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Glycobiology Advance Access published online on May 25, 2009
Glycobiology, doi:10.1093/glycob/cwp068
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© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Molecular analysis of a UDP-GlcNAc:polypeptide {alpha}-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi

Norton Heise1,2,4, Divyendu Singh1, Hanke van der Wel1, Slim O. Sassi3, Jennifer M. Johnson1, Christa L. Feasley1, Carolina M. Koeller4, Jose O. Previato4, Lucia Mendonça-Previato4 and Christopher M. West1,2,5

1 Dept. of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
3 Foundation for Applied Molecular Evolution, Gainesville, FL 32601 USA; current address: Dept. of Genetics, Harvard Medical School, Boston, MA 02114, USA
4 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-Bloco G, Cidade Universitária, Rio de Janeiro, RJ 21941–902, Brasil

2 To whom correspondence may be addressed: Norton Heise; Tel.: 55-21-2562-6589; Fax: 55-21-2280-8193; E-mail: nheise{at}biof.ufrj.br; or Chris West; Tel.: 1-405-271-4147; Fax: 1-405-271-3910; Email: Cwest2{at}ouhsc.edu

Received on April 16, 2009; accepted on May 11, 2009

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. Addition of the first sugar, {alpha}-N-acetylglucosamine (GlcNAc) linked to Threonine (Thr), is catalyzed by a polypeptide {alpha}-GlcNAc-transferase (pp-{alpha}GlcNAcT) 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-[3H]GlcNAc:Thr-peptide GlcNAc-transferase activity and UDP-[3H]GlcNAc breakdown activity. The ectodomain of TcOGNT2 was expressed and the secreted protein found to retain both activities after extensive purification away from other proteins and the endogenous activity. Product analysis showed that 3H 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-{alpha}GalNAcTs. The peptide preference and the alkaline pH optimum were indistinguishable from that 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-{alpha}GalNAcTs that initiate mucin-type O-glycosylation in animals.

Key words: polypeptide {alpha}GlcNAc-transferase / GlcNAcT / trypanosomatid / Dictyostelium / Chagas disease

5 Corresponding author: Dept. of Biochemistry & Molecular Biology, 975 NE 10th St., BRC 417, OUHSC, Oklahoma City, OK 73104, USA


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