Biochimica et Biophysica Acta 1721 (2005) 9 – 15 http://www.elsevier.com/locate/bba
Characterization of NTPDase (NTPDase1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5) activity in human lymphocytes Daniela B.R. Leala, Cristiane A. Streherb, Tiago N. Neub, Fa´bio P. Bittencourtc, Cla´udio A.M. Lealc, Jose´ E.P. da Silvac, Vera M. Morschb, Maria R.C. Schetingerb,* a
Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcellos, 2600-Anexo, 90035-003. Porto Alegre, RS, Brazil b Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, Av. Roraima, 97105-900. Santa Maria, RS, Brazil c Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Centro de Cieˆncias da Sau´de, Universidade Federal de Santa Maria, Av. Roraima, 97105-900. Santa Maria, RS, Brazil Received 10 March 2004; received in revised form 15 September 2004; accepted 28 September 2004 Available online 6 October 2004
Abstract Human lymphocytes contain NTPDase (NTPDase-1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5), a cation-dependent enzyme that hydrolyzes ATP and ADP and also other di- and triphosphate nucleosides, acting at an optimum pH of 8.0. A significant inhibition of ATP and ADP hydrolysis ( Pb0.05) was observed in the presence of 20 mM sodium azide. NTPDase inhibitors, 20 mM sodium fluoride, 0.2 mM trifluoperazine and 0.3 mM suramin, significantly decreased ATP and ADP hydrolysis ( Pb0.05) and ADP hydrolysis was only inhibited by 0.5 mM orthovanadate ( Pb0.05). ATP and ADP hydrolysis was not inhibited in the presence of 0.01 mM Ap5A (P1,P5di(adenosine-5V)pentaphosphate), 0.1 mM ouabain, 1 mM levamisole, 2 Ag/mL oligomycin, 0.1 mM N-ethylmaleimide (NEM), or 5 mM sodium azide. With respect to kinetic behavior, apparent K m values of 77.6F10.2 and 106.8F21.0 AM, and V max values of 68.9F8.1 and 99.4F8.5 (meanFS.E., n=3) nmol Pi/min/mg protein were obtained for ATP and ADP, respectively. A Chevilard plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. The presence of CD39 was determined by flow cytometry, showing a low density of 2.72F0.24% (meanFS.E.; n=30) in human peripheral lymphocytes. The study of NTPDase activity in human lymphocytes may be important to determine the immune response status against infectious agents related to ATP and ADP hydrolysis. D 2004 Elsevier B.V. All rights reserved. Keywords: NTPDase; CD39; Human lymphocyte
1. Introduction NTPDase1 (EC 3.6.1.5) is an enzyme that hydrolyzes extracellular nucleoside tri- and/or diphosphates [1]. The enzyme shows properties such as Ca2+ or Mg2+ dependence, insensitivity to specific inhibitors of P-type, F-type and Vtype ATPases, and also to alkaline phosphatase, yet the activity presents an inhibition by azide [2,3]. The * Corresponding author. Fax: +55 5522 08031. E-mail address:
[email protected] (M.R.C. Schetinger). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.09.006
NTPDase1 is a member of the ecto-nucleoside triphosphodiphosphohydrolase (E-NTPDase) family [4]. It was identified as a lymphoid cell antigen CD39 protein [5] and its increased expression leads to an enhancement of ATPase and ADPase activities in these cells [6]. CD39 had been previously shown to be a B lymphocyte activation marker and to be expressed on the surface of NK cells, cytotoxic T lymphocytes [7]. Extracellular nucleotide degradation was described 15 years ago in intact human B and T lymphocyte subpopulations and lymphoblastoid cell lines, showing evidences that there were ATPase, ADPase and AMPase
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activities in these cells [8]. In addition, the expression of ENTPDase was described in leukocytes, platelets and endothelial cells [9,10]. NTPDase plays an important role in lymphocytes as an activation marker essential for certain effector functions, since extracellular nucleotides are mediators of immune and non-immune cell function [11]. Filippini et al. [12] reported that cytolytic T-lymphocyte (CTL) ecto-ATPase activity has a protective effect against cell lysis caused by extracellular ATP that may act as a signaling compound in cytolytic mechanisms. Dombrowski et al. [13] reported that the ectoATPase activity not only serves as a protective mechanism for these lymphoid cells, but this activity is required for some lymphocyte functions. Activated lymphocytes had ecto-ATPase activity, but non-activated cells did not, and the decrease in ecto-ATPase activity inhibited the effector functions of T, B and NK cells [13]. After binding to purinergic receptors in lymphocytes, extracellular nucleotides must be hydrolyzed by ectonucleotidases such as NTPDase. In view of the properties listed above, it is important to characterize the ecto-nucleotidase activity in these cells, since lymphocytes could be related to the status of the immune response and its disorders. The objective of the present investigation is to characterize the human NTPDase-1 activity in lymphocytes and to standardize a simple and non-radioactive method for the determination the NTPDase activity.
