Experimental Parasitology 112 (2006) 76–84 www.elsevier.com/locate/yexpr
Trypanosoma rangeli: Characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity Fábio Vasconcelos Fonseca, André Luíz Fonseca de Souza, Ana Claudia Mariano, Peter F. Entringer, Katia C. Gondim, José Roberto Meyer-Fernandes ¤ Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21541-590, Rio de Janeiro, RJ, Brazil Received 12 July 2005; received in revised form 8 September 2005; accepted 20 September 2005 Available online 9 November 2005
Abstract In this work we describe the ability of living Trypanosoma rangeli 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 (1.53 § 0.12 nmol Pi/h £ 107 cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg-dependent ecto-ATPase activity was 5.24 § 0.64 nmol Pi/h £ 107 cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory eVect on the ATP hydrolysis was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2, SrCl2, and ZnCl2. The apparent Km for Mg-ATP2- was 0.53 § 0.11 mM. The optimum pH for the T. rangeli Mg-dependent ectoATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, baWlomycin A1, ouabain, furosemide, vanadate, molybdate, sodium Xuoride, tartrate, and levamizole. To conWrm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4⬘-diisothiocyanostylbene 2⬘-2⬘-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph. 2005 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Trypanosoma rangeli; Ecto-ATPase; Adenosine; Hemolymph; Lipophorin
1. Introduction Trypanosomes are digenetic parasites that usually have insects as vectors and infect humans beings and other animals as hosts. Trypanosoma rangeli, a South American trypanosome, is a harmless parasite of humans and various wild and domestic animals (Ellis et al., 1980). T. rangeli after being ingested as trypomastigotes by its vector Rhodnius prolixus, multiplies as epimastigotes in the midgut and invade the haemocoel. The epimastigotes survive in the
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hemolymph or inside the hemocytes, migrate to and complete their development in the salivary glands (Takle, 1988). Surface membrane interactions between parasites and their host cells are of critical importance for the survival of the parasite, from both the immunological and physiological viewpoints (Vannier-Santos et al., 1995). In most protozoans, glycosylated molecules, lectins or lectin-like molecules mediate adhesion or invasion to or into the host cells (Basseri et al., 2002). Lectin receptors of trypanosomatids have been extensively studied (Jacobson and Doyle, 1996). Invasion into the insect vector salivary glands by some protozoans, such as T. rangeli, is a necessary step for transmission and is likely to be mediated by speciWc receptor-ligand interactions (Basseri et al., 2002).
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The 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 intact cells (Jesus et al., 2002; Meyer-Fernandes, 2002). Cell membrane ecto-ATPases are integral membrane glycoproteins that are millimolar divalent cation-dependent, low speciWcity enzymes that hydrolyze all nucleoside triphosphates (Meyer-Fernandes, 2002; Plesner, 1995). The identity and the function of ecto-ATPases have been reviewed and the nomenclature of “E-type ATPases” was proposed to describe these enzymes (Plesner, 1995). Their physiological role is still unknown. However, several hypotheses have been suggested, such as (i) protection from cytolytic eVects of extracellular ATP (Steinberg and Di Virgilio, 1991), (ii) regulation of ectokinase substrate concentration (Plesner, 1995), (iii) termination of purinergic signaling (Weisman et al., 1996; Westfall et al., 1997), (iv) involvement in signal transduction (Dubyak and El-Moatassim, 1993), and (v) involvement in cellular adhesion (Dzhandzhugazyan and Bock, 1993; Meyer-Fernandes, 2002). In this study, we show for the Wrst time the presence of a Mg2+-dependent ecto-ATP-diphosphohydrolase activity on the surface of living cells of the T. rangeli and characterize the properties of this enzyme. 