BIOSYNTHESIS OF O-N-ACETYLGLUCOSAMINE-LINKED GLYCANS IN TRYPANOSOMA-CRUZI - CHARACTERIZATION OF THE NOVEL URIDINE AMINE-POLYPEPTIDE-N-ACETYLGLUCOSAMINYLTRANSFERASE- CATALYZING FORMATION OF N-ACETYLGLUCOSAMINE ALPHA-1-]O-THREONINE

In this study, we have characterized the activity of a uridine :polype ptide-alpha-N-acetylglucosaminyltransferase (O-alpha-GlcNAc-transferas e) from Trypanosoma cruzi. The activity is present in microsomal membr anes and is responsible for the addition of O-linked alpha-N-acetylglu cosamine to cell surface proteins. This preparation adds N-acetylgluco samine to a synthetic peptide KPPTTTTTTTTKPP containing the consensus threonine-rich dodecapeptide encoded by T. cruzi MUC gene (Di Noia, J. M., Sanchez D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24 146-24149). Incorporation of N-[H-3]acetylglucosamine is linearly depe ndent on incubation time and concentration of enzyme and substrate. Th e transferase activity has an optimal pH of 7.5-8.5, requires Mn2+, is unaffected by tunicamycin or amphomycin, and is strongly inhibited by UDP, The optimized synthetic peptide acceptor for the cytosolic O-Glc NAc-transferase (YSDSPSTST) (Haltiwanger, R. S., Holt, G. D., and Hart , G. W. (1990) J. Biol. Chem. 265, 2563-2568) is not a substrate for t his enzyme. The glycosylated KPPTTTTTTTTKPP product is susceptible to base-catalyzed beta-elimination, and the presence of N-acetylglucosami ne beta-linked to threonine is supported by enzymatic digestion and nu clear magnetic resonance data. These results describe a unique biosynt hetic pathway for T. cruzi surface mucin-like molecules, with potentia l chemotherapeutic implications.