2. Materials and methods 2.1. Chemicals Nucleotides, sodium azide, oligomycin, ouabain, orthovanadate, N-ethylmaleimide (NEM), adenylyl (3V,5V)-adenosine pentaphosphate (Ap5A), levamisole and Trizma base were purchased from Sigma (St. Louis, MO, USA). FicollHypaque (Lymphoprepk) was purchased from Nycomed Pharma (Oslo, Norway). Antibodies for flow cytometry analysis (R-Phycoerythrin (R-PE)-conjugated mouse antihuman monoclonal antibody against CD39 and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human monoclonal antibody against CD45 were purchased from BD PharMingen Technical Data Sheet (San Jose, CA, USA). All other reagents used in the experiments were of analytical grade and of the highest purity.
clinical evaluation and consisted of 60 individuals aged 35.1F5.9 years, 30 males and 30 females who had not been submitted to any pharmacological treatment during the last 30 days. The volunteers were selected randomly for the different experiments, normally three to five subjects per experiment. 2.3. Isolation of mononuclear cells from human blood Mononuclear leukocytes were isolated from human blood collected with EDTA and separated on FicollHypaque density gradients as described by Bfyum [14]. 2.4. Enzyme assays After the isolation of mononuclear cells, NTPDase activity was determined by measuring the amount of liberated inorganic phosphate using a colorimetric assay. This determination was chosen because it has been easily employed in our laboratory for various types of samples and does not require radioactive substances. Other authors have used the same colorimetric assay before, without evidencing a significant difference when compared with radioactive assays [10,15,16]. The reaction medium contained 0.5 mM CaCl2, 120 mM NaCl, 5 mM KCl, 60 mM glucose, and 50 mM Tris–HCl buffer, pH 8.0 in a final volume of 200 AL. Twenty microliters of intact mononuclear cells suspended in saline solution was added to the reaction medium (2–4 Ag protein) and preincubated for 10 min at 37 8C. The reaction was started by adding the substrate (ATP or ADP or another as indicated) at a final concentration of 2 mM and stopped with 200-AL 10% trichloracetic acid (TCA) to provide a final concentration of 5%. The enzyme incubation times and protein concentrations were chosen in order to ensure the linearity of the reactions. The samples were chilled on ice for 10 min before assaying for the release of inorganic phosphate (Pi) as described by Chan et al. [17], using malachite green as colorimetric reagent and KH2PO4 as standard. Controls with the addition of the enzyme preparation after the addition of TCA were used to correct for non-enzymatic hydrolysis of the substrate. All samples were run in duplicate or triplicate and specific activity is reported as nmol Pi released/min/mg protein in accordance with the standardization for platelets employed in our laboratory [16]. 2.5. Protein determination
2.2. Patients The sample consisted of healthy volunteers from the Hospital of the Federal University of Santa Maria (Santa Maria, RS, Brazil). All subjects gave written informed consent to participate in the study. The Human Ethics Committee of the Health Science Center, from the Federal University of Santa Maria, approved the protocol under number 13690. The group was carefully selected by
Protein was measured by the Coomassie blue method using bovine serum albumin as standard as described by Bradford [18]. 2.6. LDH measurement LDH was measured by the kinetic mode [19] using a Cobas Mira Plus apparatus (Roche DiagnosticsR).
