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

One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine residue to Set 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 a configuration as determined by NMR studies.