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A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.

The protozoan parasite Trypanosoma brucei causes human African sleeping sickness in sub-Saharan Africa. The parasite makes several essential glycoproteins, which has led to the investigation of the sugar nucleotides and glycosyltransferases required to synthesize these structures. Fucose is a common sugar in glycoconjugates from many organisms; however, the sugar nucleotide donor GDP-fucose was only recently detected in T. brucei, and the importance of fucose metabolism in this organism is not known. In this paper, we identified the genes encoding functional GDP-fucose biosynthesis enzymes in T. brucei and created conditional null mutants of TbGMD, the gene encoding the first enzyme in the pathway from GDP-mannose to GDP-fucose, in both bloodstream form and procyclic form parasites. Under nonpermissive conditions, both life cycle forms of the parasite became depleted in GDP-fucose and suffered growth arrest, demonstrating that fucose metabolism is essential to both life cycle stages. In procyclic form parasites, flagellar detachment from the cell body was also observed under nonpermissive conditions, suggesting that fucose plays a significant role in flagellar adhesion. Fluorescence microscopy of epitope-tagged TbGMD revealed that this enzyme is localized in glycosomes, despite the absence of PTS-1 or PTS-2 target sequences.

The cell surface of the epimastigote form of Trypanosoma cruzi is covered by glycoconjugates rich in galactose. The parasite cannot take up galactose through its hexose transporter, suggesting that the epimerisation of UDP-glucose to UDP-galactose may be the parasite's only route to this sugar. The T. cruzi UDP-glucose 4'-epimerase is encoded by the TcGALE gene. We were unable to make a CL-Brener strain T. cruzi epimastigote TcGALE-/- null mutant, suggesting that the gene is essential. Two TcGALE+/- single-allele knockout clones displayed aberrant morphology and haploid deficiency with respect to galactose metabolism. The morphological phenotypes included shortened flagella, increased incidence of spheromastigotes, agglutination and a novel walnut-like appearance. The reduced supply of UDP-galactose was manifest in the two clones as a six- or nine-fold reduction in the expression of galactopyranose-containing cell surface mucins and negligible or two-fold reduction in the expression of galactofuranose-containing glycoinositolphospholipids. The major loss of mucins as opposed to glycoinositolphospholipids may indicate that the latter are more important for basic parasite survival in culture. The apparent haploid deficiency suggests that epimerase levels are close to limiting, at least in the epimastigote form, and might be exploited as a potential drug target.

Glycosylation is essential for growth factor signaling through N-glycosylation of ligands and receptors and the biosynthesis of proteoglycans as co-receptors. Here, we show that protein O-GlcNAcylation is crucial for fibroblast growth factor (FGF) signaling in Drosophila . We found that nesthocker ( nst ) encodes a phosphoacetylglucosamine mutase and that nst mutant embryos exhibited low amounts of intracellular uridine 5′-diphosphate– N -acetylglucosamine (UDP-GlcNAc), which disrupted protein O-GlcNAcylation. Nst was required for mitogen-activated protein kinase (MAPK) signaling downstream of FGF but not MAPK signaling activated by epidermal growth factor. nst was dispensable for the function of the FGF ligands and the FGF receptor’s extracellular domain but was essential in the signal-receiving cells downstream of the FGF receptor. We identified the adaptor protein Downstream of FGF receptor (Dof), which interacts with the FGF receptor, as the relevant target for O-GlcNAcylation in the FGF pathway, suggesting that protein O-GlcNAcylation of the activated receptor complex is essential for FGF signal transduction.

The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei , the epimastigote form of T. cruzi , and the promastigote form of Leishmania major . Five sugar nucleotides, GDP-α- d -mannose, UDP-α- d - N -acetylglucosamine, UDP-α- d -glucose, UDP-α-galactopyranose, and GDP-β- l -fucose, were common to all three species; one, UDP-α- d -galactofuranose, was common to T. cruzi and L. major ; three, UDP-β- l -rhamnopyranose, UDP-α- d -xylose, and UDP-α- d -glucuronic acid, were found only in T. cruzi ; and one, GDP-α- d -arabinopyranose, was found only in L. major . The estimated demands for each monosaccharide suggest that sugar nucleotide pools are turned over at very different rates, from seconds to hours. The sugar nucleotide survey, together with a review of the literature, was used to define the routes to these important metabolites and to annotate relevant genes in the trypanosomatid genomes.

Galactose metabolism is essential for the survival of Trypanosoma brucei , the etiological agent of African sleeping sickness. T. brucei hexose transporters are unable to transport galactose, which is instead obtained through the epimerization of UDP-glucose to UDP-galactose catalyzed by UDP-glucose 4′-epimerase ( galE ). Here, we have characterized the phenotype of a bloodstream form T. brucei galE conditional null mutant under nonpermissive conditions that induced galactose starvation. Cellular levels of UDP-galactose dropped rapidly upon induction of galactose starvation, reaching undetectable levels after 72 h. Analysis of extracted glycoproteins by ricin and tomato lectin blotting showed that terminal β- d -galactose was virtually eliminated and poly- N -acetyllactosamine structures were substantially reduced. Mass spectrometric analysis of variant surface glycoprotein confirmed complete loss of galactose from the glycosylphosphatidylinositol anchor. After 96 h, cell division ceased, and electron microscopy revealed that the cells had adopted a morphologically distinct stumpy-like form, concurrent with the appearance of aberrant vesicles close to the flagellar pocket. These data demonstrate that the UDP-glucose 4′-epimerase is essential for the production of UDP-galactose required for galactosylation of glycoproteins and that galactosylation of one or more glycoproteins, most likely in the lysosomal/endosomal system, is essential for the survival of bloodstream form T. brucei.