptidase Y from Saccharomyces cerivisiae was characterized for its site specific N-glycosylation through mass spectrometry. The N-glycopeptides were derived using non specific proteases and are analysed directly on liquid chromatography coupled to ion trap mass spectrometer in tandem mode. The evaluation of glycan fragment ions and the Y1ions (peptide+HexNAc)+nrevealed the glycan sequence and the corresponding site of attachment. We observed the microheterogeneity in N-glycans such as Man11-15GlcNAc2at Asn13, Man8-12GlcNAc2at Asn87, Man9-14GlcNAc2at Asn168and phosphorylated Man12-17GlcNAc2as well as Man11-16GlcNAc2at Asn368. The presence of N-glycans with Man<18GlcNAc2indicated that in vacuoles the steady release of mannose/phospho mannose residues from glycans occurs initially at Asn13or Asn168followed by at Asn368. However, glycans at Asn87which comprises Man8-12residues as reported earlier remain intact suggesting its inaccessibility for a similar processing. This in turn indicates the interaction of the glycan at Asn87with the polypeptide chain implicating it in the folding of the protein.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Gnanesh Kumar
authors.initials: GK
authors.lastname: B S
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India. Electronic address: surolia@mbu.iisc.ernet.in.
authors.forename: Avadhesha
authors.initials: A
authors.lastname: Surolia
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001216:Asparagine / Q000737:chemistry; D043402:Cathepsin A / Q000737:chemistry* / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011134:Polysaccharides / Q000737:chemistry*; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D006020:Glycopeptides; D010710:Phosphates; D011134:Polysaccharides; D001216:Asparagine; D043402:Cathepsin A
keywords: Carboxypeptidase Y; LC–MS/MS; Phosphorylated N-glycans