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Ecto-ATPases in protozoa parasites: Looking for a function
Abstract
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.
... The ecto-nucleotidases are located on the plasma membrane surface, where they hydrolyze extracellular nucleotides, and have been described in different cell types (Knowles, 2011;Meyer-Fernandes, 2002;Zimmermann, 2000Zimmermann, , 2012. Early reports have described the ecto-ATPase activities of different protozoan parasites, such as Toxoplasma gondii (Nakaar et al., 1998), Leishmania tropica (Meyer-Fernandes et al., 1997), L. amazonensis (de Almeida Marques-da-Silva et al., 2008;Pinheiro et al., 2006), Leishmania braziliensis (Rezende-Soares et al., 2010), Entamoeba histolytica (Barros et al., 2000), Trypanosoma cruzi (Bernardes et al., 2000;Bisaggio et al., 2003;Fietto et al., 2004;Meyer-Fernandes et al., 2004), Trypanosoma rangeli and Trypanosoma brucei (de Souza Leite et al., 2007). ...
... These results suggest that the development of drug resistance in Leishmania might involve alterations that can interfere with its differentiation into a metacyclic infective parasite (Croft et al., 2006;Mukhopadhyay et al., 2011;Ponte-Sucre, 2003). In addition, different functions in host cell infection have been attributed to the parasite's ecto-ATPases (Bisaggio et al., 2003;Meyer-Fernandes, 2002;Paletta-Silva and Meyer-Fernandes, 2012;Sansom et al., 2008;Sansom, 2012). Here, we show an increase in Mg +2 -dependent ecto-ATPase activity in vinblastine-resistant L. amazonensis. ...
... These studies also reported an increase in parasite load in mice and a marked increase in the production of IL-4, IL-10 and Arginase-1, which decreased the synthesis of nitric oxide by macrophages and facilitated survival of the parasite Vanaerschot et al., 2011). These data corroborate the link between the increase in ecto-ATPase activity and the increases in infectivity, virulence and adhesion in various microorganisms (Berrêdo-Pinho et al., 2001;Kiffer-Moreira et al., 2007;Meyer-Fernandes, 2002;Sansom et al., 2008;Santos et al., 2009). ...
Leishmania amazonensis is a protozoan parasite that induces mucocutaneous and diffuse cutaneous lesions upon infection. An important component in treatment failure is the emergence of drug-resistant parasites. It is necessary to clarify the mechanism of resistance that occurs in these parasites to develop effective drugs for leishmaniasis treatment. Promastigote forms of Leishmania amazonensis were selected by gradually increasing concentrations of vinblastine and were maintained under continuous drug pressure (resistant cells). Vinblastine-resistant L. amazonensis proliferated similarly to control parasites. However, resistant cells showed changes in the cell shape, irregular flagella and a decrease in rhodamine 123 accumulation, which are factors associated with the development of resistance, suggesting the MDR phenotype. The Mg-dependent-ecto-ATPase, an enzyme located on cell surface of Leishmania parasites, is involved in the acquisition of purine and participates in the adhesion and infectivity process. We compared control and resistant L. amazonensis ecto-enzymatic activities. The control and resistant Leishmania ecto-ATPase activities were 16.0 ± 1.5 nmol Pi × h(-1) × 10(-7) cells and 40.0 ± 4.4 nmol Pi × h(-1) × 10(-7)cells, respectively. Interestingly, the activity of other ecto-enzymes present on the L. amazonensis cell surface, the ecto-5' and 3'-nucleotidases and ecto-phosphatase, did not increase. The level of ecto-ATPase modulation is related to the degree of resistance of the cell. Cells resistant to 10 μM and 60 μM of vinblastine have ecto-ATPase activities of 22.7 ± 0.4 nmol Pi × h(-1) × 10(-7) cells and 33.8 ± 0.8 nmol Pi × h(-1) × 10(-7)cells, respectively. In vivo experiments showed that both lesion size and parasite burden in mice infected with resistant parasites are greater than those of L. amazonensis control cells. Furthermore, our data established a relationship between the increase in ecto-ATPase activity and greater infectivity and severity of the disease caused by vinblastine-resistant L. amazonensis promastigotes. Taken together, these data suggest that ecto-enzymes could be potential therapeutic targets in the struggle against the spread of leishmaniasis, a neglected world-wide public health problem.
... Protein phosphatases may exist in soluble [4,12] or secreted forms [13][14][15][16][17][18][19][20], or even remain attached to the outer surface of the inner membrane [21][22][23] or the cell wall [24,25]. In this context, the ectophosphatases are surface membrane-bound proteins whose active site faces the extracellular medium [26][27][28]. It is noteworthy that some authors have designated secreted phosphatase activities as ectophosphatases because both activities are involved with extracellular metabolism. ...
... To establish an infection, pathogens have to evade the immune system, survive and divide in the host environment and spread to new tissues [83,84]. Ecto-enzymes are located in the plasma membrane with their active sites facing the external environment, and could be one of the parasite proteins that initially come into contact with the host cells in the invasion process [27]. This peculiar location suggests that these enzymes may be involved in virulence and infection [27,45]. ...
... Ecto-enzymes are located in the plasma membrane with their active sites facing the external environment, and could be one of the parasite proteins that initially come into contact with the host cells in the invasion process [27]. This peculiar location suggests that these enzymes may be involved in virulence and infection [27,45]. ...
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.
... Recently, high ecto-nucleotidase activity of several protozoan parasites -including Toxoplasma gondii, Entamoeba histolytica, Leishmania tropica, Leishmania amazonesis, Trypanosoma cruzi, Trypanosoma brucei, and Tritrichomonas foetus -has been shown to interfere with the extracellular signaling of the host and affect the virulence and pathogenesis of these organisms [8,9,10,11,12,13,14,15, 16,17]. Thus, it has been suggested that these enzymes play a role in the pathogenicity of these parasites by controlling the host cell response to infection, specifically by: (i) protecting the parasite from the cytolytic effects of extracellular ATP, (ii) regulating ectokinase substrate concentrations, (iii) preventing activation of signal transduction cascades associated with cellular injury, and (iv) facilitating cellular adhesion [18,19,20,21,22,23,24,25,26,27,28], reviewed in [28]. Among ecto-nucleosidases, Ecto-ATPases, or E-ATPases, are cell-surface enzymes that hydrolyze a range of extracellular nucleoside triphosphates (NTPs) and nucleoside diphosphates (NDPs). ...
... Recently, high ecto-nucleotidase activity of several protozoan parasites -including Toxoplasma gondii, Entamoeba histolytica, Leishmania tropica, Leishmania amazonesis, Trypanosoma cruzi, Trypanosoma brucei, and Tritrichomonas foetus -has been shown to interfere with the extracellular signaling of the host and affect the virulence and pathogenesis of these organisms [8,9,10,11,12,13,14,15, 16,17]. Thus, it has been suggested that these enzymes play a role in the pathogenicity of these parasites by controlling the host cell response to infection, specifically by: (i) protecting the parasite from the cytolytic effects of extracellular ATP, (ii) regulating ectokinase substrate concentrations, (iii) preventing activation of signal transduction cascades associated with cellular injury, and (iv) facilitating cellular adhesion [18,19,20,21,22,23,24,25,26,27,28], reviewed in [28]. Among ecto-nucleosidases, Ecto-ATPases, or E-ATPases, are cell-surface enzymes that hydrolyze a range of extracellular nucleoside triphosphates (NTPs) and nucleoside diphosphates (NDPs). ...
... Further studies are necessary to test this hypothesis. Apyrases are broadly distributed among the tree of life and are present in many pathogenic parasites [28]. To gain insight into the evolution of this pathogenic factor we performed phylogenetic analysis of CApy and all orthologs that we could identify from available sequences. ...
Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.
... In studying the complexity of immune system regulation, especially at the site of injury, several groups have recently begun to demonstrate a possible role for the nucleoside adenosine and adenosine nucleotidases in regulating Leishmania-specific immune responses. It was proposed that ecto-ATP diphosphohydrolases and ectonucleotidases, which are membrane-associated enzymes with their catalytic sites turned extracellular, would act to dephosphorylate adenosine nucleotides into free adenosine [18, 19]. Before discussing the role of ectonucleotidases in leishmaniasis, concepts and comments regarding the immunomodulatory roles of adenosine nucleotides and nucleosides must be stressed. ...
... Ectonucleotidases are glycoprotein enzymes present in the plasma membrane with their catalytic sites facing extracellu- larly [18, 19], which are capable of hydrolyzing extracellular nucleotides. Fundamentally important in maintaining the homeostasis of extracellular nucleotides, these enzymes are also regulatory (termination of signaling events triggered by ATP) and metabolic (generation of nutrients) in function [35]. ...
... Since Gottlieb and Dwyer [38] provided information in 1981 about the biochemistry of surface membranes of Leishmania, a large number of authors have observed nucleotidases located on the outer surface of the plasma membrane of the parasite. For example, ecto-ATPases, ecto-5 -nucleotidase, and ecto-3 -nucleotidase have all been described in Leishmania sp. [18, 19, 39, 40]. In Leishmania parasites, it was proposed that an increase in ectonucleotidase activity would increase the production of adenosine, which would consequently aid in the establishment of infection through its immunosuppressive mechanisms. ...
Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is responsible for most Leishmania-associated deaths. VL represents a serious public health problem that affects many countries. The immune response in leishmaniasis is very complex and is poorly understood. The Th1 versus Th2 paradigm does not appear to be so clear in visceral leishmaniasis, suggesting that other immunosuppressive or immune-evasion mechanisms contribute to the pathogenesis of VL. It has been demonstrated that generation of adenosine, a potent endogenous immunosuppressant, by extracellular enzymes capable to hydrolyze adenosine tri-nucleotide (ATP) at the site of infection, can lead to immune impairment and contribute to leishmaniasis progression. In this regard, this paper discusses the unique features in VL immunopathogenesis, including a possible role for ectonucleotidases in leishmaniasis.
... The signaling mechanism generated by ATP can be reverted through the action of a set of enzymes, known as ectoenzymes, which are involved in the control of extracellular nucleotide and nucleoside levels. Because the active sites of ectoenzymes face the external medium rather than the cytoplasm, the activities of these enzymes can be measured using living cells (Zimmermann, 1996;Meyer-Fernandes, 2002;Sissons et al., 2004;Bours et al., 2006;Matin & Khan, 2008;Amazonas et al., 2009;Cosentino-Gomes et al., 2009;Fonseca-de-Souza et al., 2009). ...
... The extracellular hydrolysis of ATP can be initiated by NTPDases (ectonucleoside triphosphate diphosphohydrolases) and terminated by ecto-5 0-nucleotidases (CD73; E.C. 3.1.3.5), resulting in its respective nucleoside adenosine (Zimmermann, 1996(Zimmermann, , 2000Meyer-Fernandes, 2002;Robson et al., 2006). Ecto-5 0-nucleotidase is the major enzyme responsible for the formation of extracellular adenosine from released adenine nucleotides ( Zimmermann, 2000). ...
... Colonization and infection with C. parapsilosis are dependent upon the ability of the fungus to adhere to host cells and tissues, particularly mucosal surfaces ( Trofa et al., 2008). The specific functions of ecto-ATPases and ecto-5 0-nucleotidases are not fully known, but it has been demonstrated that they participate in many relevant biological processes ( Zimmermann, 2000;Meyer-Fernandes, 2002). In C. parapsilosis, both enzymes play a role in the control of extracellular nucleotide concentrations and could have a role in limiting inflammation and immune responses from the host, favoring the establishment of infectious processes. ...
Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.
... The cell wall compo-sition is of primary importance during microbial adherence and infection establishment (Calderone et al., 1994). Cell surfaces contain enzymes whose catalytic site faces the extracellular environment Meyer-Fernandes, 2002;Gomes et al., 2006;Pinheiro et al., 2007;Peres-Sampaio et al., 2008). The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells (Goding, 2000;Meyer-Fernandes, 2002;Amazonas et al., 2009). ...
... Cell surfaces contain enzymes whose catalytic site faces the extracellular environment Meyer-Fernandes, 2002;Gomes et al., 2006;Pinheiro et al., 2007;Peres-Sampaio et al., 2008). The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells (Goding, 2000;Meyer-Fernandes, 2002;Amazonas et al., 2009). This class of enzymes includes surface ATPases (ecto-ATPases), which are transmembrane glycoproteins that hydrolyze extracellular nucleoside tri-and/or diphosphates (Zimmermann, 2001;Meyer-Fernandes, 2002). ...
... The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells (Goding, 2000;Meyer-Fernandes, 2002;Amazonas et al., 2009). This class of enzymes includes surface ATPases (ecto-ATPases), which are transmembrane glycoproteins that hydrolyze extracellular nucleoside tri-and/or diphosphates (Zimmermann, 2001;Meyer-Fernandes, 2002). ...
In this work, we describe the ability of intact cells of Candida parapsilosis to hydrolyze extracellular ATP. ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. The ecto-ATPase activity was increased in the presence of 5 mM MgCl(2), with values of V(max) and apparent K(m) for Mg-ATP(2-) increasing to 33.80 +/- 1.2 nmol Pi h(-1) 10(-8) cells and 0.6 +/- 0.06 mM, respectively. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases and Na(+)-ATPases had no effect on the C. parapsilosis Mg(2+)-stimulated ATPase activity, but extracellular impermeant compounds, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid and suramin, reduced enzyme activity in yeast living cells by 83.1% and 81.9%, respectively. ARL 67156 (6-N,N'-diethyl-d-beta-gamma-dibromomethylene ATP), a nucleotide analogue, also inhibited the ecto-ATPase activity in a dose-dependent manner. ATP was the best substrate for the yeast Mg(2+)-stimulated ecto-enzyme, but ADP, ITP, CTP, GTP and UTP were also hydrolyzed. A direct relationship between ecto-ATPase activity and adhesion to host cells was observed. In these assays, inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells. Based also on the differential expression of ecto-ATPase activities in the different isolates of C. parapsilosis, the possible role of this enzyme in fungal biology is discussed.
... They are usually located on the cell membrane with the catalytic site oriented to the extracellular milieu. Some of them are also bound to the organelle membrane, with the catalytic site oriented to the lumen of the organelle (Bernardes et al., 2000;Meyer-Fernandes, 2002;Sansom et al., 2008). The E-NTPDases are proteins characterized by having a high degree of glycosylation. ...
... Such sequential hydrolysis of extracellular ATP to adenosine was demonstrated in different parasite species, indicating that not only E-NTPDases but also ecto-5`-nucleotidases could be present in protozoa parasites, including Leishmania spp., Trypanosoma spp., Trichomonas vaginalis, Tritrichomonas foetus, Toxoplasma gondii, and Schistosoma spp. (Berredo-Pinho et al., 2001;Meyer-Fernandes, 2002;Sansom, 2012;Pimentel et al., 2016;Paes-Vieira et al., 2018). ...
Belonging to the GDA1/CD39 protein superfamily, nucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP) and inorganic phosphate (Pi). Several NTPDase isoforms have been described in different cells, from pathogenic organisms to animals and plants. Biochemical characterization of nucleotidases/NTPDases has revealed the existence of isoforms with different specificities regarding divalent cations (such as calcium and magnesium) and substrates. In mammals, NTPDases have been implicated in the regulation of thrombosis and inflammation. In parasites, such as Trichomonas vaginalis, Trypanosoma spp., Leishmania spp., Schistosoma spp. and Toxoplasma gondii, NTPDases were found on the surface of the cell, and important processes like growth, infectivity, and virulence seem to depend on their activity. For instance, experimental evidence has indicated that parasite NTPDases can regulate the levels of ATP and Adenosine (Ado) of the host cell, leading to the modulation of the host immune response. In this work, we provide a comprehensive review showing the involvement of the nucleotidases/NTPDases in parasites infectivity and virulence, and how inhibition of NTPDases contributes to parasite clearance and the development of new antiparasitic drugs.
... In contrasts to the large amount of information available about mitochondrial ATPase in mammals (5)(6)(7)(8), bacteria (9,10) and parasitic protozoa (11)(12)(13)(14)(15) , little is known about this enzyme in parasitic helminthes .We have therefore report here the purification and properties of the ATPase from adult Fasciola hepatica. ...
... The present investigation has shown that in several respects the ATPase of F .hepatica appear to be similar to those of parasitic protozoa (1,2,4,11) and parasitic helminthes (15,22,23). The recovery of a significant proportion of ATPase activity in the particulate fractions suggest that this enzyme may be associated with tegument as has been reported in the tapeworm Hymenolepis dimiuta (22). ...
ABSTRACT
Objective: The adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3.;ATPase) is a membrane -bound enzyme which transport
protons across the plasma membrane using ATP as an energy source.
Methods: The adenosine triphosphatase (ATPase ; EC: 3.6.1.3) was extracted from membrane preparations of adult Fasciola hepatica by
chloroform treatment and purified by means of ammonium sulphate fractionation, gel filtration on sephadex G-200 and DEAE- Cellulose
chromatography.
Results: The molecular weight was calculated to be 305.000 dalton by gel filtration. Kinetic experiments demonstrated a biphasic linear
lineweaver - burk relationship (km=0.142 and 1.66 mM) thus revealing the existence of two substrate binding enzyme sites.
Conclusion: In our study revealed that partial inhibition of Mg2+ dependent purified enzyme by oligomycin suggest the absence of
mitochondrial ATPase in F. hepatica. (Turkiye Parazitol Derg 2014; 38: 26-31)
Key Words: ATPase, Fasciola hepatica, gel filtration on sephadex G-200, DEAE- Cellulose chromatography.
... In contrasts to the large amount of information available about mitochondrial ATPase in mammals (5)(6)(7)(8), bacteria (9,10) and parasitic protozoa (11)(12)(13)(14)(15) , little is known about this enzyme in parasitic helminthes .We have therefore report here the purification and properties of the ATPase from adult Fasciola hepatica. ...
... The present investigation has shown that in several respects the ATPase of F .hepatica appear to be similar to those of parasitic protozoa (1,2,4,11) and parasitic helminthes (15,22,23). The recovery of a significant proportion of ATPase activity in the particulate fractions suggest that this enzyme may be associated with tegument as has been reported in the tapeworm Hymenolepis dimiuta (22). ...
A B S T RACT
The plasma membrane of cells contains enzymes whose active sites face the
external medium rather than the cytoplasm. The adenosine tri phosphatase
(ATP phosphohydrolase, EC 3.6.1.3.; ATPase) is membrane – bound enzyme
which transport protons across the plasma membrane using ATP as an enegy
source. In this work, we extracted the adenosine tri phosphatase from
promastigotes of Leishmania tropica by chloroform treatment and purified by
means of ammonium sulphate fractionation, gel filtration on sephadex G-200
and DEAE-Cellulose chromatography. Kinetic experiments demonstrated a
biphasic linear lineweaver - burk relationship (km= 0.25 and 1.1 mM) thus
revealing the existence of two substrate binding enzyme site and has an
apparent molecular weight of 365000 dalton by gel filtration. The result of
this study firmly provided the first direct evidence for the existence of Mg+2
-
dependent ATPase in L. tropica, a fact which is of great interest from the
phylogenetic point of view.
... In contrasts to the large amount of information available about mitochondrial ATPase in mammals (5)(6)(7)(8), bacteria (9,10) and parasitic protozoa (11)(12)(13)(14)(15) , little is known about this enzyme in parasitic helminthes .We have therefore report here the purification and properties of the ATPase from adult Fasciola hepatica. ...
... The present investigation has shown that in several respects the ATPase of F .hepatica appear to be similar to those of parasitic protozoa (1,2,4,11) and parasitic helminthes (15,22,23). The recovery of a significant proportion of ATPase activity in the particulate fractions suggest that this enzyme may be associated with tegument as has been reported in the tapeworm Hymenolepis dimiuta (22). ...
The present study was designed to show the role of green Tio2 nanoparticles against toxicity
of Echinococcus granulosus. The present study used 16 adult albino male rats that distributed
randomly to following groups (each group consist 5 rats); control group received ad libidium,
second group injected with 2,5 X 103 of Echinococcus granulosus protoscolices third group
injected with protoscolices and treated with 50mg/kg green Tio2 nanoparticles, fourth group
injected with protoscolices and treated with 100mg/kg green Tio2 nanoparticles. The results
show high significant increased (P < 0.05) in levels of Alanine Aminotransferase (ALT),
Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP) in group injected with
protoscolices compared with control group. Oxidative stress factor in group injected with
protoscolices show significant increased (P < 0.05) in levels of MDA (malonedialdehyied) and
significant decreased (P < 0.05) in levels of glutathione (GSH) and catalase compared with
control group. While, after used green Tio2 nanoparticles with Echinococcus granulosus, the
results showed non-significant changes (P < 0.05) in liver functions and MDA, GSH and catalase
also showed non-significant changes (P < 0.05) compared with control group. It was concluded
that green Tio2 nanoparticles has been potential role against toxicity of Echinococcus granulosus
in male rats.
... Therefore, the development of new molecules able to prevent and control these parasite infections is a constant challenge. From this point of view, virulence biomolecules, especially those secreted or ecto-localized at the parasite's plasma membrane seem to be promising targets (Meyer-Fernandes, 2002). ...
... Nucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of a variety of nucleoside tri-and diphosphates to the monophosphate forms and have been implicated in adenosine acquisition, the regulation of blood clotting, inflammatory responses, immune reactions, and microbial virulence (Sansom et al., 2008;Freitas-Mesquita and Meyer-Fernandes, 2014;Vijayamahantesh et al., 2017). Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) of the CD39 family are characterized by the presence of five "apyrase conserved regions" named ACR1 to ACR5 (Robson et al., 2006;Zimmermann et al., 2012) and are membrane bound enzymes with the active site oriented to the extracellular milieu or to the lumen of the organelle in which they are located (Bernardes et al., 2000;Meyer-Fernandes, 2002;Sansom et al., 2008). The enzyme activity is usually dependent on the presence of divalent cations such as magnesium and calcium (Handa and Guidotti, 1996;Robson et al., 2006). ...
Nucleoside Triphosphate Diphosphohydrolases (NTPDases) are enzymes that belong to the GDA1/CD39 protein superfamily. These enzymes catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP). Biochemical characterization of the nucleotidases/NTPDases from various types of cells, including those from plants, animals and pathogenic organisms, has revealed the existence of several isoforms with different specificities with respect to divalent cations (magnesium, calcium, manganese and zinc) and substrates. In mammals, the NTPDases play important roles in the regulation of thrombosis and inflammation. In parasites of the genus Leishmania, the causative agents of leishmaniasis, two NTPDase isoforms, termed NTPDase-1 and NTPDase-2 have been described. Independently of their cellular localization, whether cell-surface localized, secreted or targeted to other organelles, in some Leishmania species these NTPDases could be involved in parasite growth, infectivity and virulence. Experimental evidence has suggested that the hydrolysis of ATP and ADP by parasite ecto-nucleotidases can down-modulate the host immune response. In this context, the present work provides an overview of recent works that show strong evidence not only of the involvement of the nucleotidases/NTPDases in Leishmania spp infectivity and virulence but also of the molecular mechanisms that lead to the success of the parasitic infection.
... Ecto-nucleotidases have been observed on the surface of several microorganisms including protozoa (reviewed by Meyer-Fernandes, 2002), bacteria (Cowan et al., 2007), and fungi (Junior et al., 2005). Some of these data suggest that microbial ecto-ATPase activity could play a role in increasing the virulence of these microbial pathogens (Kiffer-Moreira et al., 2010;Giarola et al., 2013). ...
... The role of these molecules in protozoa and bacterial infections has been studied over years. In most cases, the inhibition of pathogen ecto-nucleotidase activities, or even an increase of extracellular nucleotide concentrations, is associated with reduced infection success of different parasites (Meyer-Fernandes, 2002;Bisaggio et al., 2003;Coutinho-Silva and Ojcius, 2012;Giarola et al., 2014). However, only few reports have evaluated the impact of these extracellular nucleotides in the HIV-1 life cycle. ...
... In contrasts to the large amount of information available about mitochondrial ATPase in mammals (5)(6)(7)(8), bacteria (9,10) and parasitic protozoa (11)(12)(13)(14)(15) , little is known about this enzyme in parasitic helminthes .We have therefore report here the purification and properties of the ATPase from adult Fasciola hepatica. ...
... The present investigation has shown that in several respects the ATPase of F .hepatica appear to be similar to those of parasitic protozoa (1,2,4,11) and parasitic helminthes (15,22,23). The recovery of a significant proportion of ATPase activity in the particulate fractions suggest that this enzyme may be associated with tegument as has been reported in the tapeworm Hymenolepis dimiuta (22). ...
Objective:
The adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3.;ATPase) is a membrane -bound enzyme which transport protons across the plasma membrane using ATP as an energy source.
Methods:
The adenosine triphosphatase (ATPase ; EC: 3.6.1.3) was extracted from membrane preparations of adult Fasciola hepatica by chloroform treatment and purified by means of ammonium sulphate fractionation, gel filtration on sephadex G-200 and DEAE- Cellulose chromatography.
Results:
The molecular weight was calculated to be 305.000 dalton by gel filtration. Kinetic experiments demonstrated a biphasic linear lineweaver - burk relationship (km=0.142 and 1.66 mM) thus revealing the existence of two substrate binding enzyme sites.
Conclusion:
In our study revealed that partial inhibition of Mg²⁺ dependent purified enzyme by oligomycin suggest the absence of mitochondrial ATPase in F. hepatica.
... Hence, the phenotypic characteristics of the life cycle stages that are involved in the interaction with the host may, in this context, be regarded as virulence determinants (Peres-Sampaio et al., 2008 ). Different functions in host cell infection have been attributed to the parasite's ecto-ATPases (Meyer-Fernandes, 2002; Sansom et al., 2008; Sansom, 2012; Paletta-Silva and MeyerFernandes, 2012 ). Accordingly, we showed that the Y strain (virulent ) and the Dm28c (low-virulence) strain (Contreras et al., 1988) had different Mg 2+ -dependent ecto-ATPase activities and different sensitivities to heat shock stress (Fig. 4, panel A). ...
... This result is a possibility because these parasites do not have the enzymatic machinery that is necessary for nucleoside biosynthesis and instead depend exclusively on purine salvage from the extracellular medium. This mechanism contributes to the adaptation and survival of the parasites in hostile environments (Meyer-Fernandes, 2002; De Souza Leite et al., 2007; Peres-Sampaio et al., 2008; Paletta-Silva and Meyer-Fernandes, 2012). The experiments performed with plasma membrane-enriched fractions show that the effects of heat stress on the ecto-ATPase activity from T. cruzi, necessarily, depend on an intracellular structure, as well as on heat shock proteins, and the role of this latter component remains to be elucidated. ...
... Ecto-enzymes are glycoproteins located at the external surface of the plasma membrane. The activities of these enzymes can be measured using intact cells Karnowsky, 1973, 1974;Furuya et al. 1998;Meyer-Fernandes, 2002). Ecto-nucleoside triphosphate diphoshydrolases are ecto-enzymes that hydrolyse tri-and/or di-phosphate nucleotides (Plesner, L. 1995;Zimmermann, H. 1999). ...
... Several hypotheses have been proposed for the function of ecto-ATPases in trypanosomatids, which include acquisition of adenosine from the media, necessary for normal growth, modulation of parasite infection and virulence, and involvement in cellular adhesion (Berrêdo-Pinho et al. 2001;Meyer-Fernandes, 2002;Bisaggio et al. 2003;Pinheiro et al. 2006;Peres-Sampaio et al. 2008;Santos et al. 2009;De Souza et al. 2010). However, the precise function of these enzymes is still an open question. ...
Recent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 μm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 μm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 μm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis.
... Some of these molecules are involved in triggering specific signaling pathways both in the parasite and the host cell, which are critical for parasite entry and survival [6]. Plasma membranes of cells contain enzymes that are oriented with their active sites facing the external medium rather than the cytoplasm, which are important for host-parasite interactions [7, 8]. In the case of an ectoenzyme other criteria can be included as: (1) the enzyme has to act on extracellular substrate, (2) cellular integrity is maintained during enzyme activity, (3) the products are released extracellularly, (4) the enzyme is not released to the extracellular environment; and (5) the enzyme activity can be modified by nonpenetrating reagents [7, 8] . ...
... Plasma membranes of cells contain enzymes that are oriented with their active sites facing the external medium rather than the cytoplasm, which are important for host-parasite interactions [7, 8]. In the case of an ectoenzyme other criteria can be included as: (1) the enzyme has to act on extracellular substrate, (2) cellular integrity is maintained during enzyme activity, (3) the products are released extracellularly, (4) the enzyme is not released to the extracellular environment; and (5) the enzyme activity can be modified by nonpenetrating reagents [7, 8] . Supporting this idea, the presence of surfacelocated phosphatases, called ecto or extracytoplasmic phosphatases have been characterized in several microorganisms. ...
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.
... For example, the intracellular bacterial pathogens Shigella, Mycobacteria, and Porphyromonas, as well as the Leishmania parasite, secrete ATP-utilizing enzymes (unrelated to NTPDases) that have the potential to manipulate levels of ATP and therefore interfere with purinergic signaling (Buchrieser et al., 2000;Kolli et al., 2008;Yilmaz et al., 2008;Zaborina et al., 1999). In addition, all protozoan parasites possess NTPDase homologs of the NTPDase1 family (Roberto Meyer-Fernandes, 2002); however, their role in the biology of the organism and their contribution to the pathogenesis of disease is poorly understood. Recently, an NTPDase homolog (Lpg1905) was characterized from the intracellular bacterial pathogen Legionella pneumophila, the causative agent of Legionnaire's disease (Sansom et al., 2007(Sansom et al., , 2008a. ...
... However, many intracellular eukaryotic pathogens contain NTPDase mimics of the mammalian NTPDases that control purinergic signaling. Their role in microbial physiology and their contribution to the pathogenesis of disease is poorly understood (Roberto Meyer-Fernandes, 2002;Sansom et al., 2008b). To understand the structural basis of NTPDase function from pathogenic organisms, we have determined the crystal structure of Lp1NTPDase, a prokaryotic NTPDase. ...
Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.
... Cell surface components play a key role in the survival of protozoan parasites in hostile insect and vertebrate environments and are confronted with the host immune responses [20]. In this context, the presence of enzymes with catalytic sites facing the extracellular medium, such as ecto-phosphatases and ecto-ATPase, seem to be extremely important to the living parasite [21]. Membrane-bound ecto-phosphatases have been characterized and reported to be pathogenic factors in several members of the Trypanosoma genus2223242526, the Leishmania genus272829, Leptomonas collosoma [30], Herpetomonas muscarum muscarum [31], H. samuelpessoai [32] , Phytomonas spp. ...
... [33], and Crithidia deanei [34]. Ecto-phosphatases are also supposed to be involved in nutrition [35], protection [35,36], virulence [22,28], and cellular differentiation [21,32]. Recently, our laboratory characterized ecto-phosphatase activities present in intact cells of T. rangeli [37,38]. ...
As a protozoan parasite of hematophagous insects, Trypanosoma rangeli epimastigotes are exposed to reactive oxygen species during development in hosts. In this work, we investigated the role of H(2)O(2) as a modulator of the ecto-phosphatase activity present in living T. rangeli. We observed that H(2)O(2) inhibits ecto-phosphatase activities in the short and long epimastigote forms of T. rangeli. Ecto-phosphatase activity found in the short form was more sensitive than that found in the long form. Moreover, H(2)O(2) inhibited ecto-phosphatase activity of the short form in a dose-dependent manner and this inhibition was reversible after H(2)O(2) removal. This effect was not observed for T. rangeli ecto-ATPase, another ecto-enzyme present on the external surface of T. rangeli. Cysteine, beta-mercaptoethanol, and reduced glutathione were able to revert the enzyme inhibition promoted by H(2)O(2). Catalase and glutathione peroxidase stimulated this ecto-phosphatase activity, whereas superoxide dismutase was not able to modulate this activity. The ecto-phosphatase activity was also activated by FCCP and inhibited by oligomycin. It seems that H(2)O(2) plays a fundamental role in the regulation of cellular processes of these organisms. We showed, for the first time, that these parasites can produce H(2)O(2), and it is able to regulate ecto-phosphatase activity.
... The sequential hydrolysis of extracellular ATP to adenosine indicates that not only E-NTPDases but also ecto-5 ′ -nucleotidases are present in several protozoan parasites including those belonging to the genera Leishmania and Trypanosoma [20,38,47,54]. The activity of ectonucleotidase has been demonstrated in L. amazonensis [48]. ...
Infections caused by Leishmania amazonensis are characterized by a persistent parasitemia due to the ability of the parasite to modulate the immune response of macrophages. It has been proposed that ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases) could be able to suppress the host immune defense by reducing the ATP and ADP levels. The AMP generated from E-NTPDase activity can be subsequently hydrolyzed by ecto-nucleotidases, increasing the levels of adenosine, which can reduce the inflammatory response. In the present work, we provide new information about the role of E-NTPDases on infectivity and virulence of L. amazonensis. Our data demonstrate that not only the E-NTPDase activity is differentially regulated during the parasite development but also the expression of the genes ntpd1 and ntpd2. E-NTPDase activity increases significantly in axenic amastigotes and metacyclic promastigotes, both infective forms in mammalian host. A similar profile was found for mRNA levels of the ntpd1 and ntpd2 genes. Using parasites overexpressing the genes ntpd1 and ntpd2, we could demonstrate that L. amazonensis promastigotes overexpressing ntpd2 gene show a remarkable increase in their ability to interact with macrophages compared to controls. In addition, both ntpd1 and ntpd2-overexpressing parasites were more infective to macrophages than controls. The kinetics of lesion formation by transfected parasites were similar to controls until the second week. However, twenty days post-infection, mice infected with ntpd1 and ntpd2-overexpressing parasites presented significantly reduced lesions compared to controls. Interestingly, parasite load reached similar levels among the different experimental groups. Thus, our data show a non-linear relationship between higher E-NTPDase activity and lesion formation. Previous studies have correlated increased ecto-NTPDase activity with virulence and infectivity of Leishmania parasites. Based in our results, we are suggesting that the induced overexpression of E-NTPDases in L. amazonensis could increase extracellular adenosine levels, interfering with the balance of the immune response to promote the pathogen clearance and maintain the host protection.
... Regarding the critical importance of surface membrane interactions between parasites and their host cells, the study of ecto-enzymes is relevant for the understanding of the parasite infection [26]. ...
Leishmaniasis is one of the most significant of the neglected tropical diseases, with 350 million people in 98 countries worldwide living at risk of developing one of the many forms of the disease. During the transmission of the parasite from its vector to the vertebrate host, neutrophils are rapidly recruited to the site of the sandfly bite. Using different strategies, neutrophils can often kill a large number of parasites. However, some parasites can resist neutrophil-killing mechanisms and survive until macrophage arrival at the infection site. One of the strategies for neutrophil-mediated killing is the production of neutrophil extracellular traps (NETs). Because of its ecto-localized nuclease activity, the enzyme 3’-nucleotidase/nuclease (3'NT/NU), present in different Leishmania species, was recently identified as part of a possible parasite escape mechanism from NET-mediated death. Previous studies showed that 3'NT/NU also plays an important role in the establishment of Leishmania infection by generating extracellular adenosine that favors the parasite and macrophage interaction. This study aims to deepen the knowledge about 3'NT/NU, mainly with respect to its nuclease activity that is little studied in the current literature. For this, we cloned, expressed and purified the recombinant La3'NT/NU and have confirmed its contribution to the parasite escape from NET-mediated killing.
... Parasites come across various membranes and secreted molecules for attachment and invasion of the host cells. Ectophosphatases are potentially important phosphatase group, which is the phosphatase associated with the outer cell surface having their active sites oriented towards the external medium rather than the cytoplasm [36][37][38]. 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]. ...
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.
... Outra característica das apirases é a presença de cinco domínios altamente conservados conhecidos como "regiões conservadas de apirase", abreviado como ACR1 até ACR5 e que são envolvidas no ciclo catalítico (Robson et al., 2006). Existem mais de 20 membros destas ATPases em mamíferos (Robson et al., 2006) (Meyer-Fernandes, 2002). ...