2. Materials and methods 2.1. Microorganisms The Macias strain of T. rangeli (supplied by Dr. Wanderley De Souza, UFRJ, Brazil) was used throughout this study. The parasites were grown in liver infusion tryptose medium supplemented with 20% fetal calf serum at 28 °C. Five days after inoculation, long epimastigotes, predominantly (>98%) were collected by centrifugation, washed twice and kept in 50 mM Tris–HCl buVer, pH 7.2, 100 mM sucrose, and 20 mM KCl. Cellular viability was assessed, before and after incubations, by motility, and Trypan blue dye exclusion (De Jesus et al., 2002). The viability was not aVected under the conditions employed here. 2.2. Ecto-ATPase activity measurements Intact cells were incubated for 1 h at 30 °C in 0.5 ml of a mixture containing, unless otherwise speciWed, 50 mM Tris–HCl buVer, pH 7.2, 100 mM sucrose, 20 mM KCl, 5 mM ATP, and 3.0 £ 107 cells/ml, in the absence or in the presence of 5.0 mM MgCl2. The Mg2+-dependent ectoATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. The ATPase activity was determined by measuring the hydrolysis of [32P]ATP (104 Bq/nmol ATP) (Saad-Nehme et al., 2000). The experiments were started by the addition of living cells and terminated by the addition of 1.0 ml of a cold mixture containing 25% charcoal in 1.0 M HCl. The tubes were then centrifuged at 1500g for 10 min at 4 °C. Ali-
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quots (0.5 ml) of the supernatants containing the released Pi were transferred to scintillation vials containing 9.0 ml of scintillation Xuid. The ATPase activity was calculated by subtracting the nonspeciWc ATP hydrolysis measured in the absence of cells. The ATP hydrolysis was linear with time under the assay conditions used and was proportional to the cell number. In the experiments where other nucleotides were used, the hydrolytic activities measured under the same conditions described above were assayed spectrophotometrically by measuring the release of Pi from the nucleotides (Lowry and Lopes, 1946). The values obtained for ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments where high concentrations of Mn2+, Ca2+, Sr2+, and Zn2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed, in the reaction medium containing 50 mM Tris–HCl buVer, pH 7.2, 100 mM sucrose, 20 mM KCl, and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations. 32
2.3. Lipophorin puriWcation Lipophorin was puriWed from adult, mated females of R. prolixus, fed on rabbit blood at 3-week intervals, taken from a colony kept at 28 °C and 80–90% relative humidity. Four to Wve days after a blood meal, hemolymph was collected in the presence of phenylthiourea (3–13 mg/ml), 5 mM EDTA, and a mixture of protease inhibitors prepared in 0.15 M NaCl, with Wnal concentrations of 0.1 mg/ ml soybean trypsin inhibitor (SBTI), 100 M leupeptin, and 1 mM benzamidine. The collected hemolymph was centrifuged at room temperature for 5 min at 13,000g and lipophorin was puriWed from the supernatant by KBr gradient ultracentrifugation as described elsewhere (Golodne et al., 2001). PuriWed lipophorin was collected from the top of the KBr gradient, and the bottom of the gradient, containing all other hemolymphatic proteins, was also separated. Both fractions were extensively dialyzed against 10 mM Tris, 100 mM of 3-(N-morpholino) propanesulfonic acid (MOPS), 0.15 M NaCl, pH 6.5, and stored under liquid nitrogen until use. The degree of puriWcation was monitored by SDS–PAGE (Laemmli, 1970) and protein concentration was determined according to Lowry et al. (1951) using bovine serum albumin as standard. 2.4. Statistical analysis All experiments were performed in triplicates, with similar results obtained in at least three separate cell suspensions. Apparent Km and Vmax. values were calculated using a computerized nonlinear regression analysis of the data to the Michaelis–Menten equation (Barros et al., 2000). Statistical signiWcance was determined by Student’s t test. SigniWcance was considered as P < 0.05.
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2.5. Chemicals All reagents were purchased from E. Merck (D-6100 Darmstadt, Germany) or Sigma Chemical (St. Louis, MO). [32P]ATP was prepared as described by Glynn and Chappell (1946). Distilled water was deionized using a MilliQ system of resins (Millipore, Bedford, MA) and was used in the preparation of all solutions. Concentrations of free and complexed species (Mg2+, ATP4¡, Mg-ATP2¡, and CaATP2¡) at equilibrium were calculated by using an iterative program that was modiWed (Sorenson et al., 1986) from that described by Fabiato and Fabiato (1979). 3. Results In this paper, we report the presence of an ecto-ATPase activity on the external surface of T. rangeli. The addition of MgCl2 and MnCl2, but not CaCl2, SrCl2, ZnCl2, and EDTA (a metal chelator) stimulated the ATP hydrolysis
Fig. 1. InXuence of diVerent divalent cations on the ecto-ATPase activity of intact living T. rangeli parasites. Cells were incubated for 1 h at 30 °C, in a reaction medium containing 50 mM Tris–HCl buVer, pH 7.2, 100 mM sucrose, 20 mM KCl, 3.0 £ 107 cells/ml, and 5 mM ATP [32P]ATP (speciWc activity D 104 Bq/nmol ATP), with the addition of 5 mM of each divalent cation. Data are means § SE of three determinations with diVerent cell suspensions.
(Fig. 1). In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, ATP hydrolysis was low (1.53 § 0.12 nmol Pi/h £ 107 cells) in the absence of any divalent metal (1 mM EDTA). At pH 7.2, the addition of 5 mM MgCl2 stimulated the ATP hydrolysis and the Mg2+dependent ecto-ATPase activity diVerence between total (measured in the presence of 5 mM MgCl2) minus basal ecto-ATPase activity (measured in the presence of 1 mM EDTA) present in these parasites hydrolyzed ATP at 5.24 § 0.64 nmol Pi/h £ 107 cells. The time course of ATP hydrolysis by the T. rangeli Mg2+-dependent ecto-ATPase was linear for at least 60 min (Fig. 2, A). Similarly, in assays to determine the inXuence of cell density, the Mg2+-dependent activity measured over 60 min was linear over a nearly fourfold range of cell density (Fig. 2, B). To check the possibility that the observed ATP hydrolysis was the result of secreted soluble enzymes, as seen in other parasites (Smith et al., 1997), we prepared a reaction mixture with cells that were incubated in the absence of ATP. Subsequently, the suspension was centrifuged to remove cells and the supernatant was checked for ATPase activity. This supernatant failed to show ATP hydrolysis either in the absence or in the presence of MgCl2 (data not shown). These data also rules out the possibility that the ATPase activity here described could be from lysed T. rangeli cells. The optimum pH for the ecto-ATPase activity lies in the alkaline range. In the pH range from 6.4 to 8.4, in which the cells were alive throughout the time course of reaction, the activity increased with the pH, reaching a value 90% higher at pH 8.4 as compared to pH 6.4 (Fig. 3). Similar results were obtained for Leishmania amazonensis (Berredo-Pinho et al., 2001) and Entamoeba histolytica (Barros et al., 2000) ecto-ATPases. To discard the possibility that the ATP hydrolysis was due to phosphatases or other type of ATPases with internal ATP binding sites, diVerent inhibitors for those enzymes were tested. Table 1 shows that the ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase (Berredo-Pinho et al., 2001); baWlomycin A1, a V-ATPase inhibitor (Bowman et al.,
Fig. 2. Time course (A) and cell density dependence (B) of the Mg2+-dependent ecto-ATPase activity of intact cells of T. rangeli. Cells were incubated for diVerent periods of time (A) or for 1 h (B) at 30 °C, in a reaction medium containing 50 mM Tris–HCl buVer, pH 7.2, in the absence or in the presence of 5.0 mM MgCl2. The Mg2+-dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. Data are means § SE of three determinations with diVerent cell suspensions.
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Fig. 3. EVect of pH on the ecto-ATPase activity of intact cells of T. rangeli. Cells were incubated for 1 h at 30 °C in a reaction medium containing 100 mM sucrose, 20 mM KCl, 3.0 £ 107 cells/ml, 5 mM ATP [32P]ATP (speciWc activity D 104 Bq/nmol ATP), and 50 mM Tris–HCl buVer, adjusted to pH values between 6.8 and 8.4 with HCl and Tris, in the absence or in the presence of 5.0 mM MgCl2. The Mg2+-dependent ectoATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. In this pH range the cells were viable throughout the course of the experiments. Data are means § SE of three determinations with diVerent cell suspensions. Table 1 InXuence of various agents on the ecto-ATPase activity of T. rangeli Additions
Relative ecto-ATPase activity (%)
Control Levamizole (1 mM) Molybdate (1 mM) NaF (10 mM) Tartrate (10 mM) Vanadate (1 mM) Ouabain (1 mM) BaWlomycin (1 M) NaN3 (10 mM) Oligomycin (2 g/mL) Furosemide (1 mM) AMP (10 mM) Dipyridamole (10 M) Pi (10 mM) p-NPP (5 mM) -Naphtylphosphate (5 mM) -Glycerophosphate (5 mM)
100 § 8.7 98.8 § 10.8 103.6 § 12.7 107.8 § 18.3 117.0 § 8.4 91.5 § 5.4 111.3 § 16.3 112.0 § 13.2 87.5 § 8.2 104.0 § 3.3 89.5 § 3.5 96.1 § 3.8 99.4 § 10.9 116.0 § 8.7 95.0 § 16.2 113.3 § 4.7 120.1 § 13.8
Note. ATPase activity was measured at pH 7.2 in the standard assay described in Section 2. ATPase activity is expressed as percentages of that measured under control conditions, i.e., without other additions. The ATPase (5.2 § 0.6 nmol Pi/h £ 107 cells) activity was taken as 100%. The standard errors were calculated from the absolute activity values of three experiments with diVerent cell suspensions and converted to percentage of the control values. The unpaired t test showed, in all cases with respect to ATPase activity, that there were not statistical diVerences (P > 0.05) with respect to values found for each compound.
1988); ouabain, a Na++K+-ATPase inhibitor (CarusoNeves et al., 1998a); furosemide, a Na+-ATPase inhibitor (Caruso-Neves et al., 1998b); sodium Xuoride and ammonium molybdate, two potent inhibitors of acid phosphatase activity (Dutra et al., 2000) and sodium orthovanadate, a potent inhibitor of P-ATPases and acid phosphatases (Dutra et al., 2001; Meyer-Fernandes et al.,
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1999). Levamizole, an inhibitor of alkaline phosphatase (Van Belle, 1976), and dipyridamole, a nucleoside transporter antagonist (Lemmens et al., 1996) also failed to inhibit the ATPase activity (Table 1), as well as p-nitrophenylphosphate (p-NPP), -naphtylphosphate and glycerophosphate, substrates for phosphatase activities and 5⬘AMP, a substrate for 5⬘-nucleotidase. Since we used intact cells for measuring the enzyme activities in all the experiments done in this work, it is likely that the described Mg2+-dependent ATPase activity is an ectoenzyme. To conWrm this, we applied the criterion that an authentic ectoenzyme should be inhibited by an extracellular impermeant inhibitor (Berredo-Pinho et al., 2001) such as 4, 4⬘-diisothiocyanostylbene 2⬘-2⬘-disulfonic acid (DIDS) (Berredo-Pinho et al., 2001) and possibly by an ecto-ATPase inhibitor (Meyer-Fernandes et al., 2000), such as suramin, which is also an antagonist of P2-purinergic receptors (Hourani and Chown, 1989). As shown in Fig. 4, the Mg2+-dependent ecto-ATPase activity was inhibited both by DIDS and suramin in a dose-dependent manner. Under the conditions employed, in the reaction medium containing 50 mM Tris–HCl buVer, pH 7.2, 100 mM sucrose, 20 mM KCl, and 5 mM ATP, in the absence of any divalent cation, the concentration of ATP4¡ was 2.9 mM. In the presence of 5 mM CaCl2, the concentrations of ATP4¡, and Ca-ATP2¡ were 0.51 and 4.1 mM, respectively. The changes in ATP4¡ and Ca-ATP2¡ levels had no eVect on ATPase activity (Fig. 1). These data could be indicating that Mg-ATP2¡ is the substrate for this enzyme The dependence on Mg-ATP2¡ concentration showed a normal Michaelis–Menten kinetics for this ATPase activity and the values of Vmax and apparent Km for ATP were 5.17 § 0.38 nmol Pi/h £ 107 cells and 0.53 § 0.11 mM, respectively (Fig. 5), and free Mg2+ did not increase the ecto-ATPase activity (data not shown), as also observed in L. amazonensis (Berredo-Pinho et al., 2001) and Trypanosoma cruzi (Meyer-Fernandes et al., 2004). We analyzed the speciWcity of this ecto-ATPase activity for other nucleotides. Table 2 shows that ATP and ITP were the bests substrates for this enzyme. GTP, UTP, and CTP produced lower reaction rates (Table 2). This enzyme was also able to hydrolyze ADP (Table 2). These data could be indicating that it is an ectonucleoside triphosphate diphosphohydrolase, described for other cells (Barros et al., 2000; Jesus et al., 2002; Meyer-Fernandes et al., 2000; Wang and Guidotti, 1996). Another possible explanation for the ATP hydrolysis was that 5⬘-nucleotidase, another enzyme present on the external surface of T. rangeli (Fig. 6) could be responsible this hydrolysis. However, the lack of response to ammonium molybdate (Table 1), a potent inhibitor of 5⬘-nucleotidase (Gottlieb and Dwyer, 1983), and to AMP (Table 1), the substrate for this enzyme indicated that this enzyme did not contribute for the observed ATP hydrolysis. These data conWrm that the ATP hydrolysis stimulated by MgCl2 is catalyzed by an authentic Mg-dependent ectonucleoside triphosphate diphosphohydrolase.
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Fig. 4. EVect of increasing concentrations of DIDS and suramin on the Mg2+-dependent ecto-ATPase activity of intact cells of T. rangeli. Cells were incubated for 1 h at 30 °C in the same reaction medium (Wnal volume 0.5 ml) as that described in the legend of Fig. 1, in the absence or in the presence of 5.0 mM MgCl2 with increasing concentrations of DIDS (A) or suramin (B). The Mg2+-dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. Data are means § SE of three determinations with diVerent cell suspensions.
Fig. 5. Dependence on Mg-ATP2¡ concentrations on the ecto-ATPase activity of intact cells of T. rangeli. Cells were incubated for 1 h at 30 °C in the same reaction medium (Wnal volume 0.5 ml) as that described in the legend of Fig. 1, which corresponds to Mg-ATP2¡ concentrations varying as shown on the abscissa. Curve represents the Wt of experimental data by nonlinear regression using the Michaelis–Menten equation as described under Section 2. The total amounts of ATP and MgCl2 necessary to form the desired Mg-ATP2¡ concentrations were calculated as described under Section 2. Data are means § SE of three determinations with diVerent cell suspensions.
Table 2 Substrate speciWcity of Mg-dependent ecto-ATPase activity Substrates
Relative activity
ATP ADP ITP GTP UTP CTP
100 § 10.6 37.6 § 7.8 84.8 § 3.3 51.4 § 5.2 25.2 § 3.1 17.4 § 0.7
Note. The ecto-nucleotidase activity was measured at 30 °C in medium containing the nucleotides listed (5 mM), 50 mM Hepes, pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, and 3.0 £ 107 cells/ml in the absence or in the presence of 5.0 mM MgCl2. The Mg2+-dependent ATPase (4.8 § 0.5 nmol Pi/h £ 107 cells) activity (diVerence between total (measured in the presence of 5 mM MgCl2) minus basal ecto-ATPase activity (measured in the absence of MgCl2)) was taken as 100%. The standard errors were calculated from the absolute activity values of three experiments with diVerent cell suspensions and converted to percentage of the control value. In these experiments Pi release from all nucleotides, including ATP, was measured using a spectrophotometric assay as described in Section 2.
Fig. 6. Ecto-phosphohydrolase activities of intact cells of T. rangeli. Cells were incubated for 1 h at 30 °C in the same reaction medium (Wnal volume: 0.5 ml) as that described in the legend of Fig. 1, in the presence of 5 mM of either ATP, ADP, or 5⬘AMP. Bars: total activity measured in the presence of 5 mM MgCl2. In these experiments ATP hydrolysis were measured using the same spectrophotometric assay (described in Section 2) for Pi release as that used for the other nucleotides. Data are means § SE of three determinations with diVerent cell suspensions.
