Parasitology International 62 (2013) 44–52

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An antigenic domain within a catalytically active Leishmania infantum nucleosidetriphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies

Ana Carolina Ribeiro Gomes Maia a, Gabriane Nascimento Porcino a, Michelle de Lima Detoni a,Nayara Braga Emídio a, Danielle Gomes Marconato a, Priscila Faria-Pinto a, Melissa Regina Fessel a,Alexandre Barbosa Reis b, Luiz Juliano c, Maria Aparecida Juliano c,Marcos José Marques d, Eveline Gomes Vasconcelos a,⁎a Departamento de Bioquímica, Laboratório de Estrutura e Função de Proteínas, Pós-Graduação em Imunologia e DIP/Genética e Biotecnologia, Instituto de Ciências Biológicas,Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazilb Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas & Departamento de Análises Clínicas, Escola de Farmácia, Universidade Federal de Ouro Preto,Ouro Preto, MG, Brazilc Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazild Departamento de Ciências Biológicas, Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Alfenas, MG, Brazil

⁎ Corresponding author at: Departamento de Bioquímde Juiz de Fora, Rua José Lourenço Kelmer s/n, Campus U36036‐900, Juiz de Fora, MG, Brazil. Tel.: +55 32 2102

E-mail address: eveline.vasconcelos@ufjf.edu.br (E.G

1383-5769/$ – see front matter © 2012 Elsevier Irelandhttp://dx.doi.org/10.1016/j.parint.2012.09.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 May 2012Received in revised form 6 August 2012Accepted 11 September 2012Available online 18 September 2012

Keywords:Leishmania (L.) chagasiATP diphosphohydrolaseNTPasePotato apyraseConserved domainVisceral leishmaniasis

We identified a shared B domainwithin nucleoside triphosphate diphosphohydrolases (NTPDases) of plants andparasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detectedin Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenizedpromastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, fol-lowing SDS-PAGE and silver staining was identified as a single polypeptide of 50 kDa. By Western blots, it wasrecognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptidederived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the LeishmaniaNTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantumNTPDase 1 and, also, its conserved B domain (r83–122). By immunoprecipitation assays andWestern blots, im-mune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, theimmune sera SB (44%) and SC or SD (87–99%) inhibited its activity, suggesting a direct effect on the B domain.By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respec-tively, confirming the B domain antigenicity.

© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The members of the nucleoside triphosphate diphosphohydrolase(NTPDase; EC 3.6.1.5) family (GDA1-CD39 superfamily) shareapyrase-conserved regions (ACR1 to ACR5) involved in the catalyticcycle, and include soluble or membrane-associated isoforms in thesame organism. These ubiquitous proteins, appointed as possible tar-gets for the treatment of several diseases, have the ability to hydro-lyze the nucleosides di- and triphosphate to the correspondingnucleoside monophosphate upon bivalent metal ion activation andmodulate nucleotide rate in several physiological and pathologicalconditions [1–6].

By in silico analysis, an additional conserved domain of 40 aminoacids, named B domain, which did not include the ACRs, was identified

ica, ICB, Universidade Federalniversitário, Bairro São Pedro,3217; fax: +55 32 2102 3216.. Vasconcelos).

Ltd. All rights reserved.

within NTPDase isoforms of plants and pathogenic organisms of distinctphylogenetic lineages, which seems to be conserved during host andparasite co-evolution [7,8]. By reactivity with potato apyrase [7–13], amember of the NTPDase family, or with derivative recombinant [8] orsynthetic [13,14] peptides, we identify this B domain as antigenic with-in catalytically active NTPDase isoforms from Schistosoma mansoni(ATPDase 2; r156–195) and Leishmania braziliensis (NTPDase 1; r83–122), possibly also present in Leishmania amazonensis NTPDase [15].In addition, these new biomolecules have immunostimulatory proper-ties that induce a humoral immune response in an animal model[8,10,12,14].

Several highly conserved antigens in the Leishmania genus retainthe capability to cross-protect against other species, and their charac-terization is essential to the understanding of the host–parasite rela-tionship and might be useful for the developmental of improvedtherapies or vaccines against leishmaniasis [16–20]. Leishmania spp.are intracellular protozoan parasites that cause cutaneous, mucocuta-neous and/or visceral leishmaniasis. Its flagellated promastigotes areintroduced into the skin of the vertebrate host through the bite of

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phlebotomine sandfly, and survive and multiply as nonmotileamastigotes in mammalian macrophages. The Leishmania infantum[syn. Leishmania (Leishmania) chagasi] is an etiological agent of bothhuman and canine visceral leishmaniasis, the most severe clinicalform of the disease, and the cause of important worldwide public-health problem [16–20]. Two genes codifying distinct proteins ofthe NTPDase family, annotated as ATP diphosphohydrolase (ATPDase;Gene ID 5067729) and guanosine diphosphatase (GDPase; Gene ID5067146), were found in the genome of L. infantum [17]. In this work,using biochemical procedures and polyclonal antibodies against eitherpotato apyrase, synthetic or recombinant peptides as molecular tools(a) we isolated an antigenic and catalytically active NTPDase 1 isoformfrom L. infantum promastigotes, and (b) tested their effects on the cata-lytic property of the enzyme.

2. Material and methods

2.1. Obtaining L. infantum preparation, potato apyrase, r-pot B domain,synthetic peptides, and development of polyclonal antiserum

Promastigote forms from the L. infantum MHOM/BR/1972/BH46strain were cultured in LIT medium for 7 days [20] and parasite prepa-ration was obtained as described elsewhere [13]. Potato apyrase(~50 kDa)was purified from a commercial strain of Solanum tuberosum[2,9]. The recombinant r-pot B domain (5493 Da) belonging to the con-served B domain (r78–117) from the potato apyrase (NCBI accessionnumber U58597.1) expressed as a six-histidine-linked tag polypeptidewas obtained as previously described [8]. Synthetic peptides LbB1LJ(r82–103) [13] and LbB2LJ (r102–121) (Fig. 2) belonging to the con-served B domain from the L. braziliensis NTPDase 1 (ATPDase;CAM42020.1) [17] were obtained by solid-phase synthesis [21]. Themolecular mass and purity of the synthesized peptides were confirmedby amino acid analysis and by MALDI-TOF using a Microflex-LT massspectrometer (Bruker‐Daltonics, Billerica, MA, USA). The Graphical Ab-stractwas obtained by using PSIPRED Protein Structure Prediction Serv-er. Rabbit pre-immune serum (rabbit control serum) or immune serumanti-potato apyrase [12] and mouse immune serum anti-r-pot B domain[8] or anti-synthetic peptides [14] were obtained as earlier described,using Freund's complete and incomplete adjuvants for emulsificationand inoculation with 15-day interval. To confirm the specificity of themouse polyclonal antibodies, pooled serum samples obtained fromBALB/c mice that were inoculated only with Freund's complete and in-complete adjuvants [14] in the absence of the r-pot B domain or syntheticpeptides, under the same experimental conditions, were used as controls(mouse control serum). The sera were stored at−20 °C.

