Dataset Information

Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei.

ABSTRACT: Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known ?1-4-galactosyltransferase or ?1-2- or ?1-6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian ?1-3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:?3-D-mannoside ?1-2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical "pseudohybrid" glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man3GlcNAc2, but not to triantennary Man5GlcNAc2, which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the ?3-glycosyltransferase family to catalyze ?1-2 linkages.

SUBMITTER: Damerow M

PROVIDER: S-EPMC3979372 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei.

Damerow Manuela M Rodrigues Joao A JA Wu Di D Güther M Lucia S ML Mehlert Angela A Ferguson Michael A J MA

The Journal of biological chemistry 20140218 13

Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known β1-4-galactosyltransferase or β1-2- or β1-6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian β1-3-glycosyltransferase ...[more]

PMID: 24550396

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Project description:Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

Dataset Information

Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei.

Publications

Identification and functional characterization of a highly divergent N-acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei.

Similar Datasets

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