Biosynthesis of O-N-Acetylglucosamine-linked Glycans in Trypanosoma cruzi

In this study, we have characterized the activity of a uridine diphospho-N-acetylglucosamine:polypeptide-a- N-acetylglucosaminyltransferase (O-a-GlcNAc-transfer- ase) from Trypanosoma cruzi. The activity is present in microsomal membranes and is responsible for the addi- tion of O-linked a-N-acetylglucosamine to cell surface proteins. This preparation adds N-acetylglucosamine 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, 24146 -24149). Incorporation of N-( 3 H)acetylglucosamine is linearly dependent on incubation time and concentration of enzyme and substrate. The transferase activity has an optimal pH of 7.5- 8.5, requires Mn 21 , is unaffected by tunicamycin or amphomycin, and is strongly inhib- ited by UDP. The optimized synthetic peptide acceptor for the cytosolic O-GlcNAc-transferase (YSDSPSTST) (Haltiwanger, R. S., Holt, G. D., and Hart, G. W. (1990) J. Biol. Chem. 265, 2563-2568) is not a substrate for this enzyme. The glycosylated KPPTTTTTTTTKPP product is susceptible to base-catalyzed b-elimination, and the presence of N-acetylglucosamine a-linked to thre- onine is supported by enzymatic digestion and nuclear magnetic resonance data. These results describe a unique biosynthetic pathway for T. cruzi surface mu- cin-like molecules, with potential chemotherapeutic implications.