It is well known that Trypanosomatids of the genus Trypanosoma, are unable to synthesize purines de novo and thus are dependent on exogenous sources of these essential nutrients (De Koning et al., 2002). Extracellular ATP and its degradation products ADP, AMP, and adenosine are normal components of extracellular milieu. Extracellular nucleotides do not cross the cell membrane, but rather mediate their biological actions through speciWc receptors on the cell surface, where are locally metabolized by ectonucleotidases (Dombrowski et al., 1998; El-Moatassim et al., 1992). The three diVerent enzymatic activities (ectoATPase, ecto-ADPase, and ecto-5⬘-nucleotidase) present on the surface of T. rangeli (Fig. 6) might sequentially dephosphorylate ATP to adenosine: ATP ! ADP ! AMP ! adenosine, making adenosine available to T. rangeli from nucleotides which, because of their charge, are not permeable to the plasma membrane. The physiological role of the ecto-ATPases is still unknown, but a possible involvement in cell proliferation has been proposed (Lemmens et al., 1996). T. rangeli grown in a medium supplemented with 5 mM adenosine were compared to the
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T. rangeli grown in a control medium concerning the presence of the Mg-dependent ecto-ATPase activity. As shown in Fig. 7 parasites grown in control medium showed threefold more Mg-dependent ecto-ATPase activity than the parasites grown in a medium supplemented with 5 mM adenosine. Another possible physiological role proposed for the ecto-ATPases is its involvement in cellular adhesion (Dzhandzhugazyan and Bock, 1993; Meyer-Fernandes, 2002). Carbohydrates exposed on the surface of R. prolixus salivary glands (basal lamina, muscle, and cell layers of the glands) play an important role in the interaction of those cells with T. rangeli (Basseri et al., 2002). For these reasons, we examined the eVect of diVerent carbohydrates on the ecto-ATPase activity of this parasite (Fig. 8). The Mg-
81
dependent ecto-ATPase activity was stimulated by the carbohydrates tested (glucosamine > fructose > glucose > galactose > mannose) (Fig. 8). Moreover, lipophorin, a major hemolymphatic lipoprotein (Blacklock and Ryan, 1994), is known to be somehow involved with the immediate, innate antimicrobial defenses in insects. It was shown in the cockroach Periplaneta americana that lipophorin inhibits adhesion of hemocytes in vitro (Coodin and Caveney, 1992), and in the larvae of the moth Galleria mellonella it was indicated that this eVect was basically due to one of lipophorin apoproteins, apolipophorin I (Mandato et al., 1996). Although it is generally accepted that lipophorin takes part in insect defense, the mechanisms related with this process are not known. The possibility that this lipoprotein would interact with T. rangeli and aVect the ectoATPase activity was then tested. As can be observed in Fig. 9, the addition of R. prolixus hemolymph or puriWed lipophorin stimulated the Mg-dependent ecto-ATPase activity. However, the addition of lipophorin-deWcient hemolymph, that contained free apolipophorin III, an exchangeable, lipophorin apoprotein (Blacklock and Ryan, 1994), and also all other plasma proteins, had no eVect. 4. Discussion
2+
Fig. 7. InXuence of adenosine during growth of T. rangeli on the Mg dependent ecto-ATPase activity. Parasites were cultivated as described in Section 2 for 5 days, in the absence (blank bars), or in the presence of 5 mM adenosine (black bars). Cells were incubated for 1 h at 30 °C in the same reaction medium (Wnal volume 0.5 ml) as that described in the legend of Fig. 1, in the absence or in the presence of 5.0 mM MgCl2. The Mg2+dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. Data are means § SE of three determinations with diVerent cell suspensions.
Fig. 8. InXuence of carbohydrates on the Mg2+-dependent ecto-ATPase activity of intact living T. rangeli. Cells were incubated for 1 h at 30 °C in the same reaction medium (Wnal volume: 0.5 ml) as that described in the legend of Fig. 1, in the absence (control) or in the presence of 60 mM of the carbohydrates shown on the abscissa. The Mg2+-dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. Data are means § SE of three determinations with diVerent cell suspensions.
