title: Golgi UDP-GlcNAc:polypeptide O-α-N-Acetyl-d-glucosaminyltransferase 2 (TcOGNT2) regulates trypomastigote production and function in Trypanosoma cruzi.
abstract: All life cycle stages of the protozoan parasite Trypanosoma cruzi are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of αGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-α-N-acetyl-d-glucosaminyltransferases (ppαGlcNAcTs), which are encoded by TcOGNT1 and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppαGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Carolina M
authors.initials: CM
authors.lastname: Koeller
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Hanke
authors.initials: H
authors.lastname: van der Wel
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Christa L
authors.initials: CL
authors.lastname: Feasley
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Abreu
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Juliana Dutra Barbosa
authors.initials: JD
authors.lastname: da Rocha
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Fabrício
authors.initials: F
authors.lastname: Montalvão
authors.affiliation: Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, Seropédica, Rio de Janeiro, Brazil.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Fampa
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Flávia C G
authors.initials: FC
authors.lastname: Dos Reis
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Georgia C
authors.initials: GC
authors.lastname: Atella
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Thaís
authors.initials: T
authors.lastname: Souto-Padrón
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA christopher-west@ouhsc.edu nheise@biof.ufrj.br.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: West
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil christopher-west@ouhsc.edu nheise@biof.ufrj.br.
authors.forename: Norton
authors.initials: N
authors.lastname: Heise
mesh_terms: D000818:Animals; D018507:Gene Expression Regulation, Developmental; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006056:Golgi Apparatus / Q000201:enzymology; D008018:Life Cycle Stages / Q000235:genetics; D009077:Mucins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
chemical_list: D006023:Glycoproteins; D009077:Mucins; D010455:Peptides; D011134:Polysaccharides; D015800:Protozoan Proteins
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