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. The trypanosomatids are protozoan parasites that impose a major health burden on many countries in the developing world, causing a wide range of diseases over three continents. Like most eukaryotes, the cell surfaces and endosomal/lysosomal systems of these organisms are rich in glycoconjugates, some of which play essential roles in their survival, infectivity, or virulence. The glycoconjugate repertoires of the three trypanosomatids studied here, Trypanosoma brucei, T. cruzi, and Leishmania major, are fundamentally different, reflecting their disparate life cycles, modes of infection, and disease pathologies (see references 5,27,28,38,47,65, and 69 and references therein). The monosaccharides that make up these glycoconjugates vary between the three trypanosomatid species. For example, all three contain D-mannose (Man), D-Nacetylglucosamine (GlcNAc), D-glucosamine (GlcN), D-glucose (Glc), and D-galactopyranose (Galp), while only T. cruzi and L. major contain D-galactofuranose (Galf), only T. cruzi contains D-xylose (Xyl), L-rhamnopyranose (Rha), and L-fucose (Fuc), and only L. major contains D-arabinopyranose (Ara) ( Table 1). However, a unifying theme of their glycobiology is an abundance of cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and/or non-proteinlinked GPI structures.The protein-linked GPI glycans of the leishmania are the simplest in structure, consisting of the conserved Man␣1-2Man␣1-6Man␣1-4GlcN core. Those of T. cruzi are principally Man␣1-2Man␣1-2Man␣1-6Man␣1-4GlcN, and those of T. brucei are the most complex, with ␣-D-Galp and ␤-D-Galp side chains in the bloodstream form and sialylated poly-N-acetyllactosamine (poly-LacNAc) side chains in the procyclic form (27).The non-protein-linked GPI structures include the so-called glycoinositolphospholipids, or GIPLs, that are most abundant in the leishmania and T. cruzi species (58, 64, 90) but are also found in procyclic-form T. brucei (60,73,122) and bloodstream form T. brucei (39,63). Non-protein-linked GPIs also include the more exotic leishmania-specific lipophosphoglycan (LPG) structures (38,47,64,65). The leishmania LPGs contain characteristic phosphosaccharide repeats of Galp␤1-4Man␣1-P, which in L. major can have ␤-D-Galp and ␣-D-Arap side chains (66,67). These repeats are also found in the secr...

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.

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 ␤-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.The flagellated protozoan parasite Trypanosoma brucei, transmitted by the bite of the tsetse fly, is the etiological agent of African sleeping sickness in humans and the related disease nagana in cattle. T. brucei organisms are able to survive in the blood of the host by virtue of a dense surface coat of variant surface glycoprotein (VSG) that protects the parasite from the complement pathway and undergoes antigenic variation to evade specific immune responses, making the production of a vaccine unfeasible (4). Sleeping sickness is invariably fatal if untreated, and it is estimated that up to 500,000 people are currently infected in sub-Saharan Africa (22). Current drugs are expensive, toxic, and difficult to administer, leaving an urgent need for new therapeutic agents.The bloodstream form of T. brucei is rich in galactose-containing glycoproteins, most notably the abundant VSG (11), and components of the flagellar pocket and lysomal/endosomal system, including the transferrin receptor (1, 13). T. brucei hexose transporters are unable to transport galactose (5, 18), which is instead obtained through the epimerization of UDPglucose (UDP-Glc) to UDP-galactose (UDP-Gal) via the NADH-dependent oxidoreductase UDP-Glc 4Ј-epimerase (galE) (17). Construction of conditional null mutant cell lines has demonstrated that galE is an essential gene for both bloodstream and procyclic form T. brucei, and the phenotype of procyclic form cells upon induction of galactose starvation has been reported (16,17).In this paper, we examine the phenotype of the bloodst...

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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.Typanosomatid parasites are protozoan parasites that impose a major health burden on many countries in the developing world, causing a wide range of diseases over three continents. Trypanosoma brucei is the causative agent of African sleeping sickness in humans and nagana in cattle and is spread between mammalian hosts by the bite of the tsetse fly (Glossina sp.). T. brucei transmission occurs when the bloodstream form of the parasite is ingested by a tsetse fly during feeding; the parasite then differentiates into the procyclic form in order to colonize the tsetse fly mid gut. The parasite undergoes further differentiation and migration to the salivary gland of the fly in order to infect a new mammalian host upon a subsequent blood meal. Research in several laboratories has focused on the glycobiology of these organisms to uncover potential targets for therapeutic intervention (1-5). These targets include enzymes of glycosylphosphatidylinositol biosynthesis (1, 6 -9) and enzymes of sugar nucleotide biosynthesis. For example, GDP-mannose biosynthesis has been shown to be essential for the infectivity of Leishmania mexicana (10 -13) and UDPglucose 4Ј-epimerase, the only source of UDP-galactose in T.

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 OGlcNAcylation 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 (UDPGlcNAc), 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.

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