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2.7. Flow cytometry analysis Peripheral blood cells were incubated with anti-CD39 and anti-CD45 (20 AL/106 cells) for 25 min, lysed with reagent FACS (Fluorescence Activated Cell Sorter) lysis and incubated again for 15 min in the dark. Cells were washed twice in PBS buffer pH 7.4 containing 0,02% (w/v) sodium azide and 0,2% (w/v) BSA. The cells were then resuspended in PBS buffer (pH 7,4) and immediately analyzed by FACScalibur flow cytometer using Cellquest software (Becton Dickinson, San Jose, CA, USA), without fixation. 2.8. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey–Kramer test, considering a level of significance of 5%.
Fig. 2. Effect of pH on NTPDase activity from human lymphocytes with ATP (o) or ADP (.) as substrate. The pH values tested were 6.0, 7.0, 7.5, 8.0 and 9.0. Conditions are described in Section 2. Data represent the mean of four different experiments. The activity was expressed as nmol Pi/min/ mg protein. Points represent the meanFS.E. for n=4 in triplicate.
3. Results 3.2. Conditions for incubation 3.1. Cellular integrity Cytometry analysis indicated 5% of contamination with platelets in the preparation (data not shown). The activity of lactate dehydrogenase (LDH) was used as a marker of cell integrity. The measurement of LDH activity showed that most cells (approximately 85%) were intact after the isolation procedure (data not shown). The integrity of the cells after incubation was confirmed by microscopic observations (results not shown).
Enzymatic activity was tested at temperatures of 15, 25, 37 and 45 8C, demonstrating a parallel profile for both substrates and a maximal activity at 37 8C (Fig. 1). The range of pH assayed was between 6.0 and 9.0, using the buffers MES (pH 6 and 7) and TRIS (pH 7.5, 8 and 9), and the optimum pH was found to be 8.0, which was used for the subsequent experiments (Fig. 2). The protein concentration in the incubation medium with a linear pattern of activity was in the range of 2–4 Ag protein per tube. The time course for ATP and ADP hydrolysis in human lymphocytes was linear up to 70 min in the presence of Ca2+ (0.5 mM) and 2 mM of the nucleotide (results not shown). 3.3. Cation dependence Calcium and magnesium concentrations were tested in the range of 0.5–5 mM and maximal activity was observed at the concentration of 0.5 mM for calcium and 1.0 mM for magnesium confirming the cation dependence of enzyme activity. This calcium concentration was used for the subsequent assays. Furthermore, a significant decrease ( Pb0.01) in ATP and ADP hydrolysis was observed in the presence of 5 mM EDTA, which inhibited about 73% of ATP hydrolysis and 92% of ADP hydrolysis (data not shown), confirming cation dependence. 3.4. Kinetic parameters
Fig. 1. Effect of temperature on NTPDase activity from human lymphocytes with ATP (o) or ADP (.) as substrate. Temperatures tested were 15, 25, 37 and 45 8C. Conditions are described in Section 2. Data represent the mean of four different experiments. The activity was expressed as nmol Pi/min/mg protein. Points represent the meanFS.E. for n=4 in triplicate.
ATP and ADP hydrolysis were determined using substrate in the range 50–3000 AM. The activity increased with increasing nucleotide concentrations up to 2 mM, which was sufficient to saturate the enzyme (Ca2+ fixed at
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D.B.R. Leal et al. / Biochimica et Biophysica Acta 1721 (2005) 9–15 Table 2 Substrate specificity of NTPDase from human lymphocytesa Substrate
Relative activity
ATP GTP UTP ADP GDP UDP
1.00 0.37F0.09 0.40F0.14 0.97F0.17 0.35F0.04 0.18F0.02
a Results are expressed as the meanFS.E. for at least three experiments. All substrates were used at 1.0 mM (0.5 mM Ca2+). Control apyrase activity was 58.06F13.42 nmol Pi min 1 mg of protein 1 for the ATP hydrolysis, indicating relative activity 1.00. The results were expressed in relative activity of control.
