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A metallo phosphatase activity present on the surface of Trypanosoma brucei procyclic forms
Abstract
In this work, we describe how living cells of Trypanosoma brucei procyclic forms were able to hydrolyze extracellular p-nitrophenylphosphate (pNPP). These intact parasites, which had their viability determined by motility and the Trypan blue method, presented a low level of pNPP hydrolysis in the absence of any divalent metal (0.72+/-0.07 nmol pNP/mg min). Interestingly, in the presence of 5mM MgCl(2), ectophosphatase activity of 1.91+/-0.21 nmol pNP/mg min was observed. The ectophosphatase activity was also stimulated by MnCl(2), CoCl(2) and CuCl(2) but not by CaCl(2) and CdCl(2) and was inhibited by ZnCl(2). The addition of Mg(2+), Mn(2+), Co(2+) and Cu(2+) to extracellular medium increased the ectophosphatase activity in a dose-dependent manner. At 5mM pNPP, half-maximal stimulation of pNPP hydrolysis was obtained with 0.39+/-0.05 mM MgCl(2), 0.33+/-0.03 mM MnCl(2), 1.63+/-0.12 mM CoCl(2) and 2.04+/-0.33 mM CuCl(2). In the absence of any divalent metal (basal activity) the apparent K(m) for pNPP was 0.66+/-0.09 mM, while at saturating MgCl(2) concentrations the corresponding apparent K(m) for pNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.27+/-0.03 mM. The Mg(2+)-stimulated pNPP hydrolysis was strongly inhibited by ZnCl(2) and vanadate, while the metal-independent basal phosphatase activity was less inhibited by these phosphotyrosyl phosphatase inhibitors.
... This result suggests that at least two enzymes could be involved with the ATP hydrolysis on the external surface of T. brucei brucei. Trypanosoma brucei brucei procyclic forms express surface acid phosphatases (Fernandes et al., 1997Fernandes et al., , 2003), which could contribute to ATP hydrolysis. To evaluate whether phosphate release from ATP was inXuenced by phosphatase activities, diVerent experimental approaches were followed, as presented inFig. ...
... 4 andTable 1. We showed that the increase of pH inhibited the phosphatase activity present on the external surface of T. brucei brucei (Fernandes et al., 1997Fernandes et al., , 2003 ). On the other hand the Mg 2+ -dependent ATPase activity was not modiWed by the increased of the pH (Fig. 4). ...
... For these reasons, we assign an ectolocalization for the Mg-dependent ATPase activity described here. ATP hydrolysis could not be due to a phosphatase activity present on the external surface of T. brucei brucei membrane (Fernandes et al., 1997Fernandes et al., , 2003), because as shown inTable 1 potent inhibitors for phosphatase activities were not capable of modify the Mg-dependent ecto- ATPase activity. The Mg-dependent ecto-ATPase activity described here could not be attributed to a 5-nucleotidase, since the ATP hydrolysis was not inhibited by ammonium molybdate, a potent inhibitor of 5 nucleotidase (Gottlieb and Dwyer, 1983) (Table 1). ...
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.
... Ectophosphatases play an important role in cell proliferation, differentiation, parasite-host interaction, nutrition, ROS sensing and adhesion [39,40]. Ectophosphatases have been reported in some protozoa parasites, including Tb, Tc, Eh, Gl, Tv and some Leishmania species [8,39,[41][42][43][44][45][46][47][48][49][50][51][52]. A unique ectophosphatase cloned from Tb does not have any other similar protein beyond the genus Trypanosoma [1]. ...
Phosphorylation dynamically regulates the function of proteins by maintaining a balance between protein kinase and phosphatase activity. A comprehensive understanding of the role phosphatases in cellular signaling is lacking in case of protozoans of medical and veterinary importance worldwide. The drugs used to treat protozoal diseases have many undesired effects and the development of resistance, highlights the need for new effective and safer antiprotozoal agents. In the present study we have analyzed phosphatomes of 15 protozoans of medical significance. We identified ~2000 phosphatases, out of which 21% are uncharacterized proteins. A significant positive correlation between phosphatome and proteome size was observed except for E. histolytica, having highest density of phosphatases irrespective of its proteome size. A difference in the number of phosphatases among different genera shows the variation in the signaling pathways they are involved in. The phosphatome of parasites is dominated by ser/thr phosphatases contrary to the vertebrate host dominated by tyrosine phosphatases. Phosphatases were widely distributed throughout the cell suggesting physiological adaptation of the parasite to regulate its host. 20% to 45% phosphatome of different protozoa consists of ectophosphatases, i.e. crucial for the survival of parasites. A database and a webserver “ProtozPhosDB” can be used to explore the phosphatomes of protozoans of medical significance.
... In R. aquaspersa, Co 2+ was able to promote an increase in ectophosphatase activity in a dose-dependent manner [42]. A Co 2+ -activated ectophosphatase activity was also reported in T. brucei [53]. Furthermore, cobalt is known to be capable of inducing the expression of alkaline phosphatase in bacteria [54]. ...
Ectophosphatases are surface membrane-bound proteins whose active sites face the extracellular medium. These enzymes have been reported in several microorganisms including a large number of medically relevant fungal species. An effective technique for identifying ectophosphatases is performing phosphatase activity assays using living intact cells. Biochemical characterization of these activities has shown their differential modulation by classical phosphatase inhibitors, divalent metals and pH range. The physiological roles of ectophosphatases are not well established; however, it has been suggested that these enzymes play important roles in nutrition, proliferation, differentiation, adhesion, virulence and infection. Adhesion to host cells is the first step in establishing a fungal infection and ectophosphatases may be one of the first parasite proteins that come into contact with the host cells. Several results indicate that ectophosphatase activities increase the capacity of fungi to adhere to the host cells. In this context, the present review provides an overview of recent discoveries related to the occurrence and possible roles of ectophosphatase activities in fungal cells.
