Identification and Functional Characterization of a Highly Divergent N-Acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei

Background:Trypanosoma brucei expresses a highly glycosylated surface coat that is essential for parasite survival. Results: The T. brucei gene TbGT11 encodes an N-acetylglucosaminyltransferase I, the key enzyme for initiating the biosynthesis of complex N-glycans. Conclusion:T. brucei GnTI is not a homologue of metazoan GnTI, but a highly divergent enzyme belonging to the 3-glycosyltransferase family. Significance: Deeper understanding of T. brucei N-glycosylation pathway. 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 Man(3)GlcNAc(2), but not to triantennary Man(5)GlcNAc(2), 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.