3.5. Exclusion of enzymatic associations
Fig. 3. Lineweaver–Burk plot for ATP (o) and ADP (.) hydrolysis and the inset with V versus S. ATP and ADP hydrolysis was determined using substrate in the range 50–3000 AM (n=4 in triplicate).
0.5 mM). The Lineweaver–Burk plot was used to determine the K m (Michaelis constant) and V max (maximum velocity) values. The apparent K m for ATP and ADP hydrolysis was 77.6F10.2 and 106.8F21.0 AM (meanFS.E., n=3), respectively. The V max values were 68.9F8.1 and 99.4F8.5 (meanFS.E., n=3) nmol/min/mg protein for ATP and ADP, respectively (Fig. 3). Table 1 Effects of inhibitors on ATP and ADP hydrolysis by human lymphocytesa Inhibitor
Concentration (mM)
%Control enzyme activity ATPase
ADPase
Ap5A Ouabain Azide
0.01 0.1 1 5 10 20 1 0.5 0.2 2 Ag/mL 20 0.1 0.3
96F4 92F6 96F11 96F15 81F20 52F1* 81F17 99F2 31F11** 88F1 50F7* 98F10 44F21**
100F6 88F12 99F3 88F4 82F5 55F12* 92F8 69F5* 61F11* 94F7 56F14* 91F13 63F10*
Levamisole Orthovanadate Trifluoperazine Oligomycin Fluoride NEM Suramin
The concentration used was 2 Ag/mL. a Results are expressed as meanFS.E. (n=3). Data were analyzed statistically by one-way analysis of variance (ANOVA) or Tukey–Kramer test. * Significant difference from control activity (100%) by one-way ANOVA ( Pb0.05). ** Significant difference from control activity (100%) by one-way ANOVA ( Pb0.01). Control apyrase activity was 43.5F7.56 and 39.3F7.80 nmol Pi min 1 mg protein 1 for ATP and ADP hydrolysis, respectively, indicating a control activity of 100% and the results were expressed as percentages of control activity.
Enzymatic associations were eliminated by using selective ATPase inhibitors (Table 1). Sodium azide and oligomycin (inhibitors of mitochondrial ATPase) were tested for ATP and ADP hydrolysis. Sodium azide inhibited ATP and ADP hydrolysis, in a parallel manner at a concentration of 20 mM ( Pb0.05), whereas oligomycin (2 Ag/mL) had no effect on ATP or ADP hydrolysis. The Na+/ K+-ATPase inhibitor ouabain had no effect on the enzymatic activity when tested at 0.1 mM. Orthovanadate (0.5 mM), an inhibitor of transport ATPases, acid phosphatases and phosphotyrosine phosphatases, was ineffective in inhibiting ATP hydrolysis, but significantly inhibited ( Pb0.05) ADP hydrolysis as previously reported [2]. Ap5A (0.01 mM), a specific inhibitor of adenylate kinase, did not affect ATP or ADP hydrolysis. Suramin, an inhibitor of P2 receptors that can also act as an inhibitor of NTPDase, was used at a concentration of 0.3 mM and significantly reduced ( Pb0.05) the enzymatic activity with ATP and ADP as substrate. Trifluoperazine (0.2 mM), a calcium calmodulindependent inhibitor of calcium membrane Ca2+-ATPase, significantly inhibited ATP and ADP hydrolysis ( Pb0.01 and Pb0.05, respectively). Levamisole, a specific alkaline
Fig. 4. NTPDase Chevillard plot. The concentration at which the velocities were the same for ATP and ADP was chosen for the Chevillard plot. The assay conditions are described in Section 2. The incubation time was 100 min; substrate A (ATP) at P=0 was 2 mM; substrate B (ADP) at P=2 was 2 mM. Data represent the meanFS.E. for at least three experiments.