This paper reports the presence of Mg-dependent ectoATPase present on the external surface of T. rangeli. Cellular integrity and viability were assessed, before and after the reactions, by mobility and by Trypan blue dye exclusion (De Jesus et al., 2002). The integrity of the cells was not aVected by any of the conditions used in the assays. The external location of the ATP-hydrolyzing site is supported by its sensitivity to the impermeant inhibitor DIDS (Fig. 4, A) (Berredo-Pinho et al., 2001), and to suramin (Fig. 4, B), which is a noncompetitive inhibitor of ecto-ATPases and an antagonist of P2 purinoreceptors, which mediate the physiological functions of extracellular ATP (Hourani and Chown, 1989). Also, a battery of inhibitors for other ATPases that have intracellular ATP binding sites showed no eVect on the ecto-ATPase activity (Table 1). For these reasons we assign an ectolocalization of the Mg-dependent ATPase activity described here (Plesner, 1995; Meyer-Fernandes, 2002). This ATP hydrolysis cannot be due to a phosphatase activity because as shown in Table 1, the addition of potent inhibitors for phosphatase activities were not capable of modifying the Mg-dependent ecto-ATPase activity. Similar results were obtained for others Mg-dependent ecto-ATPases present in some members of the trypanosomatid family, which also exhibit higher activity in alkaline pH and do not respond to phosphatase inhibitors (Berredo-Pinho et al., 2001). The Mg-dependent ecto-ATPase activity described here also can not be attributed to a 5⬘-nucleotidase, since ammonium molybdate, a potent inhibitor of 5⬘-nucleotidase (Gottlieb and Dwyer, 1983), did not inhibit the Mg-dependent ecto-ATPase activity. Our data are consistent with observations showing that ecto-ATPases are present in other protozoa, including
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L. amazonensis (Berredo-Pinho et al., 2001), E. histolytica (Barros et al., 2000), T. cruzi (Meyer-Fernandes et al., 2004), as well as Trichomonas vaginalis (Jesus et al., 2002). Most of the ecto-ATPases are Mg2+ or Ca2+ stimulated (Barros et al., 2000; Plesner, 1995), however, this enzyme was not stimulated by CaCl2 (Fig. 1). These data suggest that Ca-ATP2¡ is no substrate for this enzyme, as it has also been shown for the ecto-ATPases present in L. amazonensis (Berredo-Pinho et al., 2001), and T. cruzi (MeyerFernandes et al., 2004). The substrate for the enzyme described here is the complex Mg-ATP2¡ (Km D 0.53 § 0.11 mM, Fig. 5). It has been shown that the Mg2+-independent and the Mg2+-dependent ecto-ATPase activities present in E. histolytica have high speciWcity for ATP, being much less active toward other nucleoside triphosphate substrate (Barros et al., 2000). The Mg-dependent ecto-ATPase of T. rangeli characterized here also demonstrated high speciWcity for ATP, although it was able to hydrolyze ITP with high reaction rate (Table 2). The Mg2+dependent ecto-ATPase [diVerence between total (measured in the presence of 5 mM MgCl2)] minus basal ectoATPase activity (measured in the absence of MgCl2)) present in T. rangeli was also able to recognize ADP as substrate (Table 2). T. rangeli as well as L. amazonensis are pathogens which cannot synthesize purines de novo (Berredo-Pinho et al., 2001; De Koning et al., 2002). It has been postulated that these ecto-ATPases in protozoa parasites could play a role in the salvage of purines from the host cells (Meyer-Fernandes, 2002). The ability of T. rangeli to hydrolyze ATP, ADP, and AMP (Fig. 6), and the decrease of Mg-dependent ecto-ATPase activity from parasites grown in the presence of adenosine (Fig. 7), leave us also to speculate that this enzyme in T. rangeli could play a role in the salvage of purines from extracellular medium. The physiological functions of the ecto-ATPases are not known, however, many functions have been hypothesized, including roles in termination of purinergic signaling, purine recycling, and cellular adhesion (Dzhandzhugazyan and Bock, 1993; Meyer-Fernandes, 2002; Plesner, 1995). Considering the fact that in insects T. rangeli is responsible for complicated infections and that invasive form of E. histolytica presents much higher Mg-dependent ecto-ATPase activity than noninvasive form (Barros et al., 2000), one could also speculate that the presence of a Mg-dependent ecto-ATPase activity in T. rangeli might also reXect some form of evasion of the parasite from host defense mechanisms in the circulation of the insect vectors. It has been shown that lectin-like molecules on the surface of T. rangeli are involved in the attachment of the parasite to the salivary glands of R. prolixus (Basseri et al., 2002). When we investigated the possible eVect of diVerent carbohydrates on the ecto-ATPase activity present in T. rangeli, monosaccharides stimulated this enzyme more than twofold (Fig. 8). Recently, it was shown that a 46-kDa lectin isolated from root extracts of the legume Dolichos biXorus is a Nod factor binding protein as well as a nucleoside di-
