Purification, Cloning, and Characterization of an Acidic Ectoprotein Phosphatase Differentially Expressed in the Infectious Bloodstream Form of Trypanosoma brucei

Bakalara, Norbert; Santarelli, Xavier; Davis, Charles E.; Baltz, Théo

doi:10.1074/jbc.275.12.8863

Cited by 54 publications

(48 citation statements)

References 52 publications

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“…We therefore studied the expression of the GPEET kinase in bloodstream forms in culture by labeling them with [␥-32 P]ATP in the presence of exogenous GPEET. Because bloodstream form trypanosomes express a surface phosphatase (61), which may interfere with the phosphorylation reaction or immediately dephosphorylate products of an ecto-protein kinase reaction, the experiments were also carried out in the presence of the phosphatase inhibitor NaF (61). Our results show that membranes from bloodstream form trypanosomes were unable to phosphorylate GPEET (Fig.…”

Section: Resultsmentioning

confidence: 99%

Coordinate Expression of GPEET Procyclin and Its Membrane-associated Kinase in Trypanosoma brucei Procyclic Forms

Schlaeppi

Malherbe

Bütikofer

2003

Journal of Biological Chemistry

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GPEET procyclin is a major glycosylphosphatidylinositol-anchored protein of procyclic (insect stage) trypanosomes in culture and is heavily phosphorylated in the GPEET pentapeptide repeat. The phosphorylation reaction is a late event and occurs during maturation and transport of GPEET or on the parasite surface by an ecto-protein kinase. Initial biochemical characterization of the GPEET kinase activity now shows that it depends on bivalent cations for maximal activity, is stimulated by sulfhydryl group reagents, and is specific for ATP as phosphoryl donor. No kinase activity is detected in bloodstream form trypanosomes in culture, whereas strong phosphorylation is observed in early procyclic forms. In addition, the GPEET kinase activity is absent from procyclic trypanosomes that have repressed GPEET synthesis but can be induced in these same stocks by conditions, which also induce GPEET expression. However, the presence of an active kinase does not depend on the presence of (functional) GPEET because it can be detected in parasites expressing a non-phosphorylatable GPEET mutant protein and in procyclin null mutant trypanosomes. Interestingly, the presence of the glycosylphosphatidylinositol lipid moiety seems necessary for GPEET to become phosphorylated. Together, the results demonstrate that GPEET and its kinase are expressed during the same life cycle stages and that factors that induce the expression of GPEET in vitro also induce the expression of the GPEET kinase.

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Section: Resultsmentioning

confidence: 99%

Coordinate Expression of GPEET Procyclin and Its Membrane-associated Kinase in Trypanosoma brucei Procyclic Forms

Schlaeppi

Malherbe

Bütikofer

2003

Journal of Biological Chemistry

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“…The resulting recombinant protein was expressed in E. coli BL21 (DE3) from Novagen according to the manufacturer's instructions. Cells were lysed, and the recombinant protein was purified as previously described after solubilization in 6 M urea (23).…”

Section: Methodsmentioning

confidence: 99%

A Vacuolar-type H+-Pyrophosphatase Governs Maintenance of Functional Acidocalcisomes and Growth of the Insect and Mammalian Forms of Trypanosoma brucei

Lemercier¹,

Dutoya²,

Luo³

et al. 2002

Journal of Biological Chemistry

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126

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Vacuolar proton pyrophosphatases (V-H؉ -PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H ؉ -PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H ؉ -PPase gene by using double-stranded RNA interference methodology to produce inducible V-H ؉ -PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. brucei V-H ؉ -PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 ؋ 10 5 cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.Intracellular acidic vacuoles containing polyphosphate (polyP), 1 initially called volutin or polyP bodies, have been described in bacteria, algae, yeast, and protozoa (1). In trypanosomatids, these polyP vacuoles were called acidocalcisomes (2) and shown to be electron dense and contain large concentrations of PP i , calcium, magnesium, and other elements (3). Similar organelles have been identified in apicomplexan parasites (4, 5) as well as in the green algae, Chlamydomonas reinhardtii (6) and the slime mold, Dictyostelium discoideum (7). Acidocalcisomes were postulated to play an important role in the regulation of both cytosolic Ca 2ϩ concentration and intracellular pH (pH i ). For example, polyP hydrolysis (8) and activation of the Na ϩ /H ϩ antiporter (9, 10) were postulated to protect the cells against alkaline pH stress and increase the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ). In addition, these organelles possess a vacuolar-type H ϩ -translocating pyrophosphatase (V-H ϩ -PPase) (11), which is an electrogenic proton pump initially discovered in photosynthetic bacteria and plants (12). It has been shown to be associated with the plasma membrane or vacuoles in plants and with the chromatophore membranes of Rhodospirullum rubrum. The biochemical function of this enzyme in plants and unicellular eukaryotes is to couple hydrolysis of the high energy phosphate bond of PP i with H ϩ translocation from the cytosol to acidify the plant vacuole (tonoplast) or the acidocalcisome, respectively (10, 13). Working on isolated acidocalcisomes, Rodrigues et al. (14) demonstrated that the Trypanosoma brucei H ϩ -PPase was able to generate a membrane potential by PP i -dependent proton uptake. Moreover, Scott et al. (11,15) showed tha...

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“…It has been postulated that these ␤-Gal-containing structures may be involved in retaining the TfR, and other recycling components, in the T. brucei flagellar pocket and the endosomal͞lysosomal membrane system (18). In addition to the aforementioned glycoproteins, five other transmembrane glycoproteins, ISG65, ISG75 (19), the ecto-phosphatase AcP115 (20), the Golgi-and lysosome-associated glycoprotein tGLP-1 (21), and the Fla-1 glycoprotein (22), the latter associated with flagellar adhesion to the cell body (23), are known to be glycosylated, but the nature of their carbohydrate chains is unknown. There are also other membrane proteins, such as Tb-29 (24), the cysteine-rich acidic membrane protein CRAM (25), the alanine-rich protein BARP (26), and the products of expression-site-associated genes (ESAGs) 1, 2, 3, 4, 10, and 11 (26,27), that are predicted to contain carbohydrate in the form of GPI anchors and͞or N-linked oligosaccharides.…”

Section: Discussionmentioning

confidence: 99%

“…The few surviving cells failed to divide unless tetracycline was reintroduced at day 10 ( Fig. 5C) or until day [16][17][18][19][20], when the cultures spontaneously started to grow once more at normal rates (Fig. 5B).…”

Section: The T Brucei Gale Gene Is Essential To the Bloodstream Form Ofmentioning

confidence: 99%

Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei

Roper

Güther

Milne

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

100

116

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The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease Nagana. The bloodstream form of the parasite uses a dense cell-surface coat of variant surface glycoprotein to escape the innate and adaptive immune responses of the mammalian host and a highly glycosylated transferrin receptor to take up host transferrin, an essential growth factor. These glycoproteins, as well as other flagellar pocket, endosomal, and lysosomal glycoproteins, are known to contain galactose. The parasite is unable to take up galactose, suggesting that it may depend on the action of UDPglucose 4 -epimerase for the conversion of UDP-Glc to UDP-Gal and subsequent incorporation of galactose into glycoconjugates via UDP-Gal-dependent galactosyltransferases. In this paper, we describe the cloning of T. brucei galE, encoding T. brucei UDP-Glc-4 -epimerase, and functional characterization by complementation of a galE-deficient Escherichia coli mutant and enzymatic assay of recombinant protein. A tetracycline-inducible conditional galE null mutant of T. brucei was created using a transgenic parasite expressing the TETR tetracycline repressor protein gene. Withdrawal of tetracycline led to a cessation of cell division and substantial cell death, demonstrating that galactose metabolism in T. brucei proceeds via UDP-Glc-4 -epimerase and is essential for parasite growth. After several days without tetracycline, cultures spontaneously recovered. These cells were shown to have undergone a genetic rearrangement that deleted the TETR gene. The results show that enzymes and transporters involved in galactose metabolism may be considered as potential therapeutic targets against African trypanosomiasis.UDP-Gal ͉ galE ͉ epimerase T he tsetse fly-transmitted protozoan parasite Trypanosoma brucei causes human African sleeping sickness and the related cattle disease Nagana. There are 300,000-500,000 cases of the human disease per year in sub-Saharan Africa (1). The bloodstream form of the parasite divides by binary fission in the blood, lymph, and interstitial fluids of the mammalian host, and causes cachexia and anaemia in cattle and neurological disturbances in man. These conditions are fatal if not treated, and existing chemotherapies are toxic and difficult to administer. The need for new, less-toxic therapeutics is widely acknowledged (1).The bloodstream form of T. brucei is rich in galactosecontaining glycoproteins, most notably the variant surface glycoprotein (VSG) that, at 5 ϫ 10 6 homodimers per cell, forms a dense cell-surface coat. The VSG coat is a macromolecular diffusion barrier that protects the parasite from the innate immune system and also enables the parasite to undergo antigenic variation. Thus, although an individual parasite only expresses one VSG gene at a time, the parasite population can stay ahead of the host's specific immune response to expressed VSGs when a few parasites switch expression to one of several hundred genes encoding immunologically ...

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Purification, Cloning, and Characterization of an Acidic Ectoprotein Phosphatase Differentially Expressed in the Infectious Bloodstream Form of Trypanosoma brucei

Cited by 54 publications

References 52 publications

Coordinate Expression of GPEET Procyclin and Its Membrane-associated Kinase in Trypanosoma brucei Procyclic Forms

Coordinate Expression of GPEET Procyclin and Its Membrane-associated Kinase in Trypanosoma brucei Procyclic Forms

A Vacuolar-type H+-Pyrophosphatase Governs Maintenance of Functional Acidocalcisomes and Growth of the Insect and Mammalian Forms of Trypanosoma brucei

Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei

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