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phosphatase inhibitor, and NEM, a compound that modifies sulfhydryl groups, had no effects on enzymatic activity. Furthermore, sodium fluoride, a phosphatase inhibitor, significantly decreased ATP and ADP hydrolysis ( Pb0.05 and Pb0.01. respectively). 3.6. Substrate specificity The hydrolysis of various di- and triphosphate nucleosides is characteristic of NTPDases [20–23]. The nucleotides tested were hydrolyzed at a significant rate by human lymphocytes (Table 2). The NTPDase of human lymphocytes showed preference for ATP and ADP as substrates (relative activity of 0.97F0.17 for ADP considering a value of 1.00 for ATP). The relative activity for GTP, GDP and UTP hydrolysis was less than half of the ATP rate (0.37F0.09, 0.35F0.04 and 0.40F0.14, respectively) and UDP was poorly hydrolyzed (0.18F0.02). To investigate if ATP and ADP hydrolysis occurs due to only one active site able to hydrolyze both substrates or by independent catalytic sites, the competition plot described by Chevillard et al. [24] was performed (Fig. 4). To assay the different combination of substrates in the competition plot, we chose a concentration at which the rate of hydrolysis was the same when both ATP and ADP were used as substrates. Then a sequence of mixtures with different concentrations of ATP (0 to 2 mM) and ADP (0 to 2 mM) on P=0 and P=2, respectively, was prepared. The pattern of the curve obtained in the competition plot shows a horizontal straight-line profile meaning that both hydrolysis
Fig. 5. Histogram of two-color analysis of CD39 on lymphocytes illustrating a normal subject. The histogram results of flow cytometry, using R-Phycoerythrin (R-PE)-conjugated mouse anti-human monoclonal antibody against CD39 and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human monoclonal antibody against CD45. The analysis shows a low expression of CD39 on human lymphocytes (upper right hand corner of histogram).
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activities (ATP and ADP) occur at same active site on a single enzyme. 3.7. Cytometric analysis of the CD39 expression The presence of CD39 was confirmed on the gate of lymphocytes obtained from the human sample whole blood. The fluorescence detected indicated a low density, around 2.72F0.24% (mediumFS.E.; n=30). Fig. 5 illustrates the cytometry of one normal subject.
4. Discussion NTPDase-1 (EC 3.6.1.5), an ectonucleotidase, has been described in various tissues such as the heart, placenta, lung, liver, skeletal muscle, thymus, kidney, pancreas, testis, ovary, prostate, colon, and brain [2,25–27]. It is important to note that human lymphocytes present ATPase activity that is required for some lymphocyte functions and activation [7,11,28]. This study intends to standardize an easy nonradioactive method that determines NTPDase-1 activity in lymphocyte membranes. The quantification of NTPDase-1 activity in lymphocytes could help in the understanding of its immune function. The enzyme studied here, obtained from human lymphocytes, is cation-dependent. Ca2+ is the best activator for this NTPDase from human lymphocytes, and cation dependence was confirmed by the decrease in enzyme activity in the presence of EDTA. Inhibition by EDTA was also observed in previous studies [29]. The temperature of 37 8C was the best for the enzymatic assay (near to human body temperature), corroborating with results previously obtained from synaptosomes from the cerebral cortex of adult rats and in human platelets [15,26,30]. The pH curve showed a maximal enzymatic activity at alkaline pH, suggesting the presence of a NTPDase. NTPDase activity was higher at pH 8.0 than at physiological pH, but this result is in accordance with previous reports about NTPDases from other sources such as pig pancreas, synaptosomes from cerebral cortex, liver and kidney membrane-enriched fractions [27,30,31]. The exclusion of enzymatic associations was performed using ATPase inhibitors such as Ap5A, NEM, oligomycin, ouabain and levomisole, which did not affect ATP or ADP hydrolysis. Orthovanadate inhibited enzymatic activity in upper ADP hydrolysis only, as reported previously by other authors [15]. Sodium azide inhibited enzymatic activity at higher concentrations, but under this condition it appears in the literature as an inhibitor for other NTPDases [20,21,26,30,32,33]. Inhibition of ATP and ADP hydrolysis by trifluoperazine and suramin was also observed in synaptosomes and platelets [16,26]. The inhibition caused by fluoride could be explained by its reaction with cations like Ca2+, decreasing NTPDase activity. The lymphoid cells possess high ectonucleotide
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kinase activities [34]. The possibility that the nucleotides could be preferentially and rapidly transphosphorylated via ecto NDP kinase reaction was discarded with the use of Ap5A, as it did not affect ATP and ADP hydrolysis. Moreover, the adenylate kinase activity requires Mg2+ in reaction medium [22], which was not used in our experiments. Parallel profiles were observed for ATP and ADP hydrolysis under several conditions, such as cation and pH dependence and sensitivity to inhibitors, suggesting the presence of a single enzyme. The constant velocity demonstrated by Chevillard plot suggests that ATP and ADP are hydrolyzed at only one active site. This protocol was used to demonstrate the presence of a NTPDase enzyme in Sertoli cells [29] and in human platelets [15]. The enzyme described here is consistent with an NTPDase 1 (ecto-apyrase; ecto-diphosphohydrolase; CD39), because it hydrolyzes ATP and ADP almost equally well (ratio 1:1), presents an optimum pH of 8.0, activation by Ca2+ and Mg2+, and low susceptibility to ATPase inhibitors as described in the literature [1]. NTPDase is an enzyme that has an important role in cell adhesion and in controlling lymphocyte functions including antigen recognition and/or the activation of effectors’ activities of cytotoxic T cells [13]. Besides, it plays a major role in the regulation of blood flow and thrombogenesis [16,35–38]. ATP and its hydrolysis products (ADP and adenosine) at low concentrations, in the micromolar range, strongly influence vascular tone, cardiac function, platelet aggregation, and the function of lymphocytes and granulocytes [2]. It was reported that an increased expression of ecto-5Vnucleotidase and ecto-ATPase in human cytomegalovirusinfected endothelial cells may serve as a novel viral evasion strategy, affecting the regulation of coagulation, inflammation and the integrity of the endothelial layer [39]. Furthermore, it was suggested that ecto-ATPase activity is necessary to protect CD8 lymphocytes from the potential lytic effect of extracellular ATP [12]. Human lymphocyte NTPDase offers a potential therapeutic target because its catalytic site faces the extracellular space, which could be used for treatment of autoimmune diseases and transplant rejection [11]. Besides, it was reported that the administration of interleucine-2 increases CD39 expression on lymphocytes, demonstrating that this is one marker of cell activation that could be related to chronic disease [40]. Our aim is to investigate, with subsequent studies, the involvement of NTPDase-1 activity and its expression in immunossupression situations. Salazar-Gonzalez et al. [41] showed a reduced ecto-5V-nucleotidase activity on cytotoxic lymphocytes in the Acquired Immune Deficiency Syndrome. Other authors also described reduced activity of this enzyme on peripheral lymphocytes from patients with Xlinked infantile agammaglobulinemia and common variable hypogammaglobulinemia [42,43].
Thus, the identification of NTPDase-1 activity in human lymphocytes may help to understand the mechanisms involved in the immune response in physiological conditions and perhaps in the future to modulate it in pathological conditions. Besides, the density of CD39 could be used as an activation marker, with possible prognostic interest in various diseases. It would be particularly interesting to evaluate NTPDase-1 in immunosupression situations.
Acknowledgements The authors wish to thank to Dr. Joa˜o Jose´ F. Sarkis for the critical reading of the manuscript and to the Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio Grande do Sul (FAPERGS) for financial support.
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