Molecular analysis of a UDP-GlcNAc:polypeptide alpha-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi.
Trypanosoma cruzi, the causative agent of Chagas disease, is surrounded by a mucin coat that plays important functions in parasite survival/invasion and is extensively O-glycosylated by Golgi and cell surface glycosyltransferases. The addition of the first sugar, alpha-N-acetylglucosamine (GlcNAc) linked to Threonine (Thr), is catalyzed by a polypeptide alpha-GlcNAc-transferase (pp-alphaGlcNAcT) which is unstable to purification. Here, a comparison of the genomes of T. cruzi and Dictyostelium discoideum, an amoebazoan which also forms this linkage, identified two T. cruzi genes (TcOGNT1 and TcOGNT2) that might encode this activity. Though neither was able to complement the Dictyostelium gene, expression in the trypanosomatid Leishmania tarentolae resulted in elevated levels of UDP-[(3)H]GlcNAc:Thr-peptide GlcNAc-transferase activity and UDP-[(3)H]GlcNAc breakdown activity. The ectodomain of TcOGNT2 was expressed and the secreted protein was found to retain both activities after extensive purification away from other proteins and the endogenous activity. Product analysis showed that (3)H was transferred as GlcNAc to a hydroxyamino acid, and breakdown was due to hydrolysis. Both activities were specific for UDP-GlcNAc relative to UDP-GalNAc and were abolished by active site point mutations that inactivate a related Dictyostelium enzyme and distantly related animal pp-alphaGalNAcTs. The peptide preference and the alkaline pH optimum were indistinguishable from those of the native activity in T. cruzi microsomes. The results suggest that mucin-type O-glycosylation in T. cruzi is initiated by conserved members of CAZy family GT60, which is homologous to the GT27 family of animal pp-alphaGalNAcTs that initiate mucin-type O-glycosylation in animals.
Heise
Norton
N
Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. nheise@biof.ufrj.br
Singh
Divyendu
D
van der Wel
Hanke
H
Sassi
Slim O
SO
Johnson
Jennifer M
JM
Feasley
Christa L
CL
Koeller
Carolina M
CM
Previato
Jose O
JO
Mendonça-Previato
Lucia
L
West
Christopher M
CM
D000818:Animals; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism; D018503:Genome, Protozoan; D006031:Glycosylation; D007891:Leishmania / Q000378:metabolism; D009077:Mucins / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000235:genetics / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism
D009077:Mucins; D015800:Protozoan Proteins; D014530:Uridine Diphosphate; D017351:N-Acetylglucosaminyltransferases