2.2. Phosphohydrolytic activity measurement

For cation dependence analysis, ATP and ADP hydrolyses weremea-sured in L. infantum promastigote preparations (0.01 mgprotein/ml), induplicates, and in 3 different experiments, using a reaction mediumcontaining MOPS [3-(N-morpholino) propanesulfonic acid], pH 7.4, inthe absence and in the presence of either 1 mM CaCl2, 1 mM MgCl2,5 mM EDTA or 5 mM EGTA. The reaction was initiated by addition ofsubstrate, which was allowed to proceed for 60 min at 37 °C, and theamount of free inorganic phosphate (Pi) was determined spectrophoto-metrically as previously described [13]. Incubation times werechosen to ensure the linearity of the reaction with the substrate andprotein concentrations. Further, phosphohydrolytic activity measure-ments of different L. infantum promastigote preparations wereperformed using a reaction medium containing 50 mM MOPS buffer,pH 7.4, 1 mM CaCl2, 0.01 mg protein/ml and 3 mM ATP (n=9),ADP ?thyc=5?> (n=9), UDP (n=3), GDP (n=5), CTP (n=4), AMP(n=7) or CMP (n=4) substrate. The p-nitrophenylphosphate(p-NPP; 3 mM; n=6) or inorganic pyrophosphate (3 mM; n=4) wasalso used as a substrate. The effects of sodium orthovanadate

(100 μM), ouabain (1 mM), N,N′-dicyclohexylcarbodiimide (DCCD;100 μM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP;200 μM), bafilomycin A (1 μM), P1,P5-di(adenosine-5′) pentaphosphatepentasodium salt (Ap5A; 100 μM) or sodium azide (1 mM) on ATPand/or ADP hydrolysis were evaluated in four different experimentsfor each compound, in duplicates, and using the same reactionmedium.In these experiments, ATP hydrolysis measured in the absence (52±16 nmol Pi·mg−1·min−1) or presence of 2.5% (v/v) dimethyl sulfoxide(DMSO; 60±3 nmol Pi·mg−1·min−1), the solvent of DCCD, FCCP orbafilomycin A, was used as controls. The ADP hydrolysis (41±12 nmol Pi·mg−1·min−1)measured in the absence of Ap5A or sodiumazide was also used as control.

2.3. Non-denaturing polyacrylamide gel electrophoresis

The non-denaturing polyacrylamide gel electrophoresis wasperformed as modified from Detoni et al. [22], using a 6% polyacryl-amide gel and 0.1% (v/v) Triton X-100 plus 0.1% (w/v) sodiumdeoxycholate in the gel and running buffer. An aliquot of L. infantumpromastigote preparation was homogenized in 50 mM MOPS buffer,pH 7.4, and 1 mM CaCl2, supplemented with 0.2% Triton X-100 plus0.2% sodium deoxycholate. After 10 min, the detergent-homogenizedpreparation was supplemented with 0.125 M Tris–HCl, pH 6.8, 10%glycerol and 5% bromophenol blue and aliquots (100 μg of protein)were applied to the gel and subjected to electrophoresis using aMini-Protean III Cell (BioRad) apparatus for approximately 4 h at130 V in a cold room. The gel was washed for 40 min in 50 mMMOPSbuffer, pH 7.4. The lanes were cut out and separately incubated at37 °C in fresh 50 mM MOPS buffer, pH 7.4, supplemented with100 μM sodium orthovanadate, 10 mM CaCl2 and 5 mM ATP, GTP orCTP substrate. The lane incubated with ADP or UDP substrate was alsosupplemented with 1 mg/ml dodecyl nonaethylene glycol ether(C12E9), a nonionic detergent. After approximately 2 h, thewhite calci-um phosphate precipitates appeared indicating a phosphohydrolyticactivity in situ, and were photographed against a dark background.The region of the gel corresponding to the center of the active bandwas cut out and electroeluted.

Promastigote preparation (100 μg of total protein) or electroelutedactive band (originated from 100 μg of promastigote preparation) wassupplementedwith a gel loading buffer and submitted to electrophore-sis in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) on 10%polyacrylamide gels. The proteins were either stained with Coomassieblue and silver, or electroblotted onto the nitrocellulose membrane.The membrane was blocked (0.15 M phosphate buffer solution,pH 7.4, plus 0.3% Tween-20 and 2% casein), andWestern blots were in-cubated for 6 h at room temperature with either rabbit polyclonalanti-potato apyrase (diluted 1:1000) or mouse polyclonal anti-r-pot Bdomain (diluted 1:400), anti-LbB1LJ (diluted 1:200) or anti-LbB2LJ(diluted 1:200) antibodies using standard procedures [8,13]. Westernblots of promastigote preparation (100 μg of total protein) orelectroeluted active band (originated from 100 μg of promastigotepreparation) were also developed with serum samples (diluted1:100) from dogs with visceral leishmaniasis or healthy dogs (Section2.6). Signals were revealed by chemiluminescence using specificanti-IgG antibody coupled to horseradish peroxidase and Luminol assubstrate (ECLWestern Blotting System; GE Healthcare, Brazil), and ex-posed to X-ray film following the manufacturer's instructions.

Another non-denaturing gel was developed under identical exper-imental conditions, and the gel slices were also assayed in test tubesas modified from Vasconcelos et al. [23]. Briefly, only one lane wascut out and revealed with ATP for identification of the active band.The adjacent gel was quickly washed and incubated overnight in afresh 50 mM MOPS buffer, pH 7.4 for complete removal of TritonX-100 and DOC. The region of the gel corresponding to the center ofthe active band was cut out and each gel piece was assayed in testtubes by incubation in a fresh 50 mMMOPS buffer, pH 7.4, containing

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either 10 mM CaCl2 and 5 mM substrate or 1 mM CaCl2 and 3 mMsubstrate, in the presence or absence of 1 mg/ml C12E9. Gel piecesfrom the other gel regions were used as blanks. For each experimentalcondition, two gel pieces were tested in tubes, and the result repre-sents the mean plus standard deviations. The amount of free inorgan-ic phosphate (Pi) was determined spectrophotometrically [13] at thetimes indicated in Fig. 3.