Miosina pode ser precipitada pelo congelamento e descongelamento de fração
solúvel de diferentes tecidos de mamíferos. Nesse trabalho, o objetivo foi
precipitar ATPase similar à miosina a partir de fração solúvel de larva de P.
nucleorum. A fração solúvel foi congelada a -20 oC e descongelada após 48
horas. O precipitado foi recuperado por centrifugação a 45.000 g x 40 minutos e
foi lavado sucessivamente com solução tampão em pH 7,5 e 9,0. Posteriormente,
ATPase foi solubilizada pelo tratamento do precipitado com solução tampão pH
9,0 contendo 0,5 mol.L-1 de NaCl. Os principais polipeptídeos da fração ATPase
apresentaram Mr de 205 e 45 kDa. Essa fração expressou alta atividade CaATPase, mas não apresentou atividade Mg2+-ATPase e nem K+/EDTA-ATPase. A
atividade Ca2+-ATPase não foi inibida por tapsigargina 140 µ mmol.L-1, ouabaína
1,7 mmol.L-1 ou azida 1 mmol.L-1. A atividade foi sensivelmente inibida por
magnésio, mas praticamente não foi inibida por cobre ou zinco. A enzima não
hidrolisou AMP e nem PPi e hidrolisou muito pouco ADP e GTP. Nesse trabalho,
foi isolado um polipeptídeo de 205 kDa de larva de P. nucleorum, que co-purificou
com atividade Ca2+-ATPase.
... This hypothesis was investigated following previous definitions of ecto-enzymes which, according to several authors [32,39], are inhibited by impermeable inhibitors. To confirm this hypothesis, ATP hydrolysis by L. donovani was performed in the presence of an extracellular impermeant inhibitor DIDS [27,32,39] and a pharmaceutical suramin [37], which is also an ecto-ATPase inhibitor [14,34]. As shown in Fig. 3a, the Mg 2? -dependent ATPase activity of L. donovani was completely inhibited by Suramin in a dose-dependent manner, indicating its ecto-enzymatic nature. ...
In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10(7) cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2 stimulated the ATP hydrolysis to a greater extent compared with CaCl2 and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10(7) cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg(2+)-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
... Ectoapyrase activities are present in virtually all eukaryotic cells, starting with protozoa [1]. The ectoapyrases of vertebrates are called ectonucleoside triphosphate diphosphohydrolases (E-NTPDases). ...
Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, is anchored to the membrane by two transmembrane domains at the two ends of the molecule. The transmembrane domains are important for enzymatic activity, as mutants lacking one or both of these domains have a fraction of the enzymatic activity of the wild-type CD39. We investigated the interactions between the transmembrane domains by using a strain of yeast that requires surface expression of CD39 for growth. Random mutagenesis of selected amino acid residues in the N-terminal transmembrane domain revealed that the presence of charged amino acids at these positions prevents expression of functional protein. Rescue of the growth of these mutants by complementary mutations on selected residues of the C-terminal transmembrane domain indicates that there is contact between particular faces of the transmembrane domains.
... Ectoapyrase activities are present in virtually all eukaryotic cells, starting with protozoa [1]. The ectoapyrases of vertebrates are called ectonucleoside triphosphate diphosphohydrolases (E-NTPDases). ...
To generate the library of rescue mutants of CD39, the entire CD39 gene was cloned into the BamHI/HindIII site of pBluescript II vector. A primer CD39BstBI-mut, containing a sense sequence of CD39 (nucleotides 1466–1513), an NcoI and a BstBI restriction site (5′-TGGTCGCCATGGTCATCACAGGGCTGTTCATCTTTtcgAAGCCTTCGT-3′, the restriction sites are underlined, at the BstBI site the lowercase letters represent the changes from the original bases), and pVT101rev primer (5′-GCAAGGTAGACAAGC-3′), containing an antisense sequence of pVT101-U [32], was used in PCR to introduce a BstBI restriction site into CD39. The template was pVT101-CD39. The resulting PCR product was digested with NcoI and BamHI, and the corresponding fragment in pBluescript-CD39 was replaced with this NcoI/BamHI PCR fragment.
... Adenylate cyclase were also shown to be important for the virulence of animal pathogens (Markaryan et al., 2001;Munier-Lehmann et al., 2003). Given that extracellular ATP was shown to be involved in cytolysis, cell adhesion, and growth of various microbes (Roberto Meyer-Fernandes, 2002;Meyer-Fernandes et al., 2004;Li et al., 2011), these pathogenic microbes may be merely protecting themselves from ATP, a very reactive molecule. Correspondingly, a recent report by Long et al. (2014) demonstrated that conidial germination, appressorium formation and pathogenicity of rice blast fungus Magnaporthe oryzae were significantly reduced by pretreatment with nucleotidase-specific inhibitors. ...
As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded) plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs). ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling roles in animals, including acting as a DAMP during the inflammatory response and wound healing. If ATP acts outside the cell, then it is reasonable to expect that it is recognized by a plasma membrane-localized receptor. Recently, DORN1, a lectin receptor kinase, was shown to recognize extracellular ATP in Arabidopsis. DORN1 is the founding member of a new purinoceptor subfamily, P2K (P2 receptor kinase), which is plant-specific. P2K1 (DORN1) is required for ATP-induced cellular responses (e.g., cytosolic Ca²⁺ elevation, MAPK phosphorylation, and gene expression). Genetic analysis of loss-of-function mutants and overexpression lines showed that P2K1 participates in the plant wound response, consistent with the role of ATP as a DAMP. In this review, we summarize past research on the roles and mechanisms of extracellular ATP signaling in plants, and discuss the direction of future research on extracellular ATP as a DAMP signal.
... The present study was focused on characterising the ecto-phosphatase activities of intact living PS and EFF T. foetus because we were interested in the possible roles of these ecto-enzymes activities on the parasite's transformation and cytotoxicity. To establish that enzyme activities are due to ecto-enzymes, the following conditions must be met: (a) the enzymes have to act on an extracellular substrate, such as the p-NPP; (b) cellular integrity is maintained during enzyme activity; (c) the products are released extracellularly; (d) the enzymes are not released to the extracellular environment; and (e) the enzyme activities can be modified by specific non-penetrating compounds, such as, SO, AM and NaF (Meyer-Fernandes, 2002). In this study, all these criteria were followed and, therefore, our data support the presence of the ecto-phosphatase activities for both T. foetus forms. ...
... Ectoenzymes, surface molecules whose catalytic site faces the extracellular environment, have been described in different organisms [5,15,17,32] including in fungi such as Candida parapsilosis [18,24], Aspergillus fumigatus [7], Cryptococcus neoformans [13], Fosencaea pedrosoi [26,27] and Pseudallescheria boydii [25]. ...
Metarhizium anisopliae is an entomopathogenic fungus with the ability to infect a broad range of arthropods, and have evolved distinct strategies for their attachment to hosts. Here, we describe the characterisation of ecto-phosphatase activity on the conidia surface of M. anisopliae and its relevance in the host interaction process. Ecto-phosphatase activity was linear for 60 min and during this time, was linear with the increase of cell density. The optimum pH was in the acidic range and some divalent metals, such as Cu(2+), Cd(2+) and Zn(2+), inhibited ecto-phosphatase activity. The activity was also reduced by phosphatase inhibitors. Importantly, the inhibition of phosphatase activity in conidia reduced the adhesion to Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) integument and, consequently and indirectly, M. anisopliae infection. The results herein presented show, for the first time, the importance of ecto-phosphatase activity in M. anisopliae conidia and provide the first evidence of its direct involvement in adhesion and host infection.
... T. vaginalis tiene muchas proteinasas, la mayoría son del tipo cisteína (Coombs y North, 1983Meyer-Fernandes, 2002). ...
Trichomonas vaginalis is a flagellate protozoa, which infects human urogenital tract. This parasite has many proteinases specially of the cysteine present major proteinase type (CPs). Some have been related with cytoadherence, cytotoxicity, haemolysis, immune response evasion and nutrient acquisition mechanisms. Nevertheless, only four genes of CPs have been reported in T. vaginalis. Due to the importance of CPs in the pathogenicity mechanisms of T. vaginalis, the purpose of this work was the identification and partial characterization of a CP gene localized in the 3’ end of a 2891 bp insert from genomic clone 2.5.1.1 of T. vaginalis sequenced by the Sanger method. In the 5’ end of this genomic clone another an open reading frame of 951 bp which was fuond encode for 317 amino acids. This deduce sequence had homology with the ABC transporters of the GCN20 family. This ORF is incomplete at the carboxyl terminus. At the 3’ end 948 bp encoded to the complete gene of a CP precursor of 315 amino acids and theoretical molecular weight of 34.9 kDa. In this gene both a start codon (ATG) and a stop codon (TAA) used by the parasite were found.
The 5’ UTR region of the CP gene is only 17 bp, where an “Inr” consensus promoter sequence TCAY(C/T)W(A/T) was found. In the intergenic region of 975 bp (nucleotides 951 to 1926) a 3’UTR region for the CP gene, was found, which posseses four “ARE” sequences (ATTTA, TTATTTA) and a possible polyadenylation signal sequence (TAAA) recently reported for T. vaginalis. Analysis of the deduced amino acids sequence for the CP, gene showed that this CP TvCP25 do not have a signal peptide, the “pre” region is localized from the 1 to 9 residues like other CP genes reported by Mallinson. The “pro” region is from the 10 to 98 residues and the 99 residue may correspond to the start site of the mature protein of 23.6 kDa with a theoric isoelectric point of 5.08.
The TvCP25 belongs to the C1 papain family and to the cathepsin L subfamily, since it have the ERFNIN sequence in the “pro” region of this CP.
Also, this CP showed 89 % homology with the TvCP4, 80% with TvCP1, 75 % with TvCP2 and 68 % with the TvCP3 genes. The Northern blot experiments using a gene fragment of the TvCP25 as a probe showed a 1.23 Kb transcript band which is present in the different iron conditions. A theoric analysis of the mature TvCP25 protein suggests that the CP gene might encode for the 23 kDa CP present in the T. vaginalis extracts, which has a pI of 5.0.
... The plasma membrane of cells may contain enzymes whose active sites face the external medium. The activities of these enzymes, referred to as ecto-enzymes, can be measured using intact protozoa parasites (Meyer-Fernandes, 2002;Dutra et al., 2006;De Almeida-Amaral et al., 2006;Pinheiro et al., 2007;Peres-Sampaio et al., 2008). Knowledge about interactions between components of the external surface of the T. brucei brucei and the cellular elements of the host is of obvious importance for the understanding of parasite-vector interaction and the complex pathology of nagana disease. ...
Trypanosoma brucei brucei is the causative agent of animal African trypanosomiasis, also called nagana. Procyclic vector form resides in the midgut of the tsetse fly, which feeds exclusively on blood. Hemoglobin digestion occurs in the midgut resulting in an intense release of free heme. In the present study we show that the magnesium-dependent ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of procyclic T. brucei brucei is inhibited by ferrous iron and heme. The inhibition of E-NTPDase activity by ferrous iron, but not by heme, was prevented by pre-incubation of cells with catalase. However, antioxidants that permeate cells, such as PEG-catalase and N-acetyl-cysteine prevented the inhibition of E-NTPDase by heme. Ferrous iron was able to induce an increase in lipid peroxidation, while heme did not. Therefore, both ferrous iron and heme can inhibit E-NTPDase activity of T. brucei brucei by means of formation of reactive oxygen species, but apparently acting through distinct mechanisms.
... However, a more plausible hypothesis would be that, in the case of L. major and L. braziliensis, the putative secreted GDPases were responsible for the detected nucleotidasic activity or that the NTPDases are being held inside the cells due to problems in the glycosylation-dependent secretory pathway. However, in several instances the activity of E-NTPDases has been correlated with parasite virulence [17,19,22,23], acting, probably, as a protecting mechanism against the cytolytic effects of extracellular ATP [24] or by increasing parasite adhesion to the host cell [25]. The reasons for the differences observed as well as the actual involvement of these specific enzymes in the establishment of infection are under investigation in our laboratory. ...
Leishmaniasis is a parasitic disease with a variety of clinical forms, which are related to the Leishmania species involved. In the murine model, Leishmania amazonensis causes chronic non-healing lesions in Leishmania braziliensis- or Leishmania major-resistant mouse strains. In this study, we investigated the involvement of the pathway of extracellular nucleotide hydrolysis, with special focus on the role of extracellular adenosine, in the establishment of Leishmania infection. Our results show that the more virulent parasite--L. amazonensis--hydrolyzes higher amounts of ATP, ADP and AMP than the two other species, probably due to the higher expression of membrane NTPDase. Corroborating the idea that increased production of adenosine is important to lesion development and establishment of tissue parasitism, we observed that increased 5'-nucleotidase activity in L. braziliensis or addition of adenosine at the moment of infection with this parasite resulted in an increase in lesion size and parasitism as well as a delay in lesion healing. Furthermore, inhibition of adenosine receptor A2B led to decreased lesion size and parasitism. Thus, our results suggest that the conversion of ATP, a molecule with pro-inflammatory activity, into adenosine, which possesses immunomodulatory properties, may contribute to the establishment of infection by Leishmania.
... Surface membrane interactions between parasites and their host cells are of critical importance for the survival of the parasite, from both immunological and physiological viewpoints. In this context, the presence of enzymes with catalytic sites facing the extracellular medium, such as ecto-ATPases and ecto-5 -nucleotidases, seem to be extremely important to the living parasite (Meyer-Fernandes, 2002;Meyer-Fernandes et al., 2010). ...
Tritrichomonas foetus is the causative agent of sexually transmitted trichomoniasis in cattle. In females, the infection can be associated with infertility, vaginitis, endometritis, abortion or pyometra, leading to significant economic losses in cattle raising. T. foetus is devoid of the ability to synthesize purine nucleotides de novo, depending instead on salvaging purines from the host environment. Ecto-5'-nucleotidase catalyzes the final step of extracellular nucleotide degradation, the hydrolysis of nucleoside 5'-monophosphates to the corresponding nucleosides and Pi. In this work we show that living, intact cells of T. foetus were able to hydrolyze 5'AMP at a rate of 12.57 ± 1.23 nmol Pi × h(-1) × 10(-7) cells at pH 7.2 and the 5'AMP hydrolysis is due to a plasma membrane-bound ecto-enzyme activity. The apparent K(m) for 5'AMP was 0.49 ± 0.06 mM. In addition to 5'AMP, the enzyme hydrolyzed all substrate monophosphates tested except 3'AMP. No divalent metals or metal chelators were able to modulate enzyme activity. Phosphatase inhibitors did not have an effect on ecto-5'-nucleotidase activity while ammonium molybdate did inhibit the activity in a dose dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. These results indicate the existence of an ecto-5'-nucleotidase that may play a role in the salvage of purines.
... Trypanosomatid protozoans are incapable of synthesising purines de novo and must rely on their hosts for these essential nutrients (Hassan and Coombs, 1988;De Koning et al., 2000;Berrêdo-Pinho et al., 2001;Meyer-Fernandes, 2002;Meyer-Fernandes et al., 2004;Pinheiro et al., 2006;Leite et al., 2007). Leishmania species have membrane bound 3 0 -nucleotidases on their extracellular surface that hydrolyse extracellular 3 0 -nucleotides or nucleic acids to form nucleosides (Dwyer and Gottlieb, 1984;Gbenle, 1993;Lakhal-Naouar et al., 2008). ...
In this work we showed that living cells of Leishmania chagasi was able to hydrolyse 3'AMP 10 times more than 5'AMP. When parasites were grown in a low phosphate concentration (2 mM) the cellular proliferation decreased by 50% compared to cells grown in the presence of a high phosphate concentration (80 mM). However, the ecto-3'nucleotidase activity was 2-fold higher when L. chagasi was grown in a low phosphate concentration. This modulation observed on ecto-3'nucleotidase activity was not observed on ecto-5'nucleotidase activity. These results suggest that low concentration of Pi in the culture medium modulates ecto-3'nucleotidase activity that may lead to modulation of important processes for the cell. Interestingly, the macrophage-parasite interaction increased by 45% when L. chagasi were grown at low phosphate concentration compared to the parasites grown in the presence of high phosphate source. Altogether, the results described here suggest that 3'nucleotidase activity modulated by external stimuli, Pi concentration, could be involved on parasite-macrophage interaction.
... The extracellular hydrolysis of ATP can be initiated by NTPDases (ectonucleoside triphosphate diphosphohydrolases) and terminated by ecto-5 0 -nucleotidase (CD73; E.C. 3.1.3.5) (Meyer-Fernandes, 2002;Robson et al., 2006). Ecto-5 0 -nucleotidases have been described in some protozoan parasites, such as Trichomonas vaginalis (Tasca et al., 2003) and Trichomonas gallinae (Borges et al., 2007). ...
In this work, we biochemically characterized the ecto-5'-nucleotidase activity present on the surface of the living trophozoites of Giardia duodenalis. Two sequences of the 5'-nucleotidase family protein were identified in the Giardia genome. Anti-mouse CD73 showed a high reaction with the cell surface of parasites. At pH 7.2, intact cells were able to hydrolyze 5'-AMP at a rate of 10.66 ± 0.92 nmol Pi/h/10(7) cells. AMP is the best substrate for this enzyme, and the optimum pH lies in the acidic range. No divalent cations had an effect on the ecto-5'-nucleotidase activity, and the same was seen for NaF, an acid phosphatase inhibitor. Ammonium molybdate, a potent inhibitor of nucleotidases, inhibited the enzyme activity in a dose-dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. The results indicate the existence of an ecto-5'-nucleotidase that could play a role in the salvage of purines.
... To date no physiological role was allocated to ectophosphatases, however, several hypotheses have been suggested roles in protection from cytolytic effects of extracellular ATP, dephosphorylation of ectokinase or host organism kinase substrates or involvement in signal transduction and regulating cellular adhesion (reviewed in Ref. [67]). Although some ectophosphatase can dephosphorylate tyrosine-phosphorylated substrates [67,68] they do not show any sequence similarity to PTP or STPs and for this reason they were excluded from this review. In vivo experiments in mice also showed that inhibition of PTP activities can lead to a complete block of the development of cutaneous lesions, almost complete disappearance of parasites from popliteal lymph nodes and a reduction of the liver parasite load at two weeks post- treatment. ...
Protein phosphorylation is one of the most important post-translational modifications regulating various signaling processes in all known living organisms. In the cell, protein phosphatases and protein kinases play a dynamic antagonistic role, controlling the phosphorylation state of tyrosine (Tyr), serine (Ser) and threonine (Thr) side chains of proteins. The reversible phosphorylation modulates protein function, through initiating conformational changes, which influences protein complex formation, alteration of enzyme activity and changes in protein stability and subcellular localization. These molecular changes affect signaling cascades regulating the cell cycle, differentiation, cell-cell and cell-substrate interactions, cell motility, the immune response, ion-channel and transporter activities, gene transcription, mRNA translation, and basic metabolism. In addition to these processes, in unicellular parasites, like Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., additional signaling pathways have evolved to enable the survival of parasites in the changing environment of the vector and host organism. In recent years the genome of five trypanosomatid genomes have been sequenced and annotated allowing complete definition of the composition of the trypanosomatid phosphatomes. The very diverse environments involved in the different stages of the kinetoplastids' life cycle might have played a role to develop a set of trypanosomatid-specific phosphatases in addition to orthologues of many higher eukaryote protein phosphatases present in the kinetoplastid phosphatomes. In spite of their well-described phosphatomes, few trypanosomatid protein phosphatases have been characterized and studied in vivo. The aim of this review is to give an up to date scope of the research, which has been carried out on trypanosomatid protein phosphatases.
... The plasma membrane of cells may contain 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 intact cells Meyer-Fernandes, 2002;Jesus et al., 2002;Gomes et al., 2006;Pinheiro et al., 2007;Peres-Sampaio et al., 2008). Knowledge about interactions between components of the external surface of the cells and the cellular elements of the host is of obvious importance for the understanding of the complex life cycle of T. rangeli. ...
In this work, we showed that living cells of Trypanosoma rangeli express different ecto-phosphatase activities in response to different inorganic phosphate (Pi) concentrations in the culture medium. The ecto-phosphatase activity from T. rangeli grown at low-Pi concentration was inhibited by the increase of the pH, while the ecto-phosphatase of the cells grown at high Pi concentration was not modulated by the change of the pH of the medium. Okadaic acid inhibited only the ecto-phosphatase activity from cells grown at low-Pi concentration but not the ecto-phosphatase activity from cells grown at high-Pi concentration. Accordingly, phosphatase activity from T. rangeli grown at low Pi concentration was able to hydrolyze P-serine and P-threonine at high rate but not P-tyrosine. The phosphatase activity from T. rangeli grown at high-Pi concentration was able to hydrolyze P-serine, P-threonine and P-tyrosine with the same rate. The addition of anterior midgut homogenate of Rhodnius prolixus on the epimastigotes suspension inhibited the enzyme activity of T. rangeli grown at low-Pi concentration. On the other hand, anterior midgut homogenate had no effect in the ecto-phosphatase of T. rangeli maintained at high-Pi concentration. Altogether, the results described here indicate that ecto-phosphatase activities hydrolyzing phosphorylated compounds present in the extracellular medium of T. rangeli are regulated by the external Pi concentration.
... In cells, the plasma membrane may contain enzymes whose active sites face the external medium. The activities of these enzymes, referred to as ecto-enzymes, can be measured using intact cells Gomes et al., 2006;Meyer-Fernandes et al., 1997;Meyer-Fernandes, 2002). Knowledge of the interactions between components on the external surface of parasitic cells and the cellular elements of the host is of obvious importance for understanding the complex life cycle of T. rangeli. ...
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 beta-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.
Leishmania infantum, the causative agent of American Visceral Leishmaniasis (VL), is known for its ability to modulate the host immune response to its own favor. Ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) represents a family of enzymes that hydrolyze nucleotides and are involved in nucleotide-dependent biological processes. L. infantum has two ENTPDases, namely LiNTPDase1 and LiNTPDase2. Here, we used genetic tools to overexpress or abolish the expression of LiNTPDase1 and -2 to assess their role in parasite growth in culture and macrophage infection. While LiNTPDase1 or 2-overexpressing clones showed no morphological or growth changes in promastigotes, LiNTPDase2 overexpression increased macrophage adhesion and infection by 50% and 30%, respectively. The individual LiNTPDase1 and 2 knockout mutants showed lag in growth profile, which was reversed by the addition of adenine and guanine to the culture media. Moreover, the morphology of the knockout mutants even in supplemented media was changed to an amastigote-like form. The double knockout of both genes was lethal and a mechanism of compensation of deletion of one isoform was detected in these mutants. Correspondingly, the absence of LiNTPDase1 or LiNTPDase2 led to a dramatic reduction in in vitro infection (∼90%). Interestingly, nitric oxide production was decreased in both knockout mutants during infection, which suggests that both LiNTPDases can inhibit macrophage responses against the parasite. Overall, our results show important roles of LiNTPDase1 and -2 concerning in vitro macrophage infection and reinforce their use as potential targets to control Leishmania infections.
As obligate intracellular parasites, the Apicomplexa, including Toxoplasma gondii, must either synthesize or acquire essential nutrients while evading host defenses. The metabolic pathways of Toxoplasma reflect the evolutionary relationship of the Apicomplexa to plants as illustrated by the activities of the relict plastid, the apicoplast, as well as other plant-like pathways, such as amylopectin synthesis. Carbohydrate metabolism is developmentally regulated in T. gondii with specialized enzymes for different life-cycle stages. More recent studies have revealed that lipid metabolism consists of unique and conserved scavenging and biosynthetic capabilities. This chapter highlights the metabolic pathways of T. gondii and illustrates important and unique aspects of carbohydrate, lipid and nucleotide metabolism that may pose potential drug targets.