... The other line of evidence that the two ecto-phosphatases are distinct entities comes from the divalent cation requirements. The acid ectophosphatases from different trypanosomatids are, depending on the species, strongly activated by Mg 2+ , Co 2+ and/or Cu 2+ and inhibited by Zn 2+ , a divalent cation dependence that indicates a specific steric requirement for catalysis at the active site (Dick et al., 2010;Dutra et al., 2006;Fernandes et al., 2003;Fonseca-de-Souza et al., 2008). In contrast, the alkaline phosphatase activity was impaired by EDTA, being totally reversed by exogenous Ca 2+ but not by other metals (Fig. 3). ...
... It seems that these proteins are related to the regulation of T. brucei development, since these acidic phosphatases are expressed in bloodstream forms, but not in the insect procyclic form [31]. Likewise, an ectophosphatase activity on the surface of intact procyclic and bloodstream forms of T. brucei was demonstrated by Fernandes et al. [43, 44]. These enzymes show different behavior, like sensitivity to inhibitors and metal interference. ...
The interaction and survival of pathogens in hostile environments and in confrontation with host immune responses are important mechanisms for the establishment of infection. Ectophosphatases are enzymes localized at the plasma membrane of cells, and their active sites face the external medium rather than the cytoplasm. Once activated, these enzymes are able to hydrolyze phosphorylated substrates in the extracellular milieu. Several studies demonstrated the presence of surface-located ecto-phosphatases in a vast number of pathogenic organisms, including bacteria, protozoa, and fungi. Little is known about the role of ecto-phosphatases in host-pathogen interactions. The present paper provides an overview of recent findings related to the virulence induced by these surface molecules in protozoa and fungi.
The ecto-phosphatases belong to a group of enzymes closely associated with the cell surface that has its catalytic site facing the extracellular environment, where different phosphorylated substrates can be hydrolyzed. In the present work, we biochemically characterized the ecto-phosphatase activity of the freshwater microalgae Euglena gracilis, a model microorganism, ubiquitously distributed and resistant to several environmental stressors. The ecto-phosphatase activity is acidic, stimulated by copper and presents the following apparent kinetic parameters: Km = 2.52 ± 0.12 mM p-NPP and Vmax = 3.62 ± 0.06 nmol p-NP × h-1 × 106 cells. We observed that zinc, orthovanadate, molybdate, fluoride, and inorganic phosphate inhibit the ecto-phosphatase activity with different magnitudes. Fluoroaluminate complexes are also inhibitors of this ecto-phosphatase activity. They can be formed in the enzyme reaction conditions and are likely to occur in a natural environment where E. gracilis can be found. The ecto-phosphatase activity is constant through the culture growth phases and is negatively modulated after continuous subculturing in the dark when a shift from phototrophic to the heterotrophic metabolism is likely. The analysis of those biochemical parameters may contribute to understanding the role of E. gracilis ecto-phosphatase activity in natural environments.
Ecto-enzymes can be defined as membrane-bound proteins that have their active site facing the extracellular millieu. In trypanosomatids, the physiological roles of these enzymes remain to be completed elucidated; however, many important events have already been related to them, such as the survival of parasites during their complex life cycle and the successful establishment of host infection. This chapter focuses on two remarkable classes of ecto-enzymes: ecto-nucleotidases and ecto-phosphatases, summarizing their occurrence and possible physiological roles in Leishmania and Trypanosoma genera. Ecto-nucleotidases are characterized by their ability to hydrolyze extracellular nucleotides, playing an important role in purinergic signaling. By the action of these ecto-enzymes, parasites are capable of modulating the host immune system, which leads to a successful parasite infection. Furthermore, ecto-nucleotidases are also involved in the purine salvage pathway, acting in the generation of nucleosides that are able to cross plasma membrane via specialized transporters. Another important ecto-enzyme present in a vast number of pathogenic organisms is the ecto-phosphatase. These enzymes are able to hydrolyze extracellular phosphorylated substrates, releasing free inorganic phosphate that can be internalized by the cell, crossing the plasma membrane through a Pi-transporter. Ecto-phosphatases are also involved in the invasion and survival of parasite in the host cells. Several alternative functions have been suggested for these enzymes in parasites, such as participation in their proliferation, differentiation, nutrition and protection. In this context, the present chapter provides an overview of recent discoveries related to the occurrence of ecto-nucleotidase and ecto-phosphatase activities in Leishmania and Trypanosoma parasites.
Rhinocladiella aquaspersa is an etiologic agent of chromoblastomycosis, a subcutaneous chronic infectious disease. In the present work, we found that the three morphological forms of this fungus (conidia, mycelia and sclerotic bodies) expressed different levels of ecto-phosphatase activity. Our results demonstrated that surface conidial enzyme is an acid phosphatase, inhibited by sodium salts of molybdate, orthovanadate and fluoride and that the inhibition caused by orthovanadate and molybdate was irreversible. The conidial ecto-phosphatase efficiently released phosphate groups from different phosphorylated substrates, causing a higher rate of phosphate removal when p-nitrophenylphosphate was used as substrate. This ecto-enzyme of R. aquaspersa is modulated by Co(2 +) ions and inorganic phosphate (Pi). Accordingly, removal of Pi from the culture medium resulted in a marked (121-fold) increase of ecto-phosphatase activity. Surface phosphatase activity is apparently involved in fungal adhesive properties, since the attachment of R. aquaspersa to epithelial cells was reversed by the pre-treatment of the conidia with orthovanadate, molybdate and anti-phosphatase antibody. Corroborating this finding, conidia with greater ecto-phosphatase activity (grown in Pi-depleted medium) showed higher adherence to epithelial cells than fungi cultivated in the presence of Pi.
The cellular plasma membrane contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells. Ecto-phosphatases are ecto-enzymes that presumably hydrolyze extracellular phosphorylated substrates, releasing free inorganic phosphate. Although, several alternative functions have been suggested for these enzymes, such as participation in proliferation, differentiation, adhesion, virulence, and infection, little is known about the physiological roles of these enzymes in protozoa parasites. In this review, we discuss the principal features of ecto-phosphatases in protozoan parasites that are causative agents of important diseases such as Chagas' disease, leishmaniasis, amoebiasis, giardiasis, trichomoniasis and, sleeping sickness.
Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41+/-2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl2, MnCl2 and ZnCl2 were able to increase the (p-NPP) hydrolysis while CdCl2 and CuCl2 inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.
The ability of protein-tyrosine phosphatases (PTPases) to catalyze the hydrolysis of simple aromatic phosphates has been recognized for some time. However, these compounds are significantly poorer substrates than their peptide-based counterparts containing phosphotyrosine. Consequently, the effort to create potent PTPase substrates has predominately focused on the use of peptidic carriers to deliver the phosphotyrosine moiety to the enzyme active site. We now report the synthesis and evaluation of several low molecular weight aromatic phosphates that serve as robust substrates for the rat PTPase, PTP1. We initially surveyed the ability of PTP1 to catalyze the hydrolysis of a variety of phenyl phosphate structural variants. Sterically demanding substituents positioned ortho and (to a lesser extent) meta to the phosphate group severely compromise the ability of these species to serve as phosphatase substrates. However, both benzylic and negatively charged substituents para to the hydrolyzable phosphate dramatically promote hydrolytic efficiency, which appears to be augmented through a dramatic enhancement in the affinity of the substrate for PTP1. The best substrate examined in this study exhibits a K of 16 ± 3 μM. In addition, it serves as an inhibitor of the PTP1-catalyzed hydrolysis of p-nitrophenyl phosphate with a K of 4.9 ± 0.7 μM. The extraordinary structural simplicity of this compound, as well as those of several others described herein, provides a promising starting point for the design of potent PTPase inhibitors.
In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium
orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were
assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of
this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction,
confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant
of living H. m. muscarum.
ABSTRACT In the present work ectophosphatase activities of three trypanosomatid parasites of plants were characterized using intact cells. Phytomonas françai, Phytomonas mcgheei, and Herpetomonas sp. hydrolyzed p-nitro-phenylphosphate at a rate of 5.40, 7.28, and 25.58 nmol Pi/mg of protein per min, respectively. Experiments using classical inhibitors of acid phosphatases such as sodium orthovanadate (NaVO(3)) and sodium fluoride (NaF) showed a decrease in phosphatase activities. Lithium fluoride (LiF) and aluminum chloride (AlCl(3)) were also used. Although AlCl3 had no effect, LiF was able to promote a decrease in the phosphatase activities. Interestingly, the inhibition caused by LiF was enhanced by the addition of AlCl3 during the reaction, probably due to the formation of fluoroaluminate complexes. This effect was confirmed by cytochemical analysis. In this assay, electron-dense cerium phosphate deposits were visualized on the external surface of the three parasites.
Protein-tyrosine phosphatases (PTPases) are believed to exhibit restricted specificity toward phosphotyrosine. I demonstrate here that both the Yersinia PTPase and rat PTP1 can dephosphorylate alkyl phosphates such as flavin mononucleotide, pyridoxal 5'-phosphate, D-glucose 6-phosphate, DL-alpha-glycerophosphate, O-phospho-L-serine, and O-phospho-L-threonine. The kcat values for alkyl phosphates are orders of magnitude slower than those for aryl phosphates such as p-nitrophenyl phosphate and O-phospho-L-tyrosine, reflecting the intrinsic lower chemical reactivity of the alkyl phosphates. In addition, the kcat values for the PTPase-catalyzed hydrolysis of alkyl phosphates are similar to the kcat values for the PTPase-catalyzed 18O exchange reaction between inorganic phosphate and water. I conclude that the rate-limiting step for the hydrolysis of alkyl phosphates has changed to the phosphorylation of the PTPases, i.e. the formation of the phosphoenzyme intermediate. The implications of the results described in this report in terms of studying the PTPase catalytic mechanism and their potential application in developing selective PTPase inactivators are discussed.
Our previous studies have shown that exogenous ATP induces cell growth inhibition in transformed mouse fibroblasts, 3T6 cells, whereas the growth of their nontransformed counterparts, Swiss 3T3 cells, is only slightly affected. In this study a similar selective, ATP-induced growth inhibition was found in Balb/c SV40-3T3 cells and in primary cultures of adenovirus-transformed murine fibroblasts. The inhibitory activity was found in the conditioned media of ATP-treated cultures. Several lines of evidence have shown that ectoprotein kinase (ecto-PK) plays a major role in the ATP-induced growth inhibition. (a) There is a good correlation between the activity of ecto-PK and the ability of ATP to induce cell growth inhibition. (b) The removal of the ecto-PK from the cell surface prevents the ATP-induced growth inhibition. (c) Addition of the removed enzyme to the cell culture reconstitutes the ability of ATP to induced growth inhibition. (d) Serum-containing, or serum-free, conditioned media from untreated cultures gain an inhibitory activity after their phosphorylation, and dephosphorylation of conditioned media from ATP-treated cultures results in the loss of the inhibitory activity. (e) Growth medium by itself does not inhibit cell proliferation after its phosphorylation. The findings described in d and e indicate, as well, that the ATP-induced growth inhibitor is produced by the cells. The putative inhibitor was found to be a protein, with an apparent molecular mass of 13 kDa. The selectivity of the inhibition for transformed cells is due to the higher level of ecto-PK in these cells, as well as to their higher susceptibility to the inhibitor, as compared with their non-transformed counterparts.
The role of protein kinases in organisms that diverged early in the eukaryotic lineage is relatively unexplored. In this study, we determined that primitive parasitic protozoa possess multiple protein-serine kinases and inferred the presence of protein-tyrosine kinases through sensitive immunoblotting techniques. To further explore the role of protein kinases in parasite development, we examined the activity of eight renaturable protein kinases during the life cycle of the protozoan parasite Trypanosoma brucei. The activities of six protein-serine/threonine kinases were regulated during development, with several distinct patterns of regulation. In addition, an 89-kDa protein kinase was detected in dividing cells but not in nondividing cells. Our data indicate that even the most primitive eukaryotes possess a large complement of protein kinases, including protein-tyrosine kinases as well as protein-serine/threonine kinases. The data further suggest that protein kinases may play a pivotal role in regulation of proliferation and differentiation in protozoa.
