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 alpha1-->O-threonine.
In this study, we have characterized the activity of a uridine diphospho-N-acetylglucosamine:polypeptide-alpha-N-acetylglucosaminylt ransferase (O-alpha-GlcNAc-transferase) from Trypanosoma cruzi. The activity is present in microsomal membranes and is responsible for the addition of O-linked alpha-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 beta-elimination, and the presence of N-acetylglucosamine alpha-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.
Previato
J O
JO
Instituto de Microbiologia, CCS-Bloco I, Universidade Federal do Rio de Janeiro, 21944 970 Cidade Universitária, Rio de Janeiro-RJ, Brazil.
Sola-Penna
M
M
Agrellos
O A
OA
Jones
C
C
Oeltmann
T
T
Travassos
L R
LR
Mendonça-Previato
L
L
D000117:Acetylglucosamine / Q000737:chemistry*; D000818:Animals; D002412:Cations / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008861:Microsomes / Q000201:enzymology; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry*; D010455:Peptides / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis*; D015800:Protozoan Proteins / Q000737:chemistry; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014530:Uridine Diphosphate / Q000494:pharmacology
D002412:Cations; D004791:Enzyme Inhibitors; D006020:Glycopeptides; D008562:Membrane Glycoproteins; D010455:Peptides; D011134:Polysaccharides; D015800:Protozoan Proteins; D014530:Uridine Diphosphate; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine