Abstract
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.
Highlights
Sugar nucleotides are activated forms of sugars that are used as the ultimate source of sugar for the majority of glycosylation reactions
Sugar nucleotides are synthesized in the cytoplasm and transported through specific transporters into the lumen of the Golgi apparatus and/or endoplasmic reticulum, where they are used by glycosyltransferases as donor substrates in glycosylation reactions [18, 19]
The Synthesis and Role of GDP-fucose in T. brucei protease [31] and in a glycoprotein called gp72, where it is found together with rhamnose, xylose, galactopyranose, galactofuranose, and N-acetylglucosamine in unique complex glycans attached to Thr and Ser via phosphodiester linkages [32,33,34,35]. gp72 is nonessential in culture but has a role in flagellar adhesion in the epimastigote form of the parasite
Summary
Parasite Culture—T. brucei bloodstream form parasites (strain 427, variant MITaT1.2) that express T7 polymerase and tetracycline repressor protein under G418 selection [42] were cultured in HMI-9 medium [43] up to a maximum density of 3 ϫ 106 cells/ml at 37 °C with 5% CO2. Expression, and Assay of GDP-fucose Biosynthesis Genes in E. coli—For overexpression in E. coli, the TbGMD and TbGMER genes were amplified by PCR with the following forward and reverse primers to add either BamHI, EcoRI, or XhoI restriction sites (underlined): 5Ј-cccggatccATGTCAGCACGTCGACTGGC-3Ј and 5Ј-gcgaattcCTATTGCCCCGCTGCTCAACAC-3Ј for TbGMD, and 5Ј-cccggatccATGTTAGGGT-. The PCR products were digested with appropriate restriction enzymes and cloned into the pGEX 4T expression vector (Amersham Biosciences) and expressed in BL21 (DE3) E. coli cells. The primers added either HindIII or BamHI restriction sites (underlined) to the PCR products to allow cloning into the pLew100 expression vector [42] for use in constructing conditional null mutants. Cells were pelleted by centrifugation, washed in ice-cold PBS, and lysed in 70% ethanol in the presence of 10 pmol of GDP-glucose internal standard, and sugar nucleotides were extracted using EnviCarb columns [45]. Proteins were confirmed by tryptic mass fingerprinting (data not shown), and both proteins appeared relatively pure, apart from a minor contaminat-
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