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J Biol Chem, Vol. 273, Issue 24, 14982-14988, June 12, 1998

Biosynthesis of O-N-Acetylglucosamine-linked Glycans in Trypanosoma cruzi
CHARACTERIZATION OF THE NOVEL URIDINE DIPHOSPHO-N-ACETYLGLUCOSAMINE:POLYPEPTIDE N-ACETYLGLUCOSAMINYLTRANSFERASE-CATALYZING FORMATION OF N-ACETYLGLUCOSAMINE 1O-THREONINE

Jose O. Previato, Mauro Sola-Penna^§, Orlando A. Agrellos, Christopher Jones^¶, Thomas Oeltmann, Luiz R. Travassos^**, and Lucia Mendonça-Previato

From the  Instituto de Microbiologia and ^§ Faculdade de Farmácia, CCS-Bloco I, Universidade Federal do Rio de Janeiro, 21944 970 Cidade Universitária, Rio de Janeiro-RJ, Brazil, ^¶ Laboratory for Molecular Structure, National Institute for Biological Standard and Control, Potters Bar, Herts EN6 3QG, United Kingdom,  Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, and ^** Disciplina de Biologia Celular, Universidade Federal de São Paulo, 04023 062, São Paulo-SP, Brazil

In this study, we have characterized the activity of a uridine diphospho-N-acetylglucosamine:polypeptide--N-acetylglucosaminyltransferase (O--GlcNAc-transferase) from Trypanosoma cruzi. The activity is present in microsomal membranes and is responsible for the addition of O-linked -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., Sánchez D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146-24149). Incorporation of N-[³H]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 Mn²⁺, is unaffected by tunicamycin or amphomycin, and is strongly inhibited 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 -elimination, and the presence of N-acetylglucosamine -linked to threonine is supported by enzymatic digestion and nuclear magnetic resonance data. These results describe a unique biosynthetic pathway for T. cruzi surface mucin-like molecules, with potential chemotherapeutic implications.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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