Receptor-like protein tyrosine phosphatases (receptor-PTPs) represent a novel family of transmembrane proteins that are thought to play important roles in cellular regulation. They consist of a cytoplasmic catalytic region, a single transmembrane segment and an extracellular, putative ligand-binding domain, but the nature of their physiological ligands is unknown. We have recently cloned a new receptor-PTP (RPTP mu), the ectodomain of which includes an Ig-like and four fibronectin type III-like domains, suggesting that RPTP mu may be involved in cell-cell or cell-matrix interactions. To test this hypothesis, we expressed RPTP mu in insect Sf9 cells using recombinant baculovirus. We demonstrate that RPTP mu dramatically promotes cell-to-cell adhesion in a homophilic, Ca(2+)-independent manner. No adhesion is observed in Sf9 cells expressing a chimeric RPTP mu molecule containing the extracellular domain of the epidermal growth factor receptor. Furthermore, cells expressing an enzymatically inactive, point-mutated RPTP mu or a truncated form of RPTP mu, lacking the entire catalytic region, show adhesive properties indistinguishable from those of wild-type RPTP mu, indicating that the catalytic domain is not essential for RPTP mu-mediated adhesion. These results assign a physiological role for RPTP mu in signaling cell-cell recognition.
We report the first characterization of plasma-membrane-bound tyrosine phosphatase activity in the haemoprotozoan. Trypanosoma brucei. Several enzymic properties of the membrane fraction were identical to other protein tyrosine phosphatases (PTPases), such as (a) insensitivity to inhibitors of other protein phosphatases, including tetramisole, sodium tartrate and okadaic acid, (b) inhibition by sodium vanadate, and (c) activation by spermidine. Additionally, T. brucei PTPase activity presented two novel features, an acidic pH optimum at pH 4.0-5.0 and a very low Km value (2.5 nM) for the specific synthetic substrate, Tyr(P)Raytide. Higher Km values of 170 nM for Tyr(P)-RCML (RCML, reduced, carboxamidomethylated and maleylated lysozyme) and of 3 mM for the non-specific inorganic substrate p-nitrophenyl phosphate, suggested that the PTPase activity of T. brucei was substrate specific. Reconstitution experiments on bloodstream-stage membrane proteins revealed that three polypeptides of 148, 115 and 72 kDa contained vanadate-inhibitable PTPase activity. Modulator assays revealed that the 72-kDa protein was responsible for the observed spermidine stimulation, but indicated that the modulator profile of the 148-kDa protein was most similar to the whole membrane fraction. Furthermore, the PTPase activity of T. brucei was life-cycle-stage regulated. Neither the whole membrane fraction nor the reconstituted proteins of the procyclic insect stage dephosphorylated tyrosine residues.
Tyrosine phosphorylation is an important mechanism of cell regulation and has been recently implicated in defense strategies against a variety of pathogens. We have investigated the involvement of protein tyrosine kinase activity in the Leishmania attachment, invasion and survival within macrophages, as well as promastigote ability to trigger tyrosine phosphorylation, which could contribute to leishmanicidal activity. Treatment of murine macrophage monolayers with genistein, herbimycin A, tyrphostin 25 or staurosporine prior to infection decreased parasite invasion in a dose-dependent manner. Contrary, addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, phosphotyrosine and p-nitrophenyl phosphate to the interaction medium, significantly increased parasite binding and internalization, whereas phosphoserine and phosphothreonine had no effect. The phosphatase activity of intact promastigotes was greater than that of macrophages. Western blot analysis revealed tyrosine-phosphorylated bands from 198 to 28 kDa following macrophage challenge with promastigotes. Uninfected macrophages displayed no detectable tyrosine phosphorylated proteins, possibly indicating an inducible process, while in parasites it was constitutive, as seen by the presence of 42, 40 and 35 kDa phosphoproteins on the Leishmania lysates. Immunofluorescence and immunogold detection of phosphotyrosine residues in some promastigote-macrophage attachment areas, but not in the vicinity of ingested parasites, suggest that Leishmania-induced tyrosine phosphorylation is an early, local and short-lived event. Genistein treatment of Leishmania-infected cells significantly enhanced the parasite burden. This antagonist also diminished nitric oxide production in resting and interferon gamma/lipopolysaccharide-activated infected macrophages, which may account for the increased parasite survival. We propose that protein tyrosine kinase-linked pathways regulate the Leishmania promastigote invasion and the macrophage microbicidal activity.
The results presented in this paper indicate that procyclic forms of Trypanosoma brucei possess a phosphatase activity detected in the external cell surface able to hydrolyze about 0.7 nmol.mg-1.min-1 p-nitrophenylphosphate. A faster rate of hydrolysis was observed when membrane-enriched fractions were used. This activity is weakly sensitive to 1 mM NaF, 10 mM tartrate and 10 mM levamizole but strongly inhibited by 0.1 mM vanadate. Inhibition by both NaF and vanadate have a competitive character. This phosphatase activity decreases by increasing the pH from 6.8 to 8.4, a pH range in which cell viability was maintained during at least 1 hour. In the membrane-enriched fractions this phosphatase activity showed to be an acid phosphatase. In addition, intact cells could catalyze the dephosphorylation of [32P]phosphocasein phosphorylated at serine and threonine residues.
