Rules for the addition of O‐linked N ‐acetylglucosamine to secreted proteins in Dictyostelium discoideum

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 (PDT 1 RPAPGS 1 T 2 APPAHGVT 3 S 2 A) and a MUC2‐like peptide (PT 1 T 2 T 3 PIT 4 T 5 T 6 T 7 T 8 T 9 VT 10 PT 11 PT 12 PT 13 GT 14 QT 15 ), 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 T 2 and T 3 and there were no modifications on T 1 or on S 1 and S 2 . The MUC2 glycopeptide was glycosylated on T 1 , T 3 , T 5 , T 7 , T 9 , T 10 , T 11 , T 12 , T 13 and T 14 . 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 α configuration as determined by NMR studies.