Toxoplasma gondii and all other parasites of the phylum Apicomplexa reside and replicate exclusively within eukaryotic cells. It suggests that these parasites depend on the metabolism of their hosts and that they have evolved metabolic pathways reflecting their intracellular lifestyle. In addition, these parasites may display specific or alternate metabolism as documented by the recent discovery of novel metabolic pathways in the remnant and vestigial plastid (apicoplast) found in many apicomplexan parasites. While targeting unique parasite pathways is an attractive strategy, in practice the procurement of enough pure parasites for biochemical study or purification of parasite components such as enzymes is extremely difficult. Biochemical and metabolic studies of T. gondii have also been difficult to execute because of continual problems with contamination of parasite preparations with host-cell components.
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.
Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease.
Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages.
Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.
Leishmaniasis is a major vector-borne parasitic disease affecting 12 million people worldwide. With a broad range of clinical manifestations, ranging from self-healing skin ulcers to disfiguring mucosal lesions to life-threatening infections of visceral organs (liver and spleen), the disease has become a serious human health issue, particularly in developing countries. Among all of its forms, visceral leishmaniasis (VL, also known as kala-azar), caused by the Leishmania donovani complex (i.e., L. donovani and L. infantum in Old World and L. chagasi in New World), is often fatal in the absence of treatment. Although humans are the principal hosts for L. donovani, canine species are the main reservoirs of L. infantum. Canine VL affects millions of dogs and is associated with outbreaks of human VL and hence has become a major public health issue. The lack of vaccines to prevent and/or treat these infections, as well as the emergence of drug resistant parasites, is serious impediments to control leishmaniasis. Therefore, developing new prophylactic and therapeutic strategies against this disease is urgently required. However, for this to occur, a better understanding of the complex immune mechanisms generated in response to infection and defining those involved in resistance to infection is required. In this special issue on “Immunity to visceral leishmaniasis”, several selected papers will discuss these issues.
Nucleoside triphosphate diphosphohydrolases (NTPDases, GDA1_CD39 protein superfamily) play a diverse range of roles in a number of eukaryotic organisms. In humans NTPDases function in regulating the inflammatory and immune responses, control of vascular haemostasis and purine salvage. In yeast NTPDases are thought to function primarily in the Golgi, crucially involved in nucleotide sugar transport into the Golgi apparatus and subsequent protein glycosylation. Although rare in bacteria, in Legionella pneumophila secreted NTPDases function as virulence factors. In the last 2 decades it has become clear that a large number of parasites encode putative NTPDases, and the functions of a number of these have been investigated. In this review, the available evidence for NTPDases in parasites and the role of these NTPDases is summarized and discussed. Furthermore, the processes by which NTPDases could function in pathogenesis, purine salvage, thromboregulation, inflammation and glycoconjugate formation are considered, and the data supporting such putative roles reviewed. Potential future research directions to further clarify the role and importance of NTPDases in parasites are proposed. An attempt is also made to clarify the nomenclature used in the parasite field for the GDA1_CD39 protein superfamily, and a uniform system suggested.
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.
A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.
The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase. The protein is synthesized in large amounts and transported through the secretory pathway of the parasite and into the vacuolar space in an oxidized and thereby enzymatically inactive form. Complete activation of the purified enzyme is known to require dithiols (e.g. DTT); subcellular fractionation demonstrates that little if any (<5%) of the enzyme in the vacuolar space is active in the absence of DTT. Both native and epitope-tagged nucleoside triphosphate hydrolase (NTPase) were partially activated during immunoprecipitation, precluding precise assessment of enzyme activity in the vacuolar space but suggesting that protein-protein interactions may trigger activation. When infected cells were treated with DTT, the NTPase was activated in a dose-response fashion, as assessed by migration on SDS-polyacrylamide gel electrophoresis and by an increase in enzymatic activity. After activation, enzyme activity decreased with time in the presence of DTT; this inactivation was slowed by the presence of excess ATP. A rapid fall in host cell ATP was accompanied by an abrupt exit of parasites from cells. These results demonstrate that the oxidation/reduction status of the NTPase, the only parasite dense granule protein that contains disulfide bonds, is tightly controlled within the vacuolar space and may influence parasite exit from cells.
Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.
Cinemicrography of Entamoeba histolytica destruction of Chinese hamster ovary (CHO) cells shows that ameba cytopathogenicity consists of separate components: a contact-dependent cytolethal effect, and phagocytosis. Cells not in contact with amebae remain intact. Quantitation of ameba destruction of CHO cells by applying the one-hit hypothesis confirms that the cytoethal effect of amebae is contact dependent. Studies with 111Indium oxine-labeled cells provide further evidence of extracellular killing by E. histolytica and indicate that > 94% of the target cells are killed before phagocytosis. When we examined for a cytotoxin release by E. histolytica, we found no effect on CHO cells with filtrates of amebae, and a nonspecific effect of cell rounding and release with sonicates of amebae. The ameba sonicate effect was time-dose dependent, was not cytolethal, was reversible, and was inhibited by alpha II macroglobulin. Cytochalasin B altered ameba motility and morphology, and monolayer experiments confirmed that cytochalasins A, B, or D inhibited CHO cell destruction by E. histolytica. Cytochalasin D also inhibited extracellular killing of CHO cells by amebae in pellets, apparently independent of effects on ameba motility or phagocytosis. Colchicine and vinblastine, alone or in combination with cytochalasin D, did not inhibit E. histolytica cytopathogenicity, which indicates that microtubule function is not required for target cell killing by amebae.
The chicken gizzard smooth muscle extracellular ATPase (ecto-ATPase) is a low abundance, high specific activity, divalent cation-dependent, nonspecific nucleotide triphosphatase (NTPase). The ATPase is a 66-kDa glycoprotein with a protein core of 53 kDa (Stout, J.G. and Kirley, T.L. (1994) J. Biochem. Biophys. Methods 29, 61-75). In this study we evaluated the characteristics of a bank of monoclonal antibodies raised against a partially purified chicken gizzard ecto-ATPase. 18 monoclonal antibodies identified by an ATPase capture assay were tested for effects on ATPase activity as well as for their Western blot and immunoprecipitation potential. The five most promising monoclonal antibodies were used to immunopurify the ecto-ATPase. The one-step immunoaffinity purification of solubilized chicken gizzard membranes with all five of these monoclonal antibodies isolated a 66-kDa protein whose identity was confirmed by N-terminal sequence analysis to be the ecto-ATPase. Several of these monoclonal antibodies stimulated ecto-ATPase activity similar to that observed previously with lectins. Western blot analysis revealed that three of the five monoclonal antibodies recognized a major immunoreactive band at 66 kDa (53-kDa core protein), consistent with previous purification results. The other two antibodies recognized proteins of approximately 90 and 160 kDa on Western blots. The 90-kDa co-immunopurifying (and presumably associated or related) protein was identified by N-terminal analysis as LEP100, a glycoprotein that shuttles between the plasma and lysosomal membranes. The approximately 160-kDa co-immunopurifying protein was identified by N-terminal analysis as integrin, a protein involved in extracellular contacts with adhesion molecules. Extended N-terminal sequence analysis of the immunopurified 66-kDa ecto-ATPase revealed some sequence homology with mouse lysosomal associated membrane protein. Tissue distribution of the ecto-ATPase showed that the highest levels of protein were expressed in muscle tissues (cardiac, skeletal, and smooth) and brain.
We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of a virulent strain (RH) of Toxoplasma gondii. On further examination, it was found that the purified enzyme was not a single enzyme but was a mixture of two isozymes termed NTPase-I and NTPase-II.
The two isozymes had similar molecular masses of approximately 240–270 kDa by gel filtration and contained four identical subunits of molecular mass 66–67 kDa by SDS-polyacrylamide gel electrophoresis. Both forms of the NTPase were activated by dithiothreitol, and NTPase-I had a specific activity 4.5-fold higher than NTPase-II in hydrolysis of ATP. The primary difference between these isozymes lies in their ability to hydrolyze nucleoside triphosphate versus diphosphate substrates. While NTPase-II hydrolyzed ATP to ADP and ADP to AMP at almost the same rate, NTPase-I hydrolyzed ADP to AMP at a much slower rate (0.7% of the rate for ATP).
The complete cDNAs for NTPase-I and NTPase-II were sequenced and found to encode the same size predicted open reading frame of which only 16 of 628 amino acids differed between the two isozymes. Both forms of the NTPase contained an NH2-terminal hydrophobic signal peptide, consistent with our previous findings that these enzymes are secreted into the host cell vacuole occupied by the parasite. The gene encoding NTPase-II was found in all strains of Toxoplasma, while the NTPase-I was confined only to virulent strains. Expression of this highly active ATPase (NTPase-I) may contribute to intracellular survival and virulence of T. gondii.
The obligate intracellular parasite Toxoplasma gondii produces a nucleoside triphosphate hydrolase (NTPase) (nucleoside-triphosphatase, EC 3.6.1.15) activable by dithiol-containing compounds. We have isolated the genomic DNA for the NTPase from the RH strain of Toxoplasma and determined the nucleotide sequence of three tandemly arranged open reading frames termed NTP1, NTP2, and NTP3. We have also isolated and sequenced cDNAs for NTP1 and NTP3; no cDNA for NTP2 was obtained. The two cDNA clones encode proteins that are more than 97% identical at the amino acid level but significantly differ within two small domains, indicating the presence of NTPase isoforms. Both possess N-terminal signal sequences and two regions with partial homology to certain known ATP binding motifs: the glycine-rich loop common to many ATP binding proteins and the beta-phosphate binding domain found in the hexokinase-actin-hsp70 family. Antiserum against a NTP1-fusion protein immunoprecipitated NTPase activity from extracellular parasites that was increased in activity by treatment with dithiothreitol, confirming the identity of the cloned genes. By immunofluorescence, the NTPase is located in vesicular structures within the parasite, and in infected cells it is secreted into the vacuolar space and becomes partially associated with the parasitophorous vacuolar membrane. Since the vacuolar membrane is freely permeable to small molecules of < 1300 Da, host cell ATP may serve as a substrate for the NTPase and supply the energy for parasite-directed processes in the vacuolar space.
INTRODUCTION The enteric protozoan parasite Entamoeba histolytica is one of the most potent cytotoxic cells known, named by Schaudinn in 1903 for its ability to destroy human tissues (53). Infection occurs when the cyst form of the parasite is ingested with contaminated food or water. After excysting to form the trophozoite in the small intestine, the amebae can colonize the bowel lumen, invade through the intestinal epithelium to cause colitis or liver abscess, or form cysts that are excreted with the stool to start a new round of infection (28). Parasite recognition of glycoconjugates plays an important role in the pathogenesis of amebiasis. Killing of host cells by E. histolytica trophozoites in vitro occurs only upon direct contact, which is mediated by an amebic adhesin which recognizes N-and 0-linked oligosaccharides (9, 35, 36, 49). This adhesin is specifically inhibited by millimolar concentrations of galactose and N-acetyl-D-galactosamine (Gal/GalNAc) (36). Avoidance of lysis by the complement membrane attack complex is also mediated by this lectin. Here we discuss recent advances in the understanding of this novel amebic adhesin.
The insulin receptor possesses tyrosine kinase activity which is thought to mediate the biological effects of insulin upon target cells. pp120 is a liver-specific glycoprotein of apparent molecular size of 120 kDa that is phosphorylated on tyrosine residues by the receptors for insulin, insulin-like growth factor-I, and epidermal growth factor. Previously, we demonstrated that pp120 is identical to a liver-specific ecto-ATPase. In the present study, we have cloned the rat gene encoding pp120/ecto-ATPase. The gene is contained within approximately 15 kilobases of genomic DNA, and consists of nine exons interrupted by eight introns. Using the reverse transcriptase/polymerase chain reaction, we isolated cDNA clones complementary to rat liver mRNA encoding pp120/ecto-ATPase. Sequence analysis indicated the presence of two populations of cDNA's that differ by the presence or absence of a 53-base pair (bp) fragment encoding the juxta-membrane region of the cytoplasmic domain. By cloning the corresponding region of the ecto-ATPase gene, we demonstrated that the 53-bp represents exon 7 of the gene. This 53-bp exon undergoes alternative splicing, thereby giving rise to two mRNA variants. Deletion of this 53-bp cassette exon introduces a frameshift, and results in a premature chain termination codon that truncates the cytoplasmic domain. The truncated cytoplasmic domain contains 10 rather than 71 amino acid residues. Because the short isoform of ecto-ATPase lacks the putative sites for tyrosine- and serine-specific phosphorylation, this alternative splicing may have a major effect upon the physiological function of the enzyme.