and triphosphate hydrolase stimulated by carbohydrate ligands (Etzler et al., 1999). Little is known about the extracellular signaling molecules responsible for the activation of immune cell receptors in insects (Imler and HoVman, 2000; Zakarian et al., 2002), but there are indications that lipophorin may be involved. This lipophorin was shown to inhibit adhesion of hemocytes in vitro (Coodin and Caveney, 1992; Mandato et al., 1996) and, when bacteria was injected in larval G. mellonella, the circulating form of lipophorin (high density lipophorin, HDLp) was converted to a lower density form (DettloV et al., 2001). When low density lipophorin (LDLp) puriWed from the moth Manduca sexta was incubated with hemocytes from G. mellonella, it was speciWcally taken up by the cells, and this result indicated the presence of lipophorin speciWc binding sites on hemocyte surface (DettloV et al., 2001). Recently, it was demonstrated that T. rangeli takes up lipophorin from the medium, and this process also seems to be mediated by speciWc receptors in the parasite membrane (Folly et al., 2003). Herein, it was shown a stimulatory eVect of lipophorin from R. prolixus on the Mg-dependent ecto-ATPase activity (Fig. 9). It is possible that, when lipophorin was incubated in the presence of the parasites, its interaction with the putative receptor at cell surface, and/or its subsequent uptake, initiated some signal that resulted in the enhancement of the Mgdependent ecto-ATPase activity. As this enzymatic activity may be important for parasite association with insect tissues, a mechanism that signals the presence of lipophorin may be fundamental, as trypanosomatids incorporate lipoproteins from the medium (Coppens et al., 1995; Soares and de Souza, 1991). Alternatively, a direct action of the lipoprotein on the ecto-ATPase can not be discarded, and further investigations would be necessary in order to observe if Mg-dependent ecto-ATPase is an adhesion
Fig. 9. InXuence of hemolymph, lipophorin, and other hemolymphatic proteins on the Mg2+-dependent ecto-ATPase activity of intact living T. rangeli. Cells were incubated for 1 h at 30 °C in the same reaction medium (Wnal volume: 0.5 ml) as that described in the legend of Fig. 1, in the absence (control; C) or in the presence of the following additions (1 mg protein/ml): hemolymph (H), lipophorin (L) or lipophorin-depleted hemolymph (LdH) (gradient bottom). The Mg2+-dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2, minus the basal activity, measured in the absence of MgCl2. Data are means § SE of three determinations with diVerent cell suspensions.
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molecule in T. rangeli, which could be considered as a pathogenesis marker for this parasite. ApoLp-III is a soluble lipophorin apoprotein that associates with lipophorin particle under certain physiological conditions (Blacklock and Ryan, 1994), and has been implicated with various aspects of immune response (Wiesner et al., 1997; Zakarian et al., 2002). In spite of that, it appears not to be related with the enzymatic activity determined here, as the lipophorin-deWcient hemolymph, that contained ApoLp-III and all other circulating proteins, had no eVect on it. The recently identiWed family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that diVer in their substrate speciWcities and cellular locations (Zimmermann, 1999). It has been demonstrated that mammalian membrane associated ectoATPDase was homologous to human CD39, a lymphoid cell activation antigen (Wang and Guidotti, 1996).Wang and Guidotti discovered that CD39 has sequence homology with a potato apyrase and that CD39 has apyrase activity (Wang and Guidotti, 1996). This work led to the identiWcation of a family of ecto-ATPases that are related in sequence but vary in their membrane topology and tissue distribution (Goding, 2000; Plesner, 1995; Wang and Guidotti, 1996). Further characterization of cloned members of proteins related to CD39, allowed the suggestion of a unifying nomenclature. All members of the CD39-ATPdiphosphohydrolase family belong to the E-NTPDase family (Goding, 2000; Zimmermann et al., 2000). Elucidation of the primary sequence of T. rangeli ecto-ATPase will be required to positively identify this enzyme as a member of this family. Acknowledgments We acknowledge Fabiano Ferreira Esteves, Heloisa S.L. Coelho, and Lilian S. da C. Gomes for excellent technical assistance; José de S. Lima Junior and Litiane M. Rodrigues for insect care. This work was partially supported by grants from the Brazilian agencies Conselho Nacional de Desenvolvimento CientíWco e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (FINEP) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). References Barros, F.S., De Menezes, L.F., Pinheiro, A.A., Silva, E.F., Lopes, A.H., De Souza, W., Meyer-Fernandes, J.R., 2000. Ectonucleotide diphosphohydrolase activities in Entamoeba histolytica. Archives of Biochemistry and Biophysics 375, 304–314. Basseri, H.R., Tew, I.F., RatcliVe, N.A., 2002. IdentiWcation and distribution of carbohydrate moieties on the salivary glands of Rhodnius prolixus and their possible involvement in attachment/ invasion by Trypanosoma rangeli. Experimental Parasitology 100, 226–234. Berredo-Pinho, M., Peres-Sampaio, C.E., Chrispim, P.P., Belmont-Firpo, R., Dos Passos Lemos, A., Martiny, A., Vannier-Santos, M.A., Meyer-
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