A complete inactivation is observed after a 3 min pre-incubation at 70 degrees C with mitochondrial F0F1-ATPase complex depleted of the ATPase natural inhibitor protein (ammonium-Sephadex submitochondrial particles) and activated MgATP-submitochondrial particles (particles that after a 4 h-pre-incubation at 42 degrees C released the endogenous inhibitor protein). However, latent MgATP-submitochondrial particles (particles containing the inhibitor protein) pre-incubated under the same conditions are totally inactivated only after 15 min of pre-incubation. When ammonium-Sephadex particles are reconstituted with 20 micrograms/ml of purified ATPase inhibitor protein there is an increase of 15-fold in the half-time for thermal inactivation (t0.5), showing that the inhibitor protein protects the mitochondrial F0F1-ATPase complex against thermal inactivation.
Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phospho-amino-acids and 32P-casein under physiologically appropriate conditions. PNPP was hydrolysed at a rate of 80 nmol.mg-1.h-1 in the presence of 5 mM MgCl2, pH 7.2 at 30 degrees C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mM MgCl2. Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2+ (I50 = 0.43 mM). In the absence of Mg2+ (basal activity) the stimulating half concentration (S0.5) for PNPP was 1.57 mM, while at saturating MgCl2 concentrations the corresponding S0.5 for PNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mM. The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phosphothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse 32P-casein phosphorylated on serine and threonine residues but only in the presence of MgCl2. These results indicate that in the amastigote form of T. cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-amino acids and phosphoproteins under physiological conditions.
In this study we report the effects of sulfated polysaccharides on the ecto-ATPase activity of intact cells of Leishmania tropica. Increasing concentrations of dextran sulfate stimulated progressively the ecto-ATPase activity, but did not modify other ecto-enzymes present on the surface of this parasite, such as 5'nucleotidase, 3'nucleotidase and a membrane-bound acid phosphatase activity. This stimulation was not observed when other sulfated polysaccharides such as chondroitin sulfates and heparin were tested. It depends on size and charge of the dextran sulfated molecule. When the cells were incubated in the presence of dextran sulfate Mr 8,000; 40,000 and 500,000 the stimulation of the ecto-ATPase activity was 11%; 23%; and 63%, respectively, and the stimulation was not observed when desulfated dextran (Mr 40,000) was used. The effects of dextran sulfate also depend on pH of the medium. At pH 7.5, the stimulation was over 60%, whereas at pH 8.5 only 25%. The effects of dextran sulfate 500,000 on the ecto-ATPase activity was totally abolished by spermidine and partially by putrescine, two polyamines synthesized and released by Leishmania.
In the present work we have partially characterized an ecto-phosphatase activity in Crithidia deanei, using viable parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 3.55 +/- 0.47 nmol Pi/h x 10(8) cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this phosphatase activity and the value of the apparent Km for p-NPP was 5.35 +/- 0.89 mM. This phosphatase activity was inhibited by the product of the reaction, the inorganic phosphate. Experiments using classical inhibitors of acid phosphatases, such as ZnCl2 and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and ammonium molybdate, showed a decrease in this phosphatase activity, with different patterns of inhibition.
SYNOPSIS. A new culture medium (SM), based on the amino‐acid composition of tsetse hemolymph and containing fetal bovine serum, was designed for the maintenance of tsetse organs and the cultivation of various trypanosomatids. For optimum growth 20% (v/v) serum was required. The medium supported prolonged peristalsis of the alimentary tract and salivary glands of pre‐emerged Glossina morsitans morsitans. In established cultures, derived from bloodstream forms of pleomorphic Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense strains, inocula of ∼ 10 ⁶ procyclics/ml yielded 4–5 × 10 ⁷ organisms/ml after 4 or 5 days of incubation at 28 C. Bloodstream forms of a cloned monomorphic T. b. brucei strain were also able to transform into procyclics, which, however, multiplied at a lower rate, with maximum yields of ∼ 2 × 10 ⁷ after 5 days. Cultures of Trypanosoma congolense and of a nearly monomorphic Trypanosoma brucei gambiense strains could be established in SM medium only in the presence of tsetse alimentary tract. The procyclic trypomastigotes of these species, adapted to SM medium and able to grow in it without Glossina organs, gave maximum populations of ∼ 4.5 × 10 ⁷ cells/ml.
Promastigotes of Leishmania donovani , cultivated routinely in a diphasic Table's medium, multiplied actively upon being transferred into SM medium, producing yields of ∼ 4 × 10 ⁷ cells/ml.
Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.
Protein tyrosine phosphatases (PTPs) represent a diverse family of enzymes that exist as integral membrane and nonreceptor
forms. The PTPs, with specific activities in vitro 10 to 1000 times greater than those of the protein tyrosine kinases would
be expected to effectively control the amount of phosphotyrosine in the cell. They dephosphorylate tyrosyl residues in vivo
and take part in signal transduction and cell cycle regulation. Most of the transmembrane forms, such as the leukocyte common
antigen (CD45), contain two conserved intracellular catalytic domains; but their external segments are highly variable. The
structural features of the transmembrane forms suggest that these receptor-linked PTPs are capable of transducing external
signals; however, the ligands remain unidentified. A hypothesis is proposed explaining how phosphatases might act synergistically
with the kinases to elicit a full physiological response, without regard to the state of phosphorylation of the target proteins.