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 ecto-ATPase from chicken gizzard (smooth muscle) was solubilized, and the 66-kDa cell membrane ecto-ATPase protein was purified. The protein was then subjected to both enzymatic and chemical cleavage, and the resultant peptides were purified by reverse phase high pressure liquid chromatography and sequenced. Several of these internal peptide sequences were used to design oligonucleotides to screen a chicken muscle library to identify the cDNA encoding the ecto-ATPase. Two overlapping partial clones were sequenced, yielding the complete coding region and a long 3'-untranslated sequence. The deduced amino acid sequence is in agreement with the N-terminal and peptide sequences obtained from the purified protein. The chicken muscle ecto-ATPase is a slightly basic (predicted pI = 7.93) 494-amino acid protein (54.4 kDa), containing a single transmembrane domain at each end of the protein. The majority of the protein is predicted to be extracellular, making it a Type Ia plasma membrane protein. There are four putative N-glycosylation sites, a single potential cAMP/cGMP-dependent protein kinase phosphorylation site, as well as a single putative tyrosine kinase phosphorylation site. Analysis of the sequence using the BLAST programs demonstrated homology with other ecto-ATPases and ecto-apyrases, including those from the parasitic protozoan Toxoplasma gondii, potato tubers, and garden pea, as well as a guanosine diphosphohydrolase from yeast. However, the most striking homology observed was to the human and mouse lymphoid cell activation antigen 39 (CD39), a molecule now known to have apyrase activity. The chicken ecto-ATPase showed considerable amino acid sequence homology with CD39 over the entire length of the sequence, excluding about 30-40 amino acids at the extreme ends of the protein (which include the two membrane-spanning helices). The sequence homology between the gizzard ecto-ATPase and CD39 was confirmed by Western blots demonstrating immunocross-reactivity between mono- and polyclonal antibodies raised against the chicken ecto-ATPase and two commercially available monoclonal antibodies against the human CD39 protein. The results suggest that the muscle ecto-ATPase may be involved in cell adhesion, since the highly homologous CD39 protein is involved in homotypic adhesion of activated B lymphocytes.
For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote, Tetrahymena, at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme in Tetrahymena. It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+ or Mg2+ dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain, N-ethylmaleimide and p-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ecto-ATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein in Tetrahymena plasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase of Tetrahymena may play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.
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 lectin isolated from the roots of the legume, Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date. This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands. This lectin-nucleotide phosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes. LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia. These properties suggest that this protein may play a role in the rhizobium-legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant.
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.
For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote,Tetrahymena,at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme inTetrahymena.It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+or Mg2+dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain,N-ethylmaleimide andp-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ectoATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein inTetrahymenaplasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase ofTetrahymenamay play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.
Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
A variety of nucleotides and the nucleoside adenosine can act as extracellular signaling substances. Their function is terminated by extracellular degradation via surface-located enzymes. The breakdown products may be recycled. This review discusses recent developments in the cellular and molecular biology of enzymes involved in extracellular purine metabolism, including diadenosine polyphosphate hydrolase, ATP-diphosphohydrolase (apyrase), nucleotide pyrophosphatase, 5′-nucleotidase, alkaline phosphatase, NAD-glycohydrolase, and adenosine deaminase. The potential of the surface-located enzymes for ADP-ribosylation and phosphorylation of extracellular proteins is also briefly discussed. Drug Dev. Res. 39:337–352, 1996. © 1997 Wiley-Liss, Inc.
The enteric pathogen, Entamoeba histolytica, appears to cause disease by adhering to and then destroying mucosal barriers. Using an in vitro method of studying the interaction of E. histolytica with target cells (Chinese hamster ovary [CHO] and human erythrocytes [RBC]), we examined the mechanism of amebic adherence and its role in lysis of target cells. Killing and phagocytosis of target cells by amebas ceases at 4°C, allowing observation of adherence. Amebas adhere to CHO cells at 4°C, 78.9% formed rosettes (amebas with ≥3 adherent CHO cells each) at 2 h. At 37°C, cytochalasins B and D inhibit adherence of amebas to CHO cells (P < 0.0005). Amebas adhere to and kill CHO cells in media with <0.1 μM calcium and magnesium plus 10 mM EDTA, indicating that divalent cations are not required in the medium. Adherence of amebas to human RBC was not ABO blood group specific and showed greater adherence to human than bovine or sheep RBC (P < 0.005). Neither Fc nor complement receptors were found on amebas by standard rosette studies. The amebic adherence receptor is not trypsin (0.125%) sensitive nor inhibited by trypan blue (1 mM). N-acetyl-d-galactosamine (GALNAc) inhibited the adherence of amebas to CHO cells and human RBC (0.1 g/100 ml or 4.5 mM GALNAc, P < 0.005) by binding to a receptor on the amebic surface. GALNAc abolishes amebic cytolysis of target CHO cells (determined by 111Indium oxine release from CHO cells, P < 0.001) but not amebic phagocytosis of CHO cells. By suspending ameba-CHO cells rosettes in dextran, we found that GALNAc (1%) reversibly inhibits amebic adherence (P < 0.0005) and that cytochalasins decrease amebic killing of adherent CHO cells (P < 0.025).
These findings indicate that the adherence of E. histolytica to target cells requires microfilament function, is via a specific amebic receptor that has affinity for GALNAc, and is required to lyse cells. Inhibition of the adherence of E. histolytica may alter the pathogenicity of this organism.
Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATPJ to ADP and inorganic phosphate, is expressed upon cell activation, Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label, 5′-p-(fluorosulfonyl)benzoyl adenosine (5′-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide ant agonists results in the inhibition of antigen induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATPe by ecto-ATPase in lymphocyte effector function.
Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATPe) to ADP and inorganic phosphate, is expressed upon cell activation. Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide antagonists results in the inhibition of antigen-induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATPe by ecto-ATPase in lymphocyte effector function.
Although there are several reports of Leishmania parasitizing cells other than macrophages or even existing extracellularly in mammalian tissue, it is generally acknowledged that in the vertebrate host these organisms normally reside within cells of the macrophage lineage. The first description of this particular host cell–parasite interaction can probably be attributed to Cunningham, who examined biopsy material, stained with gentian violet, from a patient with “Delhi boil.” Given the now well-documented multifaceted role of macrophages in the immune response, the parasite's interplay with its host cell poses many intriguing problems for researchers. This chapter intends to update the progress that has been made in recent years in understanding the biology of the leishmania–macrophage interaction and how the parasite can attach to and enter the host cell and survive in the intracellular environment. The chapter also discusses the implications of these interactions in the induction of specific immune responses and on disease progression. It also describes those areas where future research may be of value in the rational development of new chemical and immunological therapies and vaccines.
Cell-mediated cytotoxicity involves a number of distinct mechanisms as well as the active participation of the target cell. Recently, several investigators have demonstrated that extracellular ATP can act as a cytotoxic effector.
pp120/HA4, a membrane protein found in hepatocyte plasma membranes and a substrate for the insulin receptor tyrosine kinase, was purified to homogeneity, subjected to partial proteolysis, and peptides were sequenced by Edman degradation. Six amino acid sequences were obtained, and they matched the deduced amino acid sequences of six regions of a hepatocyte membrane protein called ecto-ATPase.
Dithiothreitol-dependent MgATP2- and Mg-ADP- hydrolytic activities owing to nucleoside triphosphate hydrolase were examined kinetically in tachyzoite cells of five strains of Toxoplasma gondii with various virulence. The tachyzoites of all the strains were revealed to have a high ATP and ADP hydrolytic potency. The value of Vmax and Km obtained from each strain indicated that there were two classified types of T. gondii about ATP and ADP hydrolysis. The most virulent strain (RH) and avirulent strain (Fukaya) were classified in a type with higher ATPase than ADPase activities. Two virulent strains (C56, Beverley) and an avirulent strain (ME49) were classified in the other type with the same activities of ATPase and ADPase.
Binding and cytolysis of Chinese hamster ovary (CHO) cells by Entamoeba histolytica trophozoites is inhibitable by galactose (Gal) or N-acetyl-D-galactosamine (GalNAc). To better define the carbohydrate receptor for E. histolytica, we compared the binding and cytolytic target properties of 10 CHO glycosylation mutants. Each mutant expresses a uniquely altered array of N- and/or O-linked cell surface carbohydrates. Amebic adherence was reduced when lactosamine-containing N-linked carbohydrates were essentially absent (Lec1 mutant), almost undetectable when Gal and GalNAc residues were absent on both N- and O-linked carbohydrates (ldlD.Lec1 mutant), and enhanced for mutants with increased terminal Gal residues (Lec2 and Lec3). Parental CHO cells treated with neuraminidase to expose Gal residues behaved like Lec2 mutants. Binding of purified Gal or GalNAc lectin to parental, Lec1, ldlD.Lec1, and Lec2 mutant CHO cells corroborated the adherence results. The suitability of CHO cell mutants as targets for amebic cytolysis correlated with their glycosylation phenotype: the Lec1 mutants were less susceptible than parental CHO cells, the ldlD.Lec1 mutants were highly resistant, and the Lec2 mutants required higher concentrations of Gal for inhibition. The E. histolytica Gal or GalNAc adherence lectin bound preferentially to beta 1-6-branched, N-linked carbohydrates lacking terminal sialic acid or fucose residues. However, amebic lectin binding to either N- or O-linked cell surface carbohydrates was sufficient to initiate parasite cytolytic activity.
Adherence properties of the potentially enteropathogenic Tritrichomonas mobilensis were studied in vitro. Axenically cultivated trichomonads readily attached to isolated intestinal epithelial cells and mucus of the squirrel monkey. The kinetics and nature of T. mobilensis cytadherence were microscopically evaluated in cell-suspension assay using Chinese hamster ovary (CHO) cells and in microplate hemagglutination assay with human erythrocytes. Adherence of the parasites to target cells was concentration- and time-dependent; it was inhibited by sialic acid (N-acetylneuraminic or N-glycolylneuraminic acid) and sialyllactose. Neither trypsinization of the flagellates nor their exposure to low temperature (4 degrees C) affected their cytadherence capacities. The data indicate the presence of adhesin(s) with lectin properties on T. mobilensis. Agglutination of live protozoa by animal and plant lectins with various carbohydrate-binding specificities as well as the occurrence of an electron-dense cell coat on plasma membrane suggest marked glycosylation of the parasite surface.
A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.
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.
In the present study the parental cells and glycosylation mutants of Chinese hamster ovary (CHO) cells were used to analyze the influence of surface carbohydrates on the cytoadhesion of trichomonads. Trichomonas vaginalis and Tritrichomonas foetus were allowed to interact with host cells for 2 h at 37 degrees C. Alternatively, CHO cells were treated with 10 mM periodate prior to the assays. Both trichomonads adhered to all CHO cell clones tested. A remarkable difference could be observed between the cytoadhesion of T. vaginalis and T. foetus. Sialic acid residues present on the surface of CHO cells may favor the cytoadhesion of T. foetus while hampering that of T. vaginalis. The specificity of the parasite cytoadhesion was further investigated. Sialic acid, mannose, and galactose as well as mannose, galactose, and N-acetylglucosamine added to the interaction medium at 50, 100, and 200 mM were capable of significantly inhibiting the cytoadhesion of each trichomonad species. Periodate treatment of target cells also induced decreases in the cytoadhesion of the trichomonads. These results strongly suggest an important role for host-cell surface glycoconjugates during the cytoadhesion of trichomonads. In addition, they also point out the presence of "lectin-like" molecules on the surface of both T. vaginalis and T. foetus.
