ORIGINAL PAPER

Cysteine proteinases from promastigotes of Leishmania(Viannia) braziliensis

Karina M. Rebello & Luzia M. C. Côrtes & Bernardo A. S. Pereira &

Bernardo M. O. Pascarelli & Suzana Côrte-Real & Léa C. Finkelstein & Rosa T. Pinho &

Claudia M. d’Avila-Levy & Carlos R. Alves

Received: 3 February 2009 /Accepted: 4 September 2009 /Published online: 24 September 2009# Springer-Verlag 2009

Abstract Leishmania (Viannia) braziliensis is the majorcausative agent of American tegumentary leishmaniasis, adisease that has a wide geographical distribution and is asevere public health problem. The cysteine proteinase B(CPB) from Leishmania spp. represents an importantvirulence factor. In this study, we characterized and localizedcysteine proteinases in L. (V.) braziliensis promastigotes. Bya combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained anenriched fraction of hydrophobic proteins rich in mannoseresidues. This fraction contained two proteinases of 63 and43 kDa, which were recognized by a CPB antiserum, andwere partially sensitive to E-64 in enzymatic assays with the

peptide Glu-Phe-Leu. In confocal microscopy, the CPBhomologues localized in the peripheral region of the parasite.This data together with direct agglutination and flowcytometry assays suggest a surface localization of the CPBhomologues. The incubation of intact promastigotes withphospholipase C reduced the number of CPB-positive cells,while anti-cross-reacting determinant and anti-CPB antiserarecognized two polypeptides (63 and 43 kDa) derived fromphospholipase C treatment, suggesting that some CPBisoforms may be glycosylphosphatidylinositol-anchored.Collectively, our results suggest the presence of CPBhomologues in L. braziliensis surface and highlight the needfor further studies on L. braziliensis cysteine proteinases,which require enrichment methods for enzymatic detection.

Introduction

Leishmaniasis is a severe public health problem, which iscaused by digenetic protozoa parasites that belong to thegenus Leishmania. This disease presents a broad spectrumof clinical manifestation in humans, ranging from self-healing skin lesions to the fatal visceralizing form, dependingon the parasite species and on host immune factors (Desjeux2004). In the parasite life cycle, there are two distinctdevelopmental forms: one motile with a spindle shape, theextracellular promastigotes found within the insect vector;and one nonmotile with ovoid shape, the intracellularamastigotes found in the mammalian host cells (Alexanderand Russel 1992; Bates and Rogers 2004). Leishmania(Viannia) braziliensis is the most important etiological agentof the mucocutaneous form of the disease in the New World.The flagellated parasite causes facial disfigurement with ahigh fatality rate, being considered the most frequent causeof American tegumentary leishmaniasis in Brazil (Rangeland Lainson 2003).

K. M. Rebello : L. M. C. Côrtes : B. A. S. Pereira :C. M. d’Avila-Levy :C. R. Alves (*)Laboratório de Biologia Molecular e Doenças Endêmicas,Instituto Oswaldo Cruz (IOC), FIOCRUZ,Av. Brasil 4365, Manguinhos,21045-900 Rio de Janeiro, RJ, Brazile-mail: calves@ioc.fiocruz.br

B. M. O. PascarelliLaboratório de Patologia, Instituto Oswaldo Cruz (IOC),FIOCRUZ,Rio de Janeiro, RJ, Brazil

S. Côrte-RealLaboratório de Biologia Estrutural, Instituto Oswaldo Cruz (IOC),FIOCRUZ,Rio de Janeiro, RJ, Brazil

L. C. FinkelsteinLaboratório de Imunoparasitologia, Instituto Oswaldo Cruz (IOC),FIOCRUZ,Rio de Janeiro, RJ, Brazil

R. T. PinhoLaboratório de Imunologia Clínica, Instituto Oswaldo Cruz (IOC),FIOCRUZ,Rio de Janeiro, RJ, Brazil

Parasitol Res (2009) 106:95–104DOI 10.1007/s00436-009-1632-5

Many efforts have been done to understand Leishmaniabiochemistry and molecular biology. In this sense, severalparasite molecules have been characterized, as proteinases,which are crucial enzymes for the parasite life cycle, areabundantly expressed by the protozoa, and have beenassociated with several pathogenic processes (Sajid andMcKerrow 2002).

Cysteine proteinases (CPs) belong to one of the fourmajor groups of proteolytic enzymes produced by a varietyof organisms, including Leishmania (Mottram et al. 2004).This parasite possesses at least three classes of Clan CACPs (family C1), denoted as types I, II, and III. Type II CP,or CPA, is a cathepsin L-like proteinase that is encoded bya single copy gene. CPA has been described in Leishmaniamajor, Leishmania (L.) mexicana, and Leishmania infantum(Rafati et al. 2001; Denise et al. 2006; Williams et al.2006). Type III, or CPC, is a cathepsin B-like proteinasereported in L. mexicana, L. major, Leishmania (L.) donovani,and Leishmania chagasi, which is also encoded by a singlecopy gene (Somanna et al. 2002; Mottram et al. 2004).Finally, type I CP, or CPB, is a cathepsin L-like proteinaseencoded by genes that occur in multicopy tandem arrays,both in L. major and L. mexicana (Rafati et al. 2001;Mottram et al. 2004). Furthermore, an unusual 100 aminoacid C-terminal extension that is frequently glycosylated is adistinctive feature of these CPs (Souza et al. 1992).

In spite of the substantial advance in knowledge aboutCPs from the L. (L.) mexicana complex and to a lowerdegree from the L. (L.) donovani complex, less is knownabout these enzymes from