2.4. Immunoprecipitation assays and Western blots

An aliquot of the L. infantum promastigote preparation wassuspended in a medium containing 50 mM MOPS buffer, pH 7.4,and 1 mM CaCl2 and supplemented with 1 mg/ml C12E9. After cen-trifugation at 10,000 g for 10 min at 4 °C, rabbit immune serumanti-potato apyrase or mouse immune serum anti-r-pot B domain ata final dilution of 1:500 and 1:400, respectively, was added to the al-iquots of a high-speed supernatant (2.5 mg protein/ml) and incubat-ed for 3 h at room temperature. Assays using rabbit or mouse controlserum were run in parallel. Protein A-Sepharose was then added andincubated for an additional 2 h. The resin–antibody–antigen complexwas sedimented by centrifugation for 5 min, washed 3 times in50 mMMOPS buffer, pH 7.4, solubilized in gel loading buffer and sub-mitted to SDS-PAGE on 10% polyacrylamide gels using Mini-ProteanIII Cell (Bio-Rad). The proteins were electroblotted onto nitrocellulosemembranes, followed by a blocking step (0.15 M phosphate buffersolution, pH 7.4, plus 0.3% Tween-20 and 2% casein) using standardprocedures [8,13]. To avoid reactivity with subunits from rabbit-IgGor mouse-IgG, the Western blots of immunoprecipitated ProteinA-rabbit anti-potato apyrase antibody–antigen complex or ProteinA-mouse anti-r-pot B domain antibody–antigen complex were devel-oped with mouse immune serum anti-r-pot B domain diluted 1:800or rabbit immune serum anti-potato apyrase diluted 1:1000, respec-tively. Potato apyrase (1 μg) or total proteins (100 μg) of thepromastigote preparation were also submitted to electrophoresis in10% SDS-PAGE and electroblotted onto nitrocellulose membranes,and developed with rabbit immune serum anti-potato apyrase (dilut-ed 1:1000) or mouse immune serum anti-r-pot B domain (diluted1:800), and used as a positive control. Signals were revealed bychemiluminescence as already described in Section 2.3. The superna-tants obtained after the addition of the Protein A-Sepharose wereused for phosphohydrolytic activity measurements as already de-scribed in Section 2.2, in a pre-established reaction medium (SRM)containing 50 mM MOPS buffer, pH 7.4, 1 mM CaCl2, 100 μM sodiumorthovanadate, 0.01 mg protein/ml and 3 mM of substrate. The ex-periments were made twice in triplicates.

2.5. Effects of polyclonal antibodies on the phosphohydrolytic activity

An aliquot of L. infantum promastigote preparation was homoge-nized with 1 mg/ml C12E9 under the same experimental conditionsas already described in Section 2.4, and the aliquots of the high-speedsupernatant (2.5 mg protein/ml) were incubated for 3 h at roomtemperature with rabbit immune serum anti-potato apyrase (final di-lution 1:500) or mouse immune serum containing either anti-r-pot Bdomain or anti-synthetic peptide antibodies (final dilution 1:400).Assays using rabbit or mouse control serum were run in parallel. Analiquot of each preparation was used for phosphohydrolytic activitymeasurements in triplicate using the pre-established reaction medi-um (SRM; Section 2.4). The experiments were made twice.

2.6. Selection of serum samples from dogs

Sera from adult dogs not infected (n=17) or with visceral leish-maniasis (n=38), of both genders aging from 2- to 6-years-old,leaving in an endemic area for L. infantum, were selected fromserum collections [20]. Diagnosis of canine visceral leishmaniasis

was made by IFAT through the specific anti-Leishmania IgG reactivityusing promastigote forms of L. amazonensis (MHOM/BR/1960/BH6)and L. chagasi (MHOM/BR/1972/BH46) promastigote antigen. Infec-tion with L. chagasi was confirmed in all IFAT positive dogs by aleast one additional serological approach, including ELISA-extractand ELISA r-K39, and/or parasitological examination [20]. The seven-teen non-infected dogs, included in this work as healthy dogs fromthe endemic area, were classified by clinical exams, and confirmedby negative IFAT and negative parasitological exams for Leishmania[20]. For determination of a cut-off, 10 sera of healthy adult dogs(both genders aging from 2- to 8-years-old) from a non-endemicarea for Leishmania spp. in Brazil were examined using the sameparameters. The study protocols complied with the regulations ofthe Brazilian National Council of Research in Animals and wereapproved by the Ethical Committee for Animal Research of theUniversidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil, underprocess no. 141/2006.

2.7. Antibody analyses by enzyme-linked immunosorbent assays (ELISA)

Aliquot (10 μg/well) of either r-pot B domain, LbB1LJ or LbB2LJ in0.1 M NaHCO3, pH 9.6, was absorbed overnight onto flat-bottomedmi-crotiter plates (Costar 3590; Corning Inc., Corning, NY, USA). Followinga blocking step (0.15 M phosphate buffer solution, pH 7.2 plus 0.3%Tween-20 and 2% casein), sera diluted 1:50 (for synthetic peptides) or1:100 (for r-pot B domain) were tested in duplicate, diluted in thisblocking buffer without Tween-20, and incubated for 4 h at room tem-perature. Antibodies bound to the antigen-plate were detected usingperoxidase-conjugated sheep anti-dog IgG (anti-heavy chain specific),and o-phenylenediamine dihydrochloride (OPD)/H2O2 as substrate.The subsequent color reaction was read at 492 nm on a microplatereader (Molecular Devices Corp., Menlo Park, CA, USA). The consideredvalues of A492were themeans of 4 determinationswith a variation of nomore than 15% between them. GraphPad Prism Software (version 4)was used for statistical analysis. Themedian and the 95% confidence in-terval were calculated, and the data were analyzed using the Mann–Whitney test. P valuesb0.05 were considered significant.

3. Results and discussion

3.1. Identification of L. infantum NTPDase activity

It has been demonstrated that antibody–antigen binding is depen-dent on pH and ionic strength [24]. Recently, structural and biochem-ical studies revealed that antibodies could have an exposedmetal-binding site that requires calcium for recognition of antigen[25], and calcium in high molar concentration (1–10 mM) couldhave an inhibitory effect on the antibody–antigen binding [26].These new reports warned us about possible variable results in ourassays that associate immunological property and NTPDase activity.Because of this, in an attempt to discard interferences on the anti-body–antigen binding, reaction media containing suitable concentra-tions of nucleotide and bivalent cation were successfully used forisolation of L. braziliensis NTPDase 1 isoform by immunoprecipitationassays [8,13].