The rat liver ectoATPase has reportedly been cloned. The cDNA, a member of the carcinoembryonic antigen (CEA) gene family, was shown to increase aggregation of transfected cells, but ATPase activity was not evaluated. Using this cDNA as a probe to clone the mercurial-insensitive ectoATPase (MI-ectoATPase) of human hepatoma Li-7A cells, the cDNA obtained was that of CEA which has no ATPase activity. The probe also did not detect increased transcription when MI-ectoATPase activity was induced in Li-7A cells. It is concluded that the "rat liver ectoATPase cDNA" codes for a cell adhesion molecule but does not code for an ectoATPase. It was also discovered that expression of four CEA transcripts in Li-7A cells was markedly stimulated by a single growth modulator, EGF, and was further stimulated by a cAMP elevating agent, cholera toxin.
Toxoplasma gondii is an obligate intracellular parasite capable of invading a wide range of host cells where it residues in a specially modified compartment termed the parasitophorous vacuole. This compartment provides a protected environment for the parasite which enters a rapid growth phase shortly after invasion. To identify functional components of this interface, we have used immunolabeling and cell fractionation to localize the potent nucleoside triphosphate hydrolase (NTPase) produced by Toxoplasma. In extracellular tachyzoites, NTPase was not exposed on the cell surface but was localized in small vesicles, called dense granules, scattered throughout the cell cytoplasm. Shortly following invasion. NTPase was secreted from dense granules and occupied the lumen of the parasitophorous vacuole where it was often associated with the intravacuolar network. NTPase was exclusively found in the supernatant in cell fractionation studies of both extracellular tachyzoites lysed by freeze-thaw and following secretion into the parasitophorous vacuole. These studies provide evidence for the rapid secretion of NTPase into the parasitophorous vacuole, where it may play a key role in processing of nucleotides for purine salvage by the parasite.
Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
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).
The expression of P2 purinergic receptor subtypes in leukocytes varies with both lineage and developmental stage. Given the recent identification and cloning of at least seven distinct G protein-coupled ATP receptor subtypes (P2Y family), we investigated P2Y receptor subtype expression during myeloid cell differentiation. We observed that KG-1 myeloblasts express P2Y1 but not P2Y2 receptors (previously termed P2U receptors), whereas later myeloid progenitors, including HL-60 promyelocytes and THP-1 monocytes, expressed P2Y2 but not P2Y1 receptors. In KG-1 cells, significant activation of Ca2+ mobilization by P2Y1 receptors was only observed after preincubation with potato apyrase, an exogenous ATPase. This indicated that P2Y1 receptors are desensitized in KG-1 cells by autocrine mechanisms that may involve enhanced release of endogenous nucleotides and/or decreased expression of cell-surface ecto-nucleotidases. We compared the levels of ecto-apyrase activity and expression in KG-1 myeloblasts and HL-60 promyelocytes. Extracellular ATP was rapidly metabolized by HL-60 but not by KG-1 cells. Reverse transcription-polymerase chain reaction analysis indicated that mRNA for CD39 (cluster of differentiation), an identified ecto-apyrase, was present in HL-60 but not KG-1 cells. Ecto-apyrase activity was modestly increased with differentiation of myeloid progenitors with either phorbol 12-myristate 13-acetate (PMA) or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). Differentiation of HL-60 cells with PMA, but not DBcAMP, strongly induced ecto-5'-nucleotidase activity and CD73 mRNA expression. These observations indicate that signal transduction by extracellular ATP in myeloid leukocytes can be regulated by developmentally programmed changes in the expression of P2Y receptor subtypes and multiple ecto-nucleotidases.
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).
The Toxoplasma gondii nucleoside triphosphate hydrolase is the most active E-type ATPase yet identified, and was the first member of this new gene family to be cloned (Bermudes D, Peck KR, Afifi-Afifi M, Beckers CJM, Joiner KA. J Biol Chem 1994;269:29252-29260. Previous work also identified two isoforms of the enzyme in the virulent RH strain, and demonstrated that internal fragments of the genes encoding these isoforms were found differentially in virulent versus avirulent organisms (Asai T, Miura S, Sibley D, Okabayashi H, Tsutomu T, J Biol Chem 1995;270:11391-11397). We now show that the NTPase 1 isoform is expressed in avirulent strains, whereas virulent strains express both the NTPase 1 and NTPase 3 isoforms. The avirulent PLK strain lacks the gene for NTPase 3, explaining the absence of expression. Despite the fact that NTPase 1 and NTPase 3 are 97% identical at the amino acid level, recombinant NTPase 1 is a true apyrase, whereas recombinant NTPase 3 cleaves predominantly nucleotide triphosphates. Furthermore, native and recombinant NTPase 3 but neither native nor recombinant NTPase 1 bind to ATP-agarose, further distinguishing the two isoforms. Using chimeras between the NTP1 and NTP3 genes, we show that a block of twelve residues at the C-terminus dictates substrate specificity. These residues lie outside the regions conserved among other E-ATPases, and therefore provide new insight into substrate recognition by this class of enzymes.
It has become clear that members of both the E-NTPase and PDNPase protein family can act as ectoenzymes or exoenzymes, or both, or may even have an intracellular localization. The functional characterization of recently cloned members of the ecto-NTPase family is expected to widen the range of catalytic properties and cellular locations even further. The molecular diversity of ectonucleotidases now appears to be comparable with that of receptors for nucleotides. It seems likely that additional family members including splice variants remain to be discovered. The enormous molecular diversity and overlapping tissue distribution of the members of the E-NTPase and PDNP family make it difficult to assign specific functions to enzymes in individual tissues. However, first examples have been elaborated. The definition of their physiological substrates remains a major challenge. It is expected that the generation of heterozygous and homozygous knockout mice for each subtype followed by double-knockouts obtained by crossbreeding of single knockout mice will help to elucidate the functional roles of ectonucleotidases. These novel insights, together with the development of specific enzyme inhibitors or recombinant enzymes, or both, promise to offer important tools for basic research and therapy, and may provide new understanding of the control of cellular function at the local level.
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.
It has become clear that several families of ecto-nucleotidases function in the nervous system. They differ in their catalytic and also other functional properties and consist of several members each. At present neither the functional diversity nor the apparent redundancy of the ecto-nucleotidase pathway is understood. But the notion is further supported that nucleotidergic signaling pathways are widely distributed in the nervous system and that the ecto-nucleotidases play a significant part in it. The functional properties of the presently known ecto-nucleotidases and also of the sequenced but as yet uncharacterized potential ecto-nucleotidases need to be further investigated. An evaluation of their role in defined signaling pathways requires that the cellular location of the enzymes is determined. Knock-out mice in which individual ecto-nucleotidases have been deleted from the germline promise to be important tools to unravel their functional roles in the nervous system and also in other tissues.
In this work, we describe the ability of living cells of Entamoeba histolytica to hydrolyze extracellular ATP. In these intact parasites, whose viability was determined by motility and by the eosin method, ATP hydrolysis was low in the absence of any divalent metal (78 nmol P(i)/h/10(5) cells). Interestingly, in the presence of 5 mM MgCl(2) an ecto-ATPase activity of 300 nmol P(i)/h/10(5) cells was observed. The addition of MgCl(2) to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.23 mM MgCl(2). Both activities were linear with cell density and with time for at least 1 h. The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2) but not by SrCl(2), ZnCl(2), or FeCl(3). In fact, FeCl(3) inhibited both Mg(2+)-dependent and Mg(2+)-independent ecto-ATPase activities. The Mg(2+)-independent ATPase activity was unaffected by pH in the range between 6.4 and 8. 4, in which the cells were viable. However, the Mg(2+)-dependent ATPase activity was enhanced concomitantly with the increase in pH. In order to discard the possibility that the ATP hydrolysis observed was due to phosphatase or 5'-nucleotidase activities, several inhibitors for these enzymes were tested. Sodium orthovanadate, sodium fluoride, levamizole, and ammonium molybdate had no effect on the ATPase activities. In the absence of Mg(2+) (basal activity), the apparent K(m) for ATP(4-) was 0.053 +/- 0.008 mM, whereas at saturating MgCl(2) concentrations, the corresponding apparent K(m) for Mg-ATP(2-) for Mg(2+)-dependent ecto-ATPase activity (difference between total and basal ecto-ATPase activity) was 0.503 mM +/- 0.062. Both ecto-ATPase activities were highly specific for ATP and were also able to hydrolyze ADP less efficiently. To identify the observed hydrolytic activities as those of an ecto-ATPase, we used suramin, a competitive antagonist of P(2) purinoreceptors and an inhibitor of some ecto-ATPases, as well as the impermeant agent 4'-4'-diisothiocyanostylbenzene-2'-2'-disulfonic acid. These two reagents inhibited the Mg(2+)-independent and the Mg(2+)-dependent ATPase activities to different extents, and the inhibition by both agents was prevented by ATP. A comparison among the ecto-ATPase activities of three amoeba species showed that the noninvasive E. histolytica and the free-living E. moshkovskii were less efficient than the pathogenic E. histolytica in hydrolyzing ATP. As E. histolytica is known to have a galactose-specific lectin on its surface, which is related to the pathogenesis of amebiasis, galactose was tested for an effect on ecto-ATPase activities. It stimulated the Mg(2+)-dependent ecto-ATPase but not the Mg(2+)-independent ATPase activity.
Cell viability requires the perfect functioning of the processes controlling ATP and Ca(2+) homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with a disruption of ATP and Ca(2+) homeostasis. This study shows that 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibits Trypanosoma cruzi epimastigote cell growth. This thiol-reagent thiocyanate derivative was able to inhibit two ecto-enzymes present in this parasite. The ecto-ATPase and ecto-phosphatase activities were inhibited in a dose-dependent manner (K(i)=47.7 and 472.5 microM, respectively), but the 5'nucleotidase and 3'nucleotidase activities were not. DIDS uptake was approached by fluorescence microscopy. Pulse-chase experiments revealed the DIDS accumulation in compartments, presumably endocytic, in the posterior region of epimastigotes. In addition, we show that the T. cruzi mitochondria studied in permeabilized cells are able to accumulate and retain medium Ca(2+) in the absence of DIDS. However, in the presence of increasing concentrations of DIDS (50-200 microM), Ca(2+) transport was inhibited in a dose-dependent manner. DIDS also caused a disruption of the mitochondrial membrane potential, in the same concentration range, thus explaining its effect on Ca(2+) uptake. The presence of EGTA prevented the elimination of the mitochondrial membrane potential (DeltaPsi), supporting previous data suggesting that the binding of Ca(2+) to the mitochondrial membrane exposes buried thiols to react with DIDS. This thiocyanate derivative was also able to inhibit Ca(2+) uptake by the endoplasmic reticulum in a dose-dependent manner. Taken together, the data presented here provide further insights into the mechanisms underlying the antiproliferative actions of DIDS in T. cruzi.
Toxoplasma gondii is a protozoan parasite that infects a wide variety of warm-blooded animals and humans, in which it causes opportunistic disease. As an obligate intracellular parasite, T. gondii must invade a host cell to survive and replicate during infection. Recent studies suggest that T. gondii secretes a variety of proteins that appear to function during invasion or intracellular replication. These proteins originate from three distinct regulated secretory organelles called micronemes, rhoptries and dense granules. By discharging the contents of its secretory organelles at precise steps in invasion, T. gondii appears to timely deploy secretory proteins to their correct target destinations. Based on the timing of secretion and the characteristics of secretory proteins, an emerging theme is that T. gondii compartmentalizes its secretory proteins according to general function. Thus, it appears that micronemal proteins may function during parasite attachment to host cells, rhoptry proteins may facilitate parasite vacuole formation and host organellar association, and dense granule proteins likely promote intracellular replication, possibly by transporting and processing nutrients from the host cell. However, as more T. gondii secretory proteins are identified and characterized, it is likely that additional functions will be ascribed to each class of proteins secreted- by this fascinating invasive parasite.
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 ectoenzymes, can be measured using living cells. In this work we describe the ability of living promastigotes of Leishmania amazonensis to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (5.39 +/- 0.71 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 30.75 +/- 2.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.21 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.98 mM and free Mg(2+) did not increase the ecto-ATPase activity. In the pH range from 6.8 to 8.4, in which the cells were viable, the acid phosphatase activity decreased, while the Mg(2+)-dependent ATPase activity increased. This 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, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, 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. A comparison between the Mg(2+)-dependent ATPase activity of virulent and avirulent promastigotes showed that avirulent promastigotes were less efficient than the virulent promastigotes in hydrolyzing ATP.