title: Rules for the addition of O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum--in vivo studies on glycosylation of mucin MUC1 and MUC2 repeats.
abstract: One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine residue to Ser and Thr residues on secreted or membrane-bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O-glycosylation was used to further characterise the specificity of the UDP-GlcNAc :polypeptide N-acetylglucosaminyltransferase(s). Glutathione S-transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT1RPAPGS1T2APPAHGVT3S2A) and a MUC2-like peptide (PT1T2T3PIT4T5T6T7T8T9VT10PT11PT12PT13GT14QT15), respectively (superscript numbers indicate residues with the potential to be glycosylated). Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-acetylglucosamine attached to certain Thr residues. The MUC1 repeat was glycosylated on T2 and T3 and there were no modifications on T1 or on S1 and S2. The MUC2 glycopeptide was glycosylated on T1, T3, T5, T7, T9, T10, T11, T12, T13 and T14. Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in alpha configuration as determined by NMR studies.
authors.affiliation: Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University, Sydney, NSW, Australia.
authors.forename: E
authors.initials: E
authors.lastname: Jung
authors.forename: A A
authors.initials: AA
authors.lastname: Gooley
authors.forename: N H
authors.initials: NH
authors.lastname: Packer
authors.forename: P
authors.initials: P
authors.lastname: Karuso
authors.forename: K L
authors.initials: KL
authors.lastname: Williams
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry*; D000818:Animals; D004023:Dictyostelium / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D013058:Mass Spectrometry; D009077:Mucins / Q000737:chemistry / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009077:Mucins; D015800:Protozoan Proteins; D000117:Acetylglucosamine