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 dis coideum involves the addition of a single N-acetylglucosamine residue to Ser and Thr residues on secreted or membrane-bound proteins at an e arly 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-a cetylglucosaminyltransferase(s). Glutathione S-transferase fusion prot eins 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 T(7)T(8)T(9)VT(10)PT(11)PT(12)PT(13)GT(14)QT(15)), respectively (supe rscript numbers indicate residues with the potential to be glycosylate d). Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-ace tylglucosamine attached to certain Thr residues. The MUC1 repeat was g lycosylated on T-2 and T-3 and there were no modifications on T-1 or o n 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 in to peptide sequences similar to those reported for the addition of Gal NAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in a configuration a s determined by NMR studies.