Four major serine/threonine-specific protein phosphatase catalytic subunits are present in the cytoplasm of animal cells. Three of these enzymes, PP-1, PP-2A, and PP-2B, are members of the same gene family, while PP-2C appears to be distinct. PP-1, PP-2A, and PP-2B are complexed to other subunits in vivo, whereas PP-2C has only been isolated as a monomeric protein. PP-1, PP-2A, and PP-2C have broad and overlapping specificities in vitro, and account for virtually all measurable activity in tissue extracts toward a variety of phosphoproteins that regulate metabolism, muscle contractility, and other processes. Their precise functions in vivo are unknown, although important clues to the physiological roles of PP-1 and PP-2A are provided by the effects of okadaic acid and by the subcellular localization of PP-1. The active forms of PP-1 are largely particulate, and their association with subcellular structures is mediated by "targetting subunits" that direct PP-1 to particular locations, enhance its activity toward certain substrates, and confer important regulatory properties upon it. This concept is best established for the glycogen-bound enzymes in skeletal muscle and liver (PP-1G) and the myofibrillar form (PP-1M) in skeletal muscle. The activities of PP-1 and PP-2B are controlled by the second messengers cyclic AMP and calcium. The activity of PP-2B is dependent on calcium and calmodulin, while PP-1 is controlled in a variety of ways that depend on the form of the enzyme and the tissue. PP-1 can be inhibited by cyclic AMP in a variety of cells through the A-kinase-catalyzed phosphorylation of inhibitor-1 and its isoforms. Phosphorylation of the glycogen-binding subunit of PP-1G by A-kinase promotes translocation of the catalytic subunit from glycogen particles to cytosol in skeletal muscle, inhibiting the dephosphorylation of glycogen-metabolizing enzymes. Allosteric inhibition of hepatic PP-1G by phosphorylase a occurs in response to signals that elevate cyclic AMP or calcium, and prevents the activation of glycogen synthase in liver. PP-1 can also be activated indirectly by calcium through the ability of PP-2B to dephosphorylate inhibitor-1. This control mechanism may operate in dopaminoceptive neurones of the brain and other cells. The inactive cytosolic form of PP-1 (PP-1I) can be activated in vitro through the glycogen synthase kinase-3-catalyzed phosphorylation of its inhibitory subunit (inhibitor-2), but the physiological significance is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)
Cell fractionation of bloodstream Trypanosoma rhodesiense, using isopycnic sucrose gradient centrifugation, reveals acid phosphatase activities against a range of substrates to be associated, to varying degrees, with subcellular particle populations identified as derived from flagella pocket membrane and surface membrane. Using these same substrates (alpha and beta glycerophosphate, p-nitrophenyl phosphate and glucose-6-phosphate) at least two distinct acid phosphatase activities can be distinguished. One is thermolabile (approximately 80% inactivated after 30 min. at 60 degrees C), sensitive to tartrate (50% inhibited at 1.8 mM Na tartrate) with a pH optimum approximately 4.5 and appears to exhibit little substrate preference. The other acid phosphatase is relatively heat stable (approximately 30% inactivated), insensitive to tartrate (greater than 5.0% inhibited using 1.8 mM Na tartrate) exhibits a somewhat higher pH optimum (approximately 6.0) and is more substrate specific (6X more active toward glucose-6-PO4 than beta-glycerophosphate). Further cell fractionation experiments reveal 85% of the tartrate sensitive acid phosphatase to be associated with flagella pocket membrane and to account for 80% of the organisms hydrolytic activity toward beta-glycerophosphate. The tartrate resistant acid phosphatase however, has a much less exclusive localization being almost equally distributed between surface membrane (40%) and flagella pocket membrane (60%).
Virulent and avirulent clones of Leishmania donovani promastigotes were examined for their acid phosphatase activity. The acid phosphatase activity of whole-cell lysates of virulent clones was 1.5 to 2.0 times higher than that of avirulent clones. Pellet fractions (260,000 x g, 30 min) from sonicated promastigotes of a virulent clone and an avirulent clone contained 60 and 40% of the total enzyme activity, respectively. Membrane-bound acid phosphatase was extracted with Triton X-100 from the pellet. This membrane-bound phosphatase activity was 2.4-fold higher in virulent organisms than in avirulent organisms. The membrane acid phosphatase exhibited two distinct bands on polyacrylamide gels stained for enzyme activity. One diffuse, faster-migrating band showed identical electrophoretic mobility in both virulent and avirulent clones, although a higher enzymatic activity was observed with the extract from virulent cells. In contrast, a slower-migrating band was different between the two clones in the mobility. These results suggest that membrane-bound acid phosphatase was quantitatively and qualitatively different between virulent and avirulent promastigotes of L. donovani. In addition, virulent cells produced a relatively high level of acid phosphatase throughout the growth in culture.
Forty isolates of Leishmania, representing all major species infecting humans and one parasite of lizards, were examined for their ability to secrete an extracellular acid phosphatase activity. This enzyme, which was originally described and characterized from a Sudanese strain of L. donovani, was detected in the culture supernatants of all species of promastigotes examined except for L. major and L. tarentolae. There were quantitative differences among species in their levels of enzyme activity and in the sensitivity of the exoenzyme to inhibition by L(+) tartrate. Upon electrophoresis in nondenaturing polyacrylamide gels, extracellular acid phosphatase from L. braziliensis panamensis, L. tropica, and L. mexicana showed distinctive patterns which were similar for all isolates of a given species, while enzymes from L. donovani isolates differed from one another in relative electrophoretic mobility. Enzymes from all species appeared heterogeneous, showing either discrete multiple bands or single diffuse bands on gels stained for enzyme activity. Although the biological function of the extracellular acid phosphatase is presently unknown, the exoenzyme may be of value as a diagnostic or taxonomic characteristic.
To study the role of parasite protein kinase C (PKC) activity in the uptake of Leishmania amazonensis by mononuclear phagocytes we treated the parasites with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and/or sphingosine, before interaction assays. Promastigotes of Leishmania amazonensis were incubated with 20 ng/ml TPA and/or 50 ng/ml sphingosine before the interaction with murine peritoneal macrophages. The short treatment enhanced about 200% the parasite association with the host cells, whereas the sphingosine treatment reduced about 50% the promastigote binding, as did the prolonged TPA treatment. The binding of cells treated with both drugs was not significantly altered. Biochemical and cytochemical data indicate that the protein kinase C agonists TPA and sphingosine, respectively, increased and decreased acid phosphatase (AcP) activity. The addition of sodium tartrate, a secreted AcP inhibitor, suppressed the TPA enhancing effects, but did not affect the basal parasite binding observed in control cells. The supernatants of TPA-treated L. amazonensis promastigotes increased the parasite association by about the same extent as the TPA treatment, and this effect was also abolished by tartrate. Although TPA did not enhance the association of L. major, a species that does not secrete AcP, the supernatants of TPA-treated L. amazonensis increased it in a tartrate-sensitive manner. The results suggest that Leishmania amazonensis PKC activity may modulate its interaction with macrophages via secreted AcP.
Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.
L. Donovani promastigotes were grown to late-log and 3-day stationary phase to determine the level of protein tyrosine phosphatase activity in crude extracts and in fractions following gel filtration column chromatography. Over 90% of the activity was soluble in a low salt extraction buffer in both phases of growth. Several peaks of activity were resolved following gel filtration of the crude extracts indicating that multiple tyrosine phosphatases are present in these cells. Tyrosine phosphatase activity was lower in 3-day stationary than in late log-phase cells and a reduction in the major peak of activity, eluting in a gel fraction corresponding to an Mr
of approximately 168kDa, was observed.In vivo tyrosine phosphorylation was revealed by Western blot analysis. The degree of phosphorylation of at least two proteins differed in cells obtained from late log phase cultures as compared with 3-day stationary phase cultures. These observations indicate that changes in the balance between tyrosine phosphorylation and dephosphorylation occur with increasing culture age.
Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover,VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled.
The ability of protein-tyrosine phosphatases (PTPases) to catalyze the hydrolysis of simple aromatic phosphates has been recognized for some time. However, these compounds are significantly poorer substrates than their peptide-based counterparts containing phosphotyrosine. Consequently, the effort to create potent PTPase substrates has predominantly focused on the use of peptidic carriers to deliver the phosphotyrosine moiety to the enzyme active site. We now report the synthesis and evaluation of several low molecular weight aromatic phosphates that serve as robust substrates for the rat PTPase, PTP1. We initially surveyed the ability of PTP1 to catalyze the hydrolysis of a variety of phenyl phosphate structural variants. Sterically demanding substituents positioned ortho and (to a lesser extent) meta to the phosphate group severely compromise the ability of these species to serve as phosphatase substrates. However, both benzylic and negatively charged substituents para to the hydrolyzable phosphate dramatically promote hydrolytic efficiency, which appears to be augmented through a dramatic enhancement in the affinity of the substrate for PTP1. The best substrate examined in this study exhibits a Km of 16 +/- 3 microM. In addition, it serves as an inhibitor of the PTP1-catalyzed hydrolysis of p-nitrophenyl phosphate with a Ki of 4.9 +/- 0.7 microM. The extraordinary structural simplicity of this compound, as well as those of several others described herein, provides a promising starting point for the design of potent PTPase inhibitors.
In vitro, living bloodstream forms of Trypanosoma congolense were shown to hydrolyse p-nitrophenyl phosphate, a substrate for phosphatases. This activity appears to be from an acid phosphatase because it was enhanced at low pH values, was inhibited by the acid phosphatase inhibitor sodium fluoride, and was not inhibited by the alkaline phosphatase inhibitor tetramisole. The activity did not appear to be secreted into the surrounding medium by the living parasites although phosphatase activity could be detected in the surrounding medium when dead or dying parasites were present. Studies at various temperatures indicated that at least some of this acid phosphatase activity may be associated with the surface of the parasites, rather than with endocytic or intracellular systems. This was supported by subcellular fractionation of radiolabelled parasites which showed some cosedimentation of acid phosphatase activity with radiolabelled iodine. Histochemical studies of the parasites also supported this conclusion. Electron microscopical examination of trypanosomes incubated with lead nitrate and p-nitrophenyl phosphate showed lead phosphate deposits on the surface of the parasites in addition to the expected localisation in the flagellar pocket. We conclude that Trypanosoma congolense possesses a surface-bound acid phosphatase.
ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).
Live T. cruzi trypomastigotes and amastigotes possess ecto-protein tyrosine phosphatase activity as indicated by the ability of intact cells to catalyze dephosphorylation of tyrosine phosphorylated myelin basic protein, [32P]TyrRaytide, phosphotyrosine, or the phosphotyrosine analog p-nitrophenylphosphate (p-NPP). The dephosphorylation of myelin basic protein (MBP) and p-NPP was inhibited by sodium o-vanadate, zinc chloride and NaF, while dephosphorylation of [32P]TyrRaytide was insensitive to zinc chloride but sensitive to o-vanadate and NaF. In contrast, live cells were not able to dephosphorylate serine or threonine phosphorylated peptides ([32P]Kemptide) or proteins ([32P]RCM-lysozyme and [32P]MBP).
In the present work ecto-phosphatase activity in Herpetomonas muscarum muscarum has been characterized using live parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 4.27 nmol Pi/mg of protein.min. A pH curve was generated, in which these intact flagellates showed the highest phosphatase activity at pH 6.5. Classical inhibitors for acid phosphatase, such as sodium orthovanadate, sodium tartrate, and ammonium molybdate, were used in the experiments and showed different patterns of inhibition. Lithium fluoride, aluminum chloride, and fluoroaluminate complexes were also tested. Although lithium fluoride and fluoroaluminate complexes were capable of inhibiting the phosphatase activity, aluminum chloride stimulated this enzyme. Cytochemical analysis showed the localization of this enzyme on the parasite surface. This ecto-phosphatase activity was also significantly diminished when the parasites were treated with 10(-6) M platelet-activating factor (PAF), a potent phospholipid mediator that promoted cellular differentiation in this parasite.
