Cloning and characterisation of the UDP-glucose 4′-epimerase of Trypanosoma cruzi

Roper, Janine R.; Ferguson, Michael A. J.

doi:10.1016/j.molbiopara.2003.07.002

Cited by 30 publications

(23 citation statements)

References 32 publications

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“…2). The same is true for the Trypanosoma cruzi enzyme (35), and this almost certainly explains why GalNAc has not been found in any trypanosome glycoconjugates.…”

Section: Discussionmentioning

confidence: 69%

“…Substrate Specificity of T. brucei UDP-Glc 4Ј-EpimeraseThe recombinant enzyme was incubated with UDP-Glc, UDPGal, UDP-GlcNAc, or UDP-GalNAc, and the products were analyzed by strong anion exchange-HPLC in the presence of borate ions, a chromatographic system that allows resolution of UDP-Glc from UDP-Gal and UDP-GlcNAc from UDP-GalNAc (28,35). The HPLC profiles showed the interconversion of UDP-Glc and UDP-Gal (Fig.…”

Section: T Brucei Udp-glc 4ј-epimerase Is a Dimer In Aqueous Solution-mentioning

confidence: 99%

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The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

Roper¹,

Güther

MacRae³

et al. 2005

Journal of Biological Chemistry

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Galactose metabolism is essential in bloodstream form Trypanosoma brucei and is initiated by the enzyme UDP-Glc 4-epimerase. Here, we show that the parasite epimerase is a homodimer that can interconvert UDPGlc and UDP-Gal but not UDP-GlcNAc and UDP-GalNAc. The epimerase was localized to the glycosomes by immunofluorescence microscopy and subcellular fractionation, suggesting a novel compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T. brucei single-allele knockouts, and conditional (tetracycline-inducible) null mutants were constructed. Under non-permissive conditions, conditional null mutant cultures ceased growth after 8 days and resumed growth after 15 days. The resumption of growth coincided with constitutive re-expression epimerase mRNA. These data show that galactose metabolism is essential for cell growth in procyclic form T. brucei. The epimerase is required for glycoprotein galactosylation. The major procyclic form glycoproteins, the procyclins, were analyzed in TbGALE single-allele knockouts and in the conditional null mutant after removal of tetracycline. The procyclins contain glycosylphosphatidylinositol membrane anchors with large poly-N-acetyl-lactosamine side chains. The single allele knockouts exhibited 30% reduction in procyclin galactose content. This example of haploid insufficiency suggests that epimerase levels are close to limiting in this life cycle stage. Similar analyses of the conditional null mutant 9 days after the removal of tetracycline showed that the procyclins were virtually galactose-free and greatly reduced in size. The parasites compensated, ultimately unsuccessfully, by expressing 10-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.

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“…2). The same is true for the Trypanosoma cruzi enzyme (35), and this almost certainly explains why GalNAc has not been found in any trypanosome glycoconjugates.…”

Section: Discussionmentioning

confidence: 69%

Section: T Brucei Udp-glc 4ј-epimerase Is a Dimer In Aqueous Solution-mentioning

confidence: 99%

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

Roper¹,

Güther

MacRae³

et al. 2005

Journal of Biological Chemistry

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“…First, the substrate specificities of the T. brucei and T. cruzi enzymes differ from that of the human enzyme. The latter can interconvert UDP-GlcNAc and UDP-GalNAc, whereas the trypanosome enzymes cannot (95,97). Second, high-resolution crystal structures of the T. brucei epimerase, and comparison with those of the human counterpart (44), suggest that there is potential for selective inhibition of the parasite enzyme (4,105).…”

Section: Cruzi) Gdp-man Is Also the Precursor Of Gdp-fuc (See Below)mentioning

confidence: 99%

Sugar Nucleotide Pools of Trypanosoma brucei , Trypanosoma cruzi , and Leishmania major

2007

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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...

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“…No UDP-GalNAc was found in a nucleo- tide sugar pool of T. brucei, where GDP-Man, GDP-Fuc, UDPGlc, UDP-Gal, and UDP-GlcNAc were identified (57). Unlike the human epimerase (58), the T. brucei homolog does not have epimerase activity to convert UDP-GlcNAc to UDP-GalNAc (59,60). This may explain why GalNAc has yet to be found in any glycoconjugates of this parasite.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Nucleotide Sugar Transport in Trypanosoma brucei Alters Surface Glycosylation

Liu

Caradonna

et al. 2013

Journal of Biological Chemistry

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Background: Nucleotide sugar transporters (NSTs) provide availability of nucleotide sugars for glycosylation in the lumen of the Golgi apparatus. Results: The substrate specificities of four Trypanosoma brucei NSTs were characterized, and inhibition of TbNST4 resulted in glycosylation defects. Conclusion: TbNST4 plays an essential role in biosynthesis of glycoproteins in T. brucei. Significance: To understand the roles of TbNST(s) in the growth and pathogenesis of T. brucei.

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Cloning and characterisation of the UDP-glucose 4′-epimerase of Trypanosoma cruzi

Cited by 30 publications

References 32 publications

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

Sugar Nucleotide Pools of Trypanosoma brucei , Trypanosoma cruzi , and Leishmania major

Inhibition of Nucleotide Sugar Transport in Trypanosoma brucei Alters Surface Glycosylation

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