In order to identify the NTPDase activity in the promastigote prep-aration from the L. infantum MHOM/BR/1972/BH46 strain, the samecomposition of reaction medium was initially assayed. Using a50 mM MOPS buffer, pH 7.4, and 3 mM substrate, in the absence ofadded bivalent cation, insignificant ATPase and ADPase activitieswere observed, which were completely removed by the addition of5 mM EDTA or EGTA. ADPase activity was found in the presence of1 mM CaCl2 or 1 mM MgCl2, and the ATP hydrolysis was more effec-tive in the presence of 1 mM CaCl2 than in the presence of 1 mMMgCl2 (data not shown). Then, CaCl2 was chosen for the compositionof the reaction medium.

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In the presence of the 50 mM MOPS buffer, pH 7.4, and 1 mMCaCl2, the promastigote preparation was equally effective at thehydrolysis of either ATP or ADP, in the range of 65±38 and 59±37 nmol Pi·mg−1·min−1, respectively, with an ATPase/ADPaseactivity ratio of about 1.1. The hydrolysis of CTP (55±21 nmolPi·mg−1·min−1), UDP (35±7 nmol Pi·mg−1·min−1) or GDP (17±8 nmol Pi·mg−1·min−1) was also observed, revealing a broad phos-phohydrolytic ability.

Under these experimental conditions, insignificant hydrolysis(b1 nmol Pi·mg−1·min−1) of AMP or CMP, substrates of 5′-nucleo-tidases, was observed. Hydrolyses of inorganic pyrophosphate, a sub-strate of pyrophosphatases (4±7 nmol Pi·mg−1·min−1), and p-NPP(2±3 nmol Pi·mg−1·min−1), a substrate of phosphatases, were alsoinsignificant. Therefore, the hydrolytic activity of these enzymes didnot contribute significantly to the amount of Pi quantified in theassays.

The contribution of the well-known ATPases with the amount of Piquantified in the assayswas evaluated using several inhibitors. Additionof 100 μM sodium orthovanadate, an inhibitor of P-type cation trans-port ATPases, inhibited about 27% of the ATPase activity, suggesting adirect effect on a P-type ATPase in the promastigote preparation. Thisresult was corroborated by the 29% inhibition caused by ouabain(1 mM), an inhibitor of Na+/K+ ATPase, suggesting a contribution ofthis protein in the amount of Pi quantified in the assays. No significanteffect was observed with 100 μM dicyclohexylcarbodiimide (DCCD),an inhibitor of mitochondrial ATPase, or 200 μM carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), a protonophore thatuncouples oxidative phosphorylation in mitochondria and stimulatesMg2+ ATPase activity. Bafilomicyn A, a macrolide antibiotic inhibitorof vacuolar ATPases, was also ineffective till its 1 μM concentration. Inaddition, 100 μM Ap5A, a selective adenylate kinase inhibitor, did notsignificantly affect either ATP (14% inhibition) or ADP (8% inhibition)hydrolysis excluding the possible association of an adenylate kinaseconversing ADP in ATP and later an ATP-specific enzyme hydrolyzingATP.

On the other hand, 1 mM sodium azide, an inhibitor of either mi-tochondrial ATPase or NTPDase isoforms [6,13,27], was able to inhibit24% of the ATPase activity and 80% of the ADPase activity. Since100 μMDCCD did not affect significantly ATP hydrolysis, these resultssuggested a direct effect of sodium azide on NTPDase activity,inhibiting partially its ATPase activity and almost totally the ADPaseactivity.

Therefore, the significant hydrolysis of nucleoside di- or triphos-phate upon bivalent metal ion activation, associated to the inhibitionpromoted by sodium azide suggested that the phosphohydrolytic ac-tivity found in the L. infantum promastigote preparation is catalyzedby NTPDase isoforms.

3.2. Purification and identification of the antigenic L. infantum NTPDase 1by non-denaturing gel electrophoresis

In order to separate active NTPDase isoforms from other proteins,different concentrations and/or combinations of detergents for bothhomogenization and fractionation of parasite preparations by non-denaturing gels have been tested [9,15,23,28]. However, the NTPDasesare sensible to several non-ionic or ionic detergents, which reducedistinctly each nucleotide hydrolysis when compared to the activity ofthe crude preparation; most of them did not isolate the NTPDaseisoforms from other proteins when used alone or combined with anon-denaturing gel and, in addition, promote turbidity and interferein colorimetric measurements [6,9,15,23,28,29]. The detergent non-ionic C12E9 also did not isolate parasite NTPDase isoforms by non-denaturing gels, but has the advantage of maintaining a clear reactionmedium for colorimetric assays, and interferes less with enzyme activ-ity from homogenized-preparations [8,13,15].

Recently, we showed that plant preparations previously homoge-nized in 0.2% Triton X-100 plus 0.2% sodium deoxycholate was frac-tionated by non-denaturing gels with preservation of the apyraseactivity [22]. Now, the same detergent mixture was used and, asshown in Fig. 1, an NTPDase isoform was effectively isolated fromthe homogenized-promastigote preparation (100 μg of total protein)applied in non-denaturing gels. The enzymewas capable of catalyzingthe hydrolysis of either ATP, GTP or CTP substrate as observed by theformation of a white deposit of calcium phosphate at a single activeband upon incubation of the gel slab in 50 mM MOPS buffer, pH 7.4,100 μM sodium orthovanadate, and 5 mM substrate plus 10 mMCaCl2 (Fig. 1A, ATP, GTP and CTP). Under these experimental condi-tions, no calcium phosphate precipitate was detected when the gelslab was incubated with ADP or UDP (data not shown). It is interest-ing to observe that only after washing and addition of non-ionic de-tergent C12E9 to the reaction medium was it possible to visualizethe active band displaying an identical electrophoretic mobilityresulting from ADP or UDP hydrolysis (Fig. 1A, ADP and UDP),suggesting a greater sensitivity of nucleoside diphosphate hydrolysisto the mixture of 0.2% Triton X-100 plus 0.2% DOC.