The involvement of tyrosine phosphorylation during macrophage infection with Leishmania amazonensis amastigotes was investigated. PTK antagonists such as genistein, herbimycin A, geldanamycin and tyrphostin 25 had no significant effect on adhesion to, or entry into, murine peritoneal macrophages, but increased parasite intracellular survival. LPS-induced tyrosine phosphorylation of target host proteins assessed by immunoprecipitation and Western blot was impaired or reversed by living amastigotes soon after 60 min-infection. Such reversion was not due to parasite-secreted molecules but was contact-dependent, as assessed by cytochalasin D treatment of macrophage monolayers prior to infection. Paraformaldehyde-fixed or sodium vanadate-treated amastigotes exerted no significant effect on overall macrophage tyrosine phosphorylation. Immunoprecipitation of proteins employing 4G10 anti-phosphotyrosine antibody followed by Western blotting revealed that tyrosine phosphorylation of 120, 85, 60, 44 and 35 kDa proteins was selectively reversed by amastigote infection. Inhibition, measured by densitometry was from about 66-100% of uninfected cells. None of these proteins was immunoprecipitated from amastigote-infected macrophage lysates but all of them except for p85 were recovered after treatment of parasites with 100 microM sodium orthovanadate prior to infection, a treatment that inhibits Leishmania amastigote protein ecto-phosphatase. The 44 kDa protein was identified as ERK1 MAP kinase (MAPK) by Western blot. Amastigote infection also decreased tyrosine phosphorylation induced by zymosan particles. Vanadate treatment of amastigotes prior to infection significantly decreased parasite intracellular survival. The action of a putative leishmanial ecto-protein phosphatase (PPase) is suggested.
The effects of platelet-activating factor (PAF) on the ecto-phosphatase activity of Trypanosoma cruzi were investigated. Living parasites hydrolyzed p-nitrophenyl phosphate (p-NPP) at a rate of 5.71 +/- 0.37 nmol P(i) mg(-1) min(-1). This ecto-phosphatase activity increased to 8.70 +/- 1.12 nmol P(i) mg(-1) min(-1) when the cells were grown in the presence of 10(-9) M PAF. This effect was probably due to stimulation of the release of the ecto-phosphatase and/or the secretion of an intracellular phosphatase to the extracellular medium, as suggested by cytochemical analysis. Modulation of the ecto-phosphatase activity was also observed when PAF was added during the time course of the reaction. WEB 2086, a competitive PAF antagonist, was able to revert PAF effects when both were used at the same concentration. When PAF was added to a membrane enriched fraction preparation of T. cruzi, no alteration on the phosphatase activity was observed. This result suggests an involvement of intracellular signaling, as PAF was only effective on intact cells. Sphingosine and phorbol-12-myristate-13-acetate (PMA) were then used to investigate a possible involvement of protein kinase C (PKC) with PAF-induced phosphatase secretion. Sphingosine by itself stimulated the secretion of a phosphatase but did not significantly interfere with PAF effects on this enzyme. On the other hand, PMA was able to abrogate PAF-induced release of this phosphatase. These data are highly suggestive of a putative involvement of signal transduction mediated by a ligand of mammalian origin (PAF), through PKC and a specific receptor located on the cell surface of the human parasite Trypanosoma cruzi.
We purified an ecto-phosphatase of 115 kDa (TryAcP115) specifically expressed by bloodstream forms of Trypanosoma brucei. The corresponding gene coded for a 45-kDa protein potentially including a signal peptide, a membrane-spanning domain and an N-terminal domain containing 8 N-glycosylation sites. There was no significant sequence homology with other phosphatases. Antiserum to the Escherichia coli recombinant N-terminal domain, Petase7, recognized a protein of 55 kDa in Western blots after deglycosylation of the TryAcP115 protein by N-glycosidase F. Immunofluorescence and trypsin treatment of living parasites showed that TryAcP115 was localized to the surface of the parasite and that its N-terminal domain was oriented extracellularly. The recombinant N-terminal domains, expressed in E. coli and Leishmania amazonensis, harbored phosphatase activity against Tyr(P)-Raytide, Ser(P)-neurogranin, and ATP. The enzymatic properties of native TryAcP115 and the recombinant proteins for the substrate Tyr(P)-Raytide were virtually identical and included: (i) K(m) and V(max) values of 15 nM and 200 pmol/min/mg, (ii) no requirement for divalent cations, and (iii) sensitivity to vanadate, sodium fluoride, and tartrate, but insensitivity to okadaic acid and tetramisole. Although the function of TryAcP115 remains unknown, a differentially expressed, unique ecto-phosphatase could regulate growth or influence parasite-host interactions and might provide a useful target for chemotherapy.
In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl(2) to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46mM MgCl(2). The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2), but not by SrCl(2). The Mg(2+)-dependent ATPase presents two apparent K(m) values for Mg-ATP(2-) (K(m1)=0.03 mM and K(m2)=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg(2+)-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60-70% of the Mg(2+)-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg(2+)-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2',2'-disulfonic acid (DIDS) 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. This ecto-ATPase was stimulated by more than 90% by 50mM D-galactose. Since previous results showed that D-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg(2+)-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity.
The plasma membrane of cells contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells. Cell membrane ecto-ATPases are integral membrane glycoproteins that are millimolar divalent cation-dependent, low specificity enzymes that hydrolyze all nucleoside triphosphates. Their physiological role is still unknown. However, several hypotheses have been suggested such as; (i). protection from cytolytic effects of extracellular ATP, (ii). regulation of ectokinase substrate concentration, (iii). termination of purinergic signaling, (iv). involvement in signal transduction, and (v). involvement in cellular adhesion. In this review, the biochemical properties and possible functions of the ecto-ATPases of different protozoa are summarized.
Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [(32)P]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T. brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3-5 nmol pNP min(-1) mg(-1), linearly for up to at least 30 min. The activity was not significantly increased by Mg(2+), Mn(2+), Ca(2+) and Co(2+), but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn(2+) and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5-5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min(-1) mg(-1)) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a K(m) value of 0.29 mM with pNPP and was insensitive to the Fe(2+)/H(2)O(2)/ascorbate system.
- A P Lemos
- A L F Souza
- A A S Pinheiro
- M Berrêdo-Pinho
- J R Meyer-Fernandes
Lemos, A.P., Souza, A.L.F., Pinheiro, A.A.S., Berrêdo-Pinho, M., Meyer-Fernandes, J.R., 2002. Ecto-phosphatase
activity on the cell surface of Crithidia deanei. Z. Naturforsch. 57, 500–505.
- E C Fernandes
E.C. Fernandes et al. / Veterinary Parasitology 118 (2003) 19-28