Trypanosoma rangeli: A possible role for ecto-phosphatase activity on cell proliferation

Here we demonstrate for the first time that growth of Trypanosoma rangeli, a protozoa parasite, is strongly dependent on the presence of inorganic phosphate (Pi) in the culture medium and that the replacement of the inorganic phosphate in the culture medium by β-glycerophosphate, a substrate for phosphatases lead the cells to achieve its maximal growth. The ecto-phosphatase activity present on the external surface of T. rangeli decreased during the growth phase of the parasite, suggesting that this enzyme could be important for the development. Accordingly, the inhibition of this ecto-phosphatase activity by sodium orthovanadate also inhibited the proliferation of T. rangeli. Parasites maintained in a Pi-starved culture medium (2 mM Pi) had 4-fold more ecto-phosphatase activity as compared to parasites maintained in a Pi-supplemented culture medium (50 mM Pi). Altogether, these results presented here suggest that this ecto-phosphatase activity leads to hydrolysis of phosphorylated compounds present in the extracellular medium, which could contribute to the acquisition of inorganic phosphate during the development of T. rangeli epimastigotes.     

Trypanosoma rangeli: An alkaline ecto-phosphatase activity is involved with survival and growth of the parasite

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca²⁺ present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (P_i). Levamisole at the IC₅₀ spared significantly parasite growth when β-GP was the sole source of P_i and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.     

Identification and Characterization of an Ecto-Pyrophosphatase Activity in Intact Epimastigotes of Trypanosoma rangeli

In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in Trypanosoma rangeli that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H⁺-PPase) in the epimastigote forms of T. rangeli. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H⁺-PPase of Arabidopsis thaliana. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of T. rangeli. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl₂. In the presence of MgCl₂, this activity was inhibited by millimolar concentrations of CaCl₂. The ecto-PPase activity of T. rangeli decreased with increasing cell proliferation in vitro, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of T. rangeli that possibly plays an important role in phosphate metabolism of this protozoan.     

Elevated extracellular inorganic phosphate inhibits ecto-phosphatase activity in breast cancer cells: Regulation by hydrogen peroxide

For cells to obtain inorganic phosphate, ectoenzymes in the plasma membrane, which contain a catalytic site facing the extracellular environment, hydrolyze phosphorylated molecules. In this study, we show that increased Pi levels in the extracellular environment promote a decrease in ecto-phosphatase activity, which is associated with Pi-induced oxidative stress. High levels of Pi inhibit ecto-phosphatase because Pi generates H₂O₂. Ecto-phosphatase activity is inhibited by H₂O₂, and this inhibition is selective for phospho-tyrosine hydrolysis. Additionally, it is shown that the mechanism of inhibition of ecto-phosphatase activity involves lipid peroxidation. In addition, the inhibition of ecto-phosphatase activity by H₂O₂ is irreversible. These findings have new implications for understanding ecto-phosphatase regulation in the tumor microenvironment. H₂O₂ stimulated by high Pi inhibits ecto-phosphatase activity to prevent excessive accumulation of extracellular Pi, functioning as a regulatory mechanism of Pi variations in the tumor microenvironment.     

Giardia lamblia: Characterization of ecto-phosphatase activities

Ecto-phosphatase activities of Giardia lamblia were characterized in intact cells, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 8.4 ± 0.8 nmol p-NP/h/10⁷ cells. The ecto-phosphatase activities were inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium fluoride and sodium molybdate and by inorganic phosphate, the final product of the reaction. Experiments using a classical inhibitor of phosphotyrosine phosphatase, sodium orthovanadate, also showed that the ecto-phosphatase activity was inhibited in a dose-dependent manner. Different phosphorylated amino acids were used as substrates for the G. lamblia ecto-phosphatase activities the highest rate of phosphate release was achieved using phosphotyrosine. Not only p-NPP hydrolysis but also phosphotyrosine hydrolysis was inhibited by sodium orthovanadate. Phosphotyrosine but not phospho-serine or phospho-threonine inhibited the p-nitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites.     

Role of β-GP-derived pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells

The permissive effect of β-GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto-ALP at physiological pH range. β-GP present in the culture medium for 8 days exerted a stimulatory effect on ⁴⁵Ca incorporation into matrix cell layers while the ecto-ALP activity level measured on intact cells with a saturating concentration of p cells grown either in the presence or absence of β-GP. In both types of cultures, β-GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of β-GP on ⁴⁵Ca incorporation and generation of Pi was similar (kϕ = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and β-GP hydrolyses, which were likely catalyzed by the same ecto-enzyme. The rate of ⁴⁵Ca incorporation into matrix cell layers, which was high (0.90 μmol/4h/mg cell protein) in cells grown in the absence of β-GP, was inhibited by 50% by levamisole. In cells grown in the absence of β-GP, the ⁴⁵Ca incorporation rate increased progressively after β-GP addition, reaching after 12 h the value of cultures grown in the presence of β-GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of β-GP-derived Pi rapidly led to high ⁴⁵Ca incorporation rate which was unaffected by levamisole. β-GP removal from cultures grown in its presence reduced by 50% the ⁴⁵Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto-ALP to hydrolyze β-GP in cultured fetal calvaria cells, yet it influenced the β-GP-stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels. © 1996 Wiley-Liss, Inc.     

Leishmania amazonensis: characterization of an ecto-phosphatase activity

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.     

Leishmania amazonensis: effects of heat shock on ecto-ATPase activity

In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress.     

Characterization of an ecto-phosphatase activity in malpighian tubules of hematophagous bug Rhodnius prolixus

We have characterized a phosphatase activity present on the external surface of intact Malpighian tubules in Rhodnius prolixus. This phosphatase hydrolyses the substrate p-nitrophenyl phosphate at a rate of 3.38 +/- 0.07 nmol Pi x mg(-1) x min(-1). Phosphatase activity decreased with the increase of the pH from 6.4 to 7.6 pH, a range in which tubules cellular integrity was maintained for at least 1 h. Classical inhibitors of acid phosphatase, such as ammonium molybdate, fluoride, vanadate, mpV-PIC, and bpV-PHEN, caused different patters of inhibition. The ecto-phosphatase present an apparent Km of 1.67 +/- 0.34 mM and Vmax of 5.71 +/- 0.37 nmol Pi x mg(-1) x min(-1) for p-NPP. Zinc chloride inhibited 78.2% of ecto-phosphatase activity, with Ki of 0.35 mM. Such inhibition was reversed by incubation with cysteine and GSH, but not DTT, serine, and GSSG, showing that cysteine residues are important for enzymatic activity. Phosphatase activity increased 141% three days after blood meal, and returned to basal levels 2 days later. These results suggest that ecto-phosphatase activity could be involved in a diuretic mechanism, essential in the initial days after a blood meal for the control of Rhodnius homeostasis.     

Trypanosoma brucei brucei: biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.     

Preferential dephosphorylation of protein bound phosphorylthreonine and phosphorylserine residues by cytosol and mitochondrial "casein phosphatases".

The partial purification of a rat liver cytosol protein phosphatase is described, resulting in a preparation active on casein but not on phosvitin, cytosol phosphopeptides, ATP, ADP and p-nitrophenylphosphate, which, on the contrary, are still dephosphorylated by the protein phosphatase purified from rat liver mitochondria. Moreover the activity of the former enzyme on casein appears to involve only a limited amount of phosphoric sites which are also preferentially phosphorylated by soluble protein kinase. The isolation and evaluation of ³²P-serine and ³²P-threonine from protein-kinase-dependently labelled phosvitin and casein, before and after incubation with the two enzymes, led to the conclusion that mitochondrial protein phosphatase hydrolyzes more actively the phosphorylserine residues, while the cytosol “casein phosphatase” promotes a preferential breakdown of the ³²P-threonine residues.     

Molecular characterization of calcineurin B from the non-virulent Trypanosoma rangeli kinetoplastid indicates high gene conservation

Calcineurin B, the regulatory subunit of calcineurin, a serine/threonine protein phosphatase, is highly conserved throughout the evolutionary scale including trypanosomatids such as Trypanosoma cruzi, and Leishmania major. Thus, in these flagellates the protein is required for mammalian host cell invasion and virulence and stress responses. With the aim of determining the presence of calcineurin B in Trypanosoma rangeli, a non-virulent trypanosome for mammals, the respective gene was amplified by PCR, cloned and sequenced. Two sequences of 531 bp in length showing a nucleotide polymorphism (314A>C) were obtained in spite of a single-copy gene was revealed by Southern blot. These sequences, probably the alleles from the gene, showed a 79 % of identity with those from T. cruzi and clustered as the sister group of this trypanosome species in a Maximum Parsimony analysis. Deduced amino acid sequence comparison with trypanosomatids and other organisms through the phylogenetic scale as well as the obtained protein structural homology model suggested the presence of the four potential EF-hand regions and the corresponding calcium binding sites of the last three of these domains. Having assessed the expression of this protein in T. rangeli epimastigotes, and taking into account the following facts: (i) calcineurin inhibitors have inhibitory effect on the in vitro replication of T. cruzi, (ii) L. major promastigote growth is inhibited by chelating agents, and (iii) T. rangeli does not seem to productively infect mammalian cells, it is hypothesized herein that the function of this protein in T. rangeli is required for epimastigote growth.     

Phosphate relieves chromium toxicity in Arabidopsis thaliana plants by interfering with chromate uptake

Soil contamination by hexavalent chromium [Cr(VI) or chromate] due to anthropogenic activities has become an increasingly important environmental problem. Mineral nutrients such as phosphate (Pi), sulfate and nitrate have been reported to attenuate Cr(VI) toxicity, but the underlying mechanisms remain to be clarified. Here, we show that chromate activates the expression of low-Pi inducible reporter genes AtPT1 and AtPT2 in Arabidopsis thaliana transgenic seedlings. Primary-root growth was inhibited by 60 % in AtPT2::uidA-expressing seedlings upon exposure to 140-μM Cr(VI). However, increasing the Pi and sulfate supply to the seedlings that were experiencing Cr(VI) toxicity completely and partially restored the root growth, respectively. This effect correlated with the Cr(VI)-induced AtPT2::uidA expression being completely reversed by addition of Pi. To evaluate whether the nutrient supply may affect the endogenous level of Cr in plants grown under toxic Cr(VI) levels, the contents of Cr were measured (by ICP-MS analyses) in seedlings treated with Cr and with or without Pi, sulfate or nitrate. It was found that Cr accumulation increases tenfold in plants treated with 140-μM Cr(VI) without modifying the phosphorus concentration in the plant. In contrast, the supply of Pi specifically decreased the Cr content to levels similar to those found in seedlings grown in medium without chromate. Taken together, these results show that in A. thaliana seedlings the uptake of Cr(VI) is reduced by Pi. Moreover, our data indicate that Pi and sulfate supplements may be useful in strategies for handling Cr-contaminated soils.     

Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests

The major storage proteins in cereals, prolamins, have an abundance of the amino acids glutamine and proline. Storage pests need specific digestive enzymes to efficiently hydrolyze these storage proteins. Therefore, post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored-product pest, Tenebrio molitor (yellow mealworm). Three distinct PGP activities were found in the anterior and posterior midgut using the highly-specific chromogenic peptide substrate N-benzyloxycarbonyl-L-Ala-L-Ala-L-Gln p-nitroanilide. PGP peptidases were characterized according to gel elution times, activity profiles in buffers of different pH, electrophoretic mobility under native conditions, and inhibitor sensitivity. The results indicate that PGP activity is due to cysteine and not serine chymotrypsin-like peptidases from the T. molitor larvae midgut. We propose that the evolutionary conservation of cysteine peptidases in the complement of digestive peptidases of tenebrionid stored-product beetles is due not only to the adaptation of insects to plants rich in serine peptidase inhibitors, but also to accommodate the need to efficiently cleave major dietary proteins rich in glutamine.     

Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis

Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.     

Response to Phosphate Deprivation in Brassica nigra Suspension Cells : Enhancement of Intracellular, Cell Surface, and Secreted Phosphatase Activities Compared to Increases in Pi-Absorption Rate

Suspension cells of Brassica nigra responded to Pi deprivation by increasing their potential for Pi influx and by raising the active levels of intracellular, cell surface, and secreted acid phosphatases. These responses, however, were temporally distinct. Phosphate influx capacity increased 15-fold in parallel to a 10-fold decrease in endogenous Pi during 7 days of culture in basal growth medium. In contrast, intracellular and cell surface phosphatase activities changed only after alterations in cellular phosphorus status had been in place for a number of days. Even in nutrient sufficient cells the secretion of phosphatase remained relatively high as did the activities of the other phosphatases. The cell surface acid phosphatase had a K_m of approximately 10 times that of the influx process and molybdate was a much stronger inhibitor of this phosphatase activity. From these results it appears that Pi absorption and the production or activation of phosphatases are regulated in a distinct manner. In addition, Pi uptake into Brassica nigra cells does not appear to directly involve the cell surface phosphatase under Pi-deficient conditions.