After electrophoresis of the eluted active band (originated from100 μg of total protein) in SDS-PAGE followed by Coomassieblue-staining, no polypeptide was detected showing a low yield(data not shown). On the other hand, the same Coomassie blue-stained gel submitted to silver staining showed a single polypeptideof approximately 50 kDa (Fig. 1B, P, B), confirming its purity andthat it is not a major protein as compared to the total protein(100 μg) from the promastigote preparation (Fig. 1B, P, A). The hy-drolysis of nucleoside di- or triphosphate (Fig. 1A) confirmed thepresence of an NTPDase isoform of approximately 50 kDa (Fig. 1B,P, B) in the promastigote preparation.

By Western blots of the L. infantum promastigote preparation(100 μg of total protein) a polypeptide of approximately 50 kDa wasrecognized by polyclonal immune serum anti-potato apyrase(Fig. 1B, Wb-R, 1). In addition, the isolated polypeptide of 50 kDawas also recognized by immune serum anti-potato apyrase (Wb-R,2) and polyclonal immune serum anti-r-pot B domain (Fig. 1B,Wb-R, 3). In previous works, by in silico analysis we showed that aputative NTPDase (ATPDase; Gene ID 5067729) annotated in theL. infantum genome [17], of a predicted molecular mass of 47 kDaand glycosylation sites, has high homology (31% identity and 45%similarity over 404 amino acids) with potato apyrase [7,8,10]. Highhomology (55% identity and 60% similarity over 40 amino acids)was also found between the conserved B domain from the potato ap-yrase (r78–117) and its L. infantum ATPDase counterpart (r83–122)[7,8,10]. The putative guanosine diphosphatase (GDPase; Gene ID5067146), also belonging to the NTPDase family, and annotated inthe genome of this parasite [17], has been predicted to have a molec-ular mass of 74 kDa, and only 33% identity and 47% similarity over228 amino acids of potato apyrase, which were mostly restricted tothe conserved regions (ACRs) from the NTPDase family [7,8]. In addi-tion, no significant similarity was found between the B domain fromeither potato apyrase or L. infantum ATPDase and its GDPase counter-part (r236–275) [7,8]. These results identified the L. infantum NTPDase1 and confirmed the presence of shared epitopes with potato apyraseand, in addition, the presence of the conserved B domain within thisparasite isoform.

Definitive proof was obtained by reactivity between the L. infantumNTPDase 1 of 50 kDa and both immune sera anti-LbB1LJ (Wb-R, 4) andanti-LbB2LJ (Fig. 1B,Wb-R, 5). It is interesting to observe that these spe-cific antibodies produced against B domain from Leishmania spp., ofhigher affinity and sensitivity, were capable of reacting with an addi-tional band of approximately 47 kDa (Fig. 1B, Wb-R; 4 and 5), whichwas not visualized on the silver stained gel (Fig. 1B, P, B). As shown inthe detached mode in Fig. 2, the amino acid sequence of LbB1LJ (86%identity and 95% similarity over 22 amino acids) or LbB2LJ (80% identity

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and 90% similarity over 20 amino acids) derived from the conserved Bdomain from L. braziliensis NTPDase 1 (ATPDase; CAM42020.1) [17]has high identity with its L. infantum NTPDase 1 counterpart, and nosignificant similaritywith putative LeishmaniaGDPase isoform counter-parts (Fig. 2). Thus, the results strongly suggested that this polypeptideof approximately 50 kDa, named in this work as NTPDase 1, corre-sponds to the hypothetical ATPDase (Gene ID 5067729) annotated inthe L. infantum genome [17]. It is possible that the L. infantum NTPDase1 is subject to posttranslational processing such as glycosylation, andthis band of lower molecular mass represents its deglycosylated form.

The isolated L. infantum NTPDase 1 of approximately 50 kDa, andthe polypeptide of approximately 47 kDa were also reactive withserum samples from naturally L. infantum-infected dogs with symp-tomatic visceral leishmaniasis, and a representative result is shownin Fig. 1 (Wb-A, B). These serum samples also react with antigenic

proteins from the promastigote preparation (Fig. 1B, Wb-A, A),whereas no reactivity was found when the isolated NTPDase 1 wastested with serum samples of healthy dogs (Fig. 1B, Wb-A, C). The re-sults suggested that the L. infantum NTPDase 1 is antigenic, like whatwas demonstrated for Toxoplasma gondii NTPases 1 and 2, S. mansoniATPDases 1 and 2, and L. amazonensis and L. braziliensis NTPDaseisoforms [7–13,30,31].

Additionally, gel pieces containing active NTPDase 1 were obtainedfrom a non-denaturing gel, which was previously washed overnightfor a complete removal of Triton X-100 and DOC, and assayed in testtubes as described in Section 2.3. (Fig. 3). Each gel piece was incubatedin a fresh 50 mM MOPS buffer, pH 7.4, containing 10 mM CaCl2and 5 mM ATP (Fig. 3A) or ADP (Fig. 3B) in the presence or absenceof detergent C12E9. The free inorganic phosphate produced was deter-mined spectrophotometrically at the times indicated in Fig. 3. Inthe complete absence of detergents (white square or circle), insig-nificant ATP (0.14±0.08 nmol Pi·mg−1·min−1) or ADP (0.13±0.06 nmol Pi·mg−1·min−1) hydrolysis was detected (Fig. 3, A and B).On the other hand, in the presence of C12E9 (black square or circle),the ATPase and ADPase activities increased to 5.95±2.04 and 3.91±1.09 nmol Pi·ml−1·min−1, respectively, suggesting that in the pres-ence of this non-ionic detergent the ATPase/ADPase activity ratio isabout 1.5 (Fig. 3, A and B). This reaction medium, which was alsoused to induce and visualize calcium phosphate deposits on non-denaturing gels (Fig. 1A), contains approximately 4.8 mM Ca-ATP, andfree Ca2+ (5.2 mM) and ATP (0.2 mM) concentrations, as calculatedby an interactive computer program [32] available in the world wideweb. This experimental condition favors the presence of a stable ion–nucleotide complex, the possible substrate of the parasite NTPDaseisoforms [29].

The reaction medium composed of 50 mM MOPS buffer, pH 7.4,1 mM CaCl2 and 3 mM substrate, used in initial studies described inthis work (Section 3.1), was also tested in gel slices assayed in testtubes, in the absence (white square or circle) or presence (blacksquare or circle) of C12E9 (Fig. 3, A and B). Using ATP as substrate,this reaction medium presumably contains 900 μM Ca-ATP, andfree Ca2+ (100 μM) and ATP (2.1 mM) concentrations [32]. In the ab-sence of C12E9, the ATPase and ADPase activities from the pureNTPDase 1 were also low, of approximately 0.16±0.04 and 0.24±0.12 nmol Pi·ml−1·min−1, respectively (Fig. 3, A and B). In the pres-ence of this non-ionic detergent, the ATPase and ADPase activ-ities increased to approximately 3.46±0.08 and 3.04±0.83 nmolPi·ml−1·min−1, respectively (Fig. 3, A and B). The ATP hydrolysisdecreased by approximately 42%, and the ADPase activity did notdiffer significantly from that obtained using 5 mM CaCl2 and 10 mM

Fig. 1. (A) Purification of the L. infantum NTPDase 1 by a non-denaturing gel. Aliquots(100 μg of total proteins) from a homogenized-detergent promastigote preparationwere separated by non-denaturing polyacrylamide gel electrophoresis. The gel waswashed in 50 mM MOPS buffer, pH 7.4, and the lanes cut out were immersed in afresh buffer containing 5 mM substrate, 100 μM sodium orthovanadate, and 10 mMCaCl2, supplemented (ADP or UDP) or not (ATP, GTP or CTP) with non ionic detergentC12E9. After 2 h of incubation at 37 °C, white deposits of calcium phosphate appearedas a result of nucleotide hydrolysis catalyzed by the enzyme. The gel was photographedagainst a dark background. (B) Identification of the protein extracted from an activeband of non-denaturing gels. The corresponding region of the active band (A) was ex-cised from non-denaturing gels. The proteins of the aliquot of the promastigote prepa-ration (P, lane A; Wb-R, lane 1; 100 μg) or excised active band (P, lane B; Wb-R, lanes 2,3, 4, and 5) were separated by SDS-PAGE on 10% polyacrylamide gel and subjected toCoomassie blue-silver staining (P, lanes A and B) or electroblotting onto the nitrocellu-lose membrane. The Western blots were developed with immune serum anti-potatoapyrase (Wb-R, lanes 1 and 2; dilution 1:1000), anti-r-pot B domain (Wb-R, lane 3;dilution 1:400), anti-LbB1LJ (Wb-R, lane 4; dilution 1:200) or anti-LbB2LJ (Wb-R,lane 5; dilution 1:200). In addition, an aliquot of the promastigote preparation(Wb-A, lane A; 100 μg of protein) and excised active band (Wb-A, lanes B and C)were tested with a serum sample diluted 1:100 from either a dog with visceral leish-maniasis (Wb-A, lanes A and B) or a healthy dog (Wb-A, lane C). The membraneswere revealed by chemiluminescence.

Fig. 2. The primary amino acid sequences of the synthetic peptides LbB1LJ and LbB2LJ. Each peptide belonging to the B domain from the L. braziliensis NTPDase 1 was aligned with itsputative Leishmania NTPDase counterpart. The identical amino acid residues are shown as gray columns. GenBank accession numbers of the amino acid sequences are: L. braziliensisATPDase, CAM42020.1; L. infantum ATPDase, CAM66723.1; L. braziliensis GDPase, XP_001562788.1; and L. infantum GDPase, CAM66031.1.

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substrate, and the ATPase/ADPase activity ratio is about 1.1. Theseresults suggested the effectiveness of C12E9 for the refolding of thepure L. infantum NTPDase 1, which was previously homogenized inthe mixture of 0.2% Triton X-100 and 0.2% DOC and isolated bynon-denaturing gels (Fig. 1, A and B). Somemembers of the NTPDasefamily are regulated by different stages of oligomerization, whichaffect both the catalytic activity level and the preference for thenucleosides di- and triphosphate. Several detergents dissociate

Fig. 3. ATPase or ADPase activity of the L. infantum NTPDase 1 in a gel slice assayed in atest tube using distinct reaction media. One lane from a non-denaturing gel similar tothat shown in Fig. 1A was revealed with ATP. The adjacent gel was washed and incu-bated overnight with fresh 50 mM MOPS buffer, pH 7.4, for a complete removal ofTriton X-100 and DOC. The region of the gel corresponding to the active band wascut out and each gel piece was tested for ATP (A) or ADP (B) hydrolysis. The reactionmedium containing fresh 50 mM MOPS buffer, pH 7.4, 10 mM CaCl2 and 5 mM sub-strate (A, white square), added with C12E9 (B, black square) or, alternatively,50 mM MOPS buffer, pH 7.4, 1 mM CaCl2 and 3 mM substrate (C, white circle),added with C12E9 (D, black circle). Gel piece from the other region was used asblank. The free inorganic phosphate produced was determined spectrophotometricallyat the times indicated. For each experimental condition, two gel pieces were tested intubes, and the result represents the mean plus standard deviation.

oligomers and induce modifications in the kinetic properties ofthese proteins [6,29,33,34]. Our assays did not discard a possibleoligomerization of the L. infantum NTPDase 1, and the kinetic param-eters of this enzyme should be better investigated.

Under our experimental conditions, the reactionmediumwith 1 mMCaCl2 and 3 mM substrate seemingly contains a stable ion–nucleotidecomplex in a micromolar concentration higher than the pure proteinwhich is capable of hydrolyzing and, in addition, a simultaneous lowerfree Ca2+ concentration that enables us to access the L. infantumNTPDase 1 activity in biochemical and immunological assays. In addition,the ATPase/ADPase activity ratio of approximately 1.1 found in thepromastigote preparation (Section 3.1) is possibly resulting from theL. infantum NTPDase 1 in its native form, and closer to in vivo conditions.In view of the results obtained, we established a suitable reaction medi-um (SRM) containing 50 mM MOPS buffer, pH 7.4, 1 mM CaCl2 and3 mM substrate, supplemented with 100 μM sodium orthovanadateand 1 mg/ml C12E9, which was used in subsequent assays.

3.3. Identification of a catalytically active isoform, the NTPDase 1, inL. infantum promastigotes by immunoprecipitation assays

The polyclonal anti-potato apyrase and polyclonal anti-r-pot B do-main antibodies immobilized on Protein A-Sepharose were tested fortheir ability to immunoprecipitate NTPDase 1 from the detergent-homogenized promastigote preparation using the SRM. Anti-potatoapyrase antibodies immunodepleted approximately 95% of theATPase and 75% of the ADPase activity, and anti-r-pot B domain anti-bodies immunodepleted approximately 37% of the ATPase and 43% ofthe ADPase activity compared to the controls (Table 1).

The immunoprecipitated resin–antibody–antigen complexes werewashed and subjected to electrophoresis andWestern blots. Both poly-clonal anti-potato apyrase (Wb-I, lane A) and polyclonal anti-r-pot Bdomain (Wb-I, lane B) antibodies immobilized on Protein A-Sepharoseimmunoprecipitated a band of approximately 50 kDa, the same bandidentified in the detergent-homogenized promastigote preparationby cross-immunoreactivity of rabbit polyclonal anti-potato apyrase(Wb-A) or mouse polyclonal anti-r-pot B domain (Wb-B) antibodies(Fig. 4; Li lanes). As a positive control, potato apyrase (Wb-A andWb-B, A lanes) was recognized by the same immune sera (Fig. 4).

Therefore, the immunoprecipitation assays using anti-potatoapyrase or anti-r-pot B domain antibodies, allowed both depletion

Table 1Depletion of NTPDase 1 activity from the detergent-homogenized promastigote prepa-ration by polyclonal antibodies immobilized on Protein A-Sepharose.

Experimental conditions ATPase activitya ADPase activitya

nmol Pi·mg−1·min−1

Rabbit control serumb 58±10 55±12Rabbit immune serum anti-potato apyraseb 3±1 (5) 14±5 (25)Mouse control serumc 56±3 44±2Mouse immune serum anti-r-pot B Domainc 35±3 (63) 25±3 (57)

a In parenthesis is the percentage of hydrolytic activity compared to the control.b Rabbit serum diluted 1:500.c Mouse serum diluted 1:400.

Fig. 4. Identification of the active NTPDase 1 isoform from L. infantum promastigoteforms by immunoprecipitation assays using immune serum anti-potato apyrase oranti-r-pot B domain. Potato apyrase (1 μg; Cb, Wb-A and Wb-B, A lanes), an aliquotof promastigote preparation (100 μg of protein; Cb, Wb-A andWb-B, Li lanes) or eitherimmunoprecipitated Protein A-rabbit anti-potato apyrase antibody–antigen (Wb-I,lane A) or Protein A-mouse anti-r-pot B domain antibody–antigen (Wb-I, lane B) com-plex isolated from the promastigote preparation was submitted to electrophoresis in10% SDS-PAGE, and electroblotted onto the nitrocellulose membrane. The Westernblots were developed with rabbit polyclonal anti-potato apyrase (Wb-A, lanes A andLi; Wb-I, lane B; dilution 1:1000) or mouse polyclonal anti-r-pot B domain (Wb-B,lanes A and Li; Wb-I, lane A; dilution 1:800) antibodies. The gel (Cb) was stainedwith Coomassie blue, and the membranes (Wb) were revealed by chemiluminescence.

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of the phosphohydrolytic activity and recognition of the polypeptideof approximately 50 kDa, thus confirming the identity of the activeNTPDase 1 in L. infantum promastigote forms also by other methods.As previously suggested [7,8,10], polyclonal or monoclonal antibodiesproduced against either potato apyrase or its B domain are potentialmolecular tools for initial isolation and structural studies of homologNTPDase isoforms from several pathogenic organisms of distinct phy-logenetic lineages not yet investigated.

3.4. Effects of polyclonal anti-potato apyrase, anti-r-pot B domain oranti-synthetic peptide antibodies on the phosphohydrolytic activity ofthe L. infantum NTPDase 1

The effects of polyclonal antibodies on the phosphohydrolytic activityof the L. infantumNTPDase 1were studied using a promastigote prepara-tion and SRM. It is interesting to observe that immune serum anti-potatoapyrase immunodepletes approximately 75–95% of the NTPDase ac-tivity (Table 1), but it did not inhibit NTPDase 1 activity from thehomogenized-L. infantum promastigote preparation (Table 2). In con-trast, polyclonal anti-r-pot B domain antibodies were capable ofinhibiting approximately 44% of the ATPase or ADPase activity compared

Table 2NTPDase 1 activity inhibition by polyclonal antibodies produced against potato apyrase,r-pot B domain, LbB1LJ or LbB2LJ.

Experimental conditions ATPase activitya ADPase activitya

nmol Pi·mg−1·min−1

Rabbit control serumb 45±6 33±4Rabbit immune serum anti-potato apyraseb 45±7 35±1Mouse control serumc 64±14 38±6Mouse immune serum anti-r-pot B Domainc 36±5 (56) 21±4 (55)Mouse immune serum anti-LbB1LJc 0.6±0.8 (1) 3±3 (8)Mouse immune serum anti-LbB2LJc 5±7 (8) 5±4 (13)

a In parenthesis is the percentage of hydrolytic activity compared to the control.b Rabbit serum diluted 1:500.c Mouse serum diluted 1:400.

to the control (Table 2). The immune serum anti-potato apyrase possiblycontains a greater diversity of antibodies against other epitopes alsosharedwith the L. infantumNTPDase 1 [7] and aminor amount of specificanti-B domain antibodies, the true inhibitors of the phosphohydrolyticactivity, allowing an effective immunodepletion but not an effectiveNTPDase 1 activity inhibition.

Definitive proof was obtained using the polyclonal antibodiesanti-LbB1LJ or anti-LbB2LJ peptides, which inhibited almost totallyand similarly ATPase (92–99%) or ADPase (87–92%) activity, andin higher rates than the immune serum anti-r-pot B domain(Table 2). Therefore, the inhibition promoted by the immuneserum anti-LbB1LJ or anti-LbB2LJ, which is specific for LeishmaniaNTPDase 1 isoforms (Fig. 2), reinforces the specific inhibition ofthe B domain (r82–121) from the L. infantum NTPDase 1, a domainpossibly involved in the catalytic cycle of this NTPDase isoformand a new target for inhibitor design. In addition, antibodies raisedagainst this specific domain could inhibit enzyme activity and causethe death of the parasite.

Specific inhibitors have not been found for several NTPDaseisoforms [1,3,6,29,35], and the effect of selective anti-NTPDase anti-body is an approach that has been applied in studies of the functionof members of this protein family [33,34,36,37]. As previously dem-onstrated, rabbit polyclonal anti-NTPase antibodies did not inhibitATPase activity from T. gondii NTPase isoforms, while the monoclonalantibody was capable of inhibiting almost totally the ATPase andADPase activities, and yet of identifying a distinct pattern of NTPaseisoforms among virulent and avirulent parasite strains [33]. In addi-tion, both T. gondii NTPase II activity and tachyzoite replication ininfected host cells were significantly inhibited by monoclonal anti-bodies, suggesting that antibodies against this enzyme could have aprotective effect against T. gondii infection [37]. A polyclonal immuneserum against Trypanosoma cruzi NTPDase 1 significantly inhibited(50%) the infectivity of trypomastigotes in vitro [36]. Monoclonal an-tibodies produced against NTPDase 3 from Langerhans islet cells ofthe human pancreas were capable of inhibiting 60 to 90% of the en-zyme activity depending on the experimental conditions used, andthe results suggested the relevance of these antibodies for the studyof insulin secretion [34].

The enzyme activities of parasite NTPDase isoforms are involvedwith infectivity, purine salvage, and disease pathogenesis regulatingnucleotide rates in several physiological processes such as purinergicsignaling systems [4–6,29,33,35–37], suggesting that these proteinsare important for parasite survival. Thus, the use of inhibitory anti-bodies against the conserved B domain could be used in further stud-ies of the structure of the L. infantum NTPDase 1, and they havepotential antileishmanial applications. Site-directed mutagenesiswithin the B domain from an active recombinant of the L. infantumNTPDase 1 could identify the amino acid residues that likely interactwith the antibodies.

3.5. The conserved B domain within the L. infantum NTPDase 1 is antigenic

As shown in Fig. 1B (Wb-A, B), the pure L. infantum NTPDase 1 isrecognized by serum samples from dogs with visceral leishmaniasissuggesting its antigenicity. IgG antibody level was quantified inserum samples from dogs using r-pot B domain or synthetic peptidesas coating antigen in ELISA (Fig. 5). Using as cut-off the IgG reactivityof healthy dogs domiciled in the non-endemic area, 14 (37%), 17(45%) or 19 (50%) of the 38 naturally infected dogs (INF) were sero-positive for the r-pot B domain, LbB1LJ and LbB2LJ, respectively, andonly 2 (12%) of the 17 healthy dogs also domiciled in the endemicarea (HEA) were seropositive for the r-pot B domain or LbB1LJ(Fig. 5). The IgG antibody reactivity against the r-pot B domain(Pb0.001), LbB1LJ (Pb0.01) or LbB2LJ (Pb0.05) from the INF groupwas significantly higher than the HEA group (Fig. 5). These resultsconfirmed the L. infantum NTPDase 1 antigenicity, and identified its

Fig. 5. IgG antibody reactivity against r-pot B domain, LbB1LJ or LbB2LJ. IgG antibody level was quantified in serum samples from infected dogs (INF; n=38) domiciled in an en-demic area, using r-pot B domain (DomB), LbB1LJ or LbB2LJ as the coating antigen in ELISA, and compared to healthy dogs (HEA; n=17) domiciled in the same endemic area. TheIgG antibody levels are expressed as the optical density of the serum samples. The horizontal line represents the mean. The dotted horizontal line represents the mean of the opticaldensity of the serum samples from healthy dogs (n=10) domiciled in a non-endemic area plus 2 standard deviations. Values greater than the cut-off level for r-pot B domain(0.339±0.032; 0.404), LbB1LJ (0.274±0.030; 0.334) or LbB2LJ (0.212±0.054; 0.320) were considered to be seropositive. The statistical significance of group differences was de-termined using Mann–Whitney test. P value is ***0.001, **0.01 or *0.05.

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conserved B domain (r82–121) as rich in B cell epitopes, which couldbe involved in both host immunomodulation and parasite survival aspreviously suggested [7,8,10].

In canine visceral leishmaniasis, increased IgG levels are detectedprior to the appearance of the first clinical signals, being relevant hall-marks of the distinct clinical status of this disease, and several promis-ing antigens and diagnosis methods have been described [16,18–20].The B domains within parasite NTPDase isoforms could elicit specificimmune responses in distinct diseases due to the different parasitelife-cycles [7,8]. Thus, the biomolecules shown in this work, or deriva-tives of them, could be useful as a composition to improve diagnosisand/or monitoring of the clinical status during the development of ca-nine visceral leishmaniasis.

Numerous researchers have focused their attention on the NTPDaseactivity, but the antigenic properties of parasite NTPDase isoforms haveonly been reported in T. gondii [30,31,33,37,38], S. mansoni [7–9,11,12],L. (L.) amazonensis [15] and L. (V.) braziliensis [8,13]. The studies aboutthe antigenic T. gondiiNTPase isoforms aremost advanced and, recently,have demonstrated that pre-immunization of BALB/c mice with a re-combinant form of NTPase II, elicits a strong humoral and cellular im-mune response inducing partial protection against virulent T. gondiistrains, indicating this protein as an effective candidate for the develop-ment of a vaccine against toxoplasmosis [38].

Different vaccine candidates with potential to trigger immuno-protective mechanisms against visceral canine leishmaniasis havebeen proposed as an important tool for controlling this zoonotic dis-ease [18,19]. Recently, by immunohistochemistry, we showed thatantibody produced against potato apyrase [39] or a synthetic peptidederivative from the B domain from the S. mansoni ATPDase 2 isoform[14] did not recognize mammalian NTPDases, corroborating the loweridentity found between mammalian NTPDases and either potato apy-rase or parasite B domain [7,8]. Thus, the biomolecules shown in thiswork, or its derivatives, could also be tested in formulations of eitherprophylactic or immunotherapy experimental protocols. These hy-potheses are currently under investigation in our laboratory.

In conclusion, using suitable approaches we reported for the firsttime an antigenic and catalytically active NTPDase 1 isoform of ap-proximately 50 kDa from L. infantum promastigotes, and identifiedan antigenic conserved domain (r82–121) rich in B cell epitopes.We reinforced the use of specific inhibitory antibodies against thisconserved domain for further studies of the structure and functionof parasite NTPDases and, in addition, showed the potential applica-tion of the r-pot B domain and synthetic peptides LbB1LJ andLbB2LJ, or antibodies raised against them, in immunological studiesof canine visceral leishmaniasis such as diagnosis, and prophylacticor immunotherapy experimental protocols.

Acknowledgments

This work was supported in part by grants from the Fundação deAmparo a Pesquisa do Estado de Minas Gerais (FAPEMIG; processesCBB-APQ-01384-09 and CBB-APQ 00754‐09). All authors contributedequally to this work. Maia ACRG, Porcino GN, Detoni ML, Emídio NBand Marconato DG were recipients of IC and Doctor Degree fellow-ships from the BIC/UFJF, PROQUALI/UFJF, FAPEMIG and CAPES/REUNI.

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