Cloning of Trypanosoma brucei and Leishmania major Genes Encoding the GlcNAc-Phosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis That Is Essential to the African Sleeping Sickness Parasite

Chang, Tunhan; Milne, Kenneth G.; Güther, Maria Lucia S.; Smith, Terry; Ferguson, Michael A. J.

doi:10.1074/jbc.m208374200

Cited by 73 publications

(99 citation statements)

References 52 publications

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“…replacement of one allele followed by introduction of tetracycline-inducible ectopic copy and then replacement of the second allele) (7,15,50). However, using the same constructs, we failed to obtain even a single allele replacement in bloodstream form T. brucei.…”

Section: The T Brucei Gdp-fucose Biosynthesis Enzymes Are Active In mentioning

confidence: 71%

“…RT-PCR performed with mRNA from day 10 cells showed that these cells were re-expressing the TbGMD ORF (data not shown). This kind of escape from tetracycline control of gene expression is a common feature of conditional null mutants of essential genes in bloodstream form T. brucei (7,15). The procyclic form mutants grew at the same rate for the first 7 days with and without tetracycline (Fig.…”

Section: Loss Of Tbgmd Expression Results In Depletion Of Gdp-fucose-tomentioning

confidence: 99%

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The de Novo Synthesis of GDP-fucose Is Essential for Flagellar Adhesion and Cell Growth in Trypanosoma brucei

Turnock

Izquierdo

Ferguson

2007

Journal of Biological Chemistry

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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.

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Section: The T Brucei Gdp-fucose Biosynthesis Enzymes Are Active In mentioning

confidence: 71%

Section: Loss Of Tbgmd Expression Results In Depletion Of Gdp-fucose-tomentioning

confidence: 99%

The de Novo Synthesis of GDP-fucose Is Essential for Flagellar Adhesion and Cell Growth in Trypanosoma brucei

Turnock

Izquierdo

Ferguson

2007

Journal of Biological Chemistry

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“…The low steady-state concentrations of the cellular GDP-Man pools and the very high rates of flux through them suggests that inhibition of GDP-Man biosynthesis would have rapid and profound effects on glycoconjugate biosynthesis, and consequently on viability and/or virulence, in these organisms. Certainly, GDP-Man and Dol-P-Man biosynthesis may be assumed to be essential in bloodstream form T. brucei, where disrupting GPI anchor biosynthesis (which is dependent on Dol-P-Man made from GDP-Man) is known to be lethal for the parasite (15,26,60,74). Since Man is present in every known type of trypanosomatid glycoconjugate carbohydrate chain, with the exception of the O-linked side chains of the T. cruzi mucins, GDP-Man biosynthesis has potential as a therapeutic target against all three parasites, so long as parasite-selective inhibitors can be found.…”

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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“…Phosphatidylinositol glycan, class L is an enzyme that catalyzes the deacetylation of N-acetyl-D-glucosaminylphosphatidylinositol in glycosylphosphatidylinositol anchor biosynthesis. The enzymes from a variety of species have a section of sequence, AHPDDE, that carries the metal binding histidine and aspartate residues, as well as the catalytic general base aspartate (11,31).…”

Section: Figmentioning

confidence: 99%

The Crystal Structure of 1-D-myo-Inosityl 2-Acetamido-2-deoxy-α-D-glucopyranoside Deacetylase (MshB) from Mycobacterium tuberculosis Reveals a Zinc Hydrolase with a Lactate Dehydrogenase Fold

Maynes

Garen

Cherney

et al. 2003

Journal of Biological Chemistry

100

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Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)-amido-2-deoxy-␣-D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn 2؉ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a Cterminal domain consisting of two ␤-sheets and two ␣-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD ؉ co-factor of lactate dehydrogenase. The Zn 2؉ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn 2؉ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol Sconjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol Sconjugate amidase based on the structure of MshB.

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Cloning of Trypanosoma brucei and Leishmania major Genes Encoding the GlcNAc-Phosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis That Is Essential to the African Sleeping Sickness Parasite

Cited by 73 publications

References 52 publications

The de Novo Synthesis of GDP-fucose Is Essential for Flagellar Adhesion and Cell Growth in Trypanosoma brucei

The de Novo Synthesis of GDP-fucose Is Essential for Flagellar Adhesion and Cell Growth in Trypanosoma brucei

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

The Crystal Structure of 1-D-myo-Inosityl 2-Acetamido-2-deoxy-α-D-glucopyranoside Deacetylase (MshB) from Mycobacterium tuberculosis Reveals a Zinc Hydrolase with a Lactate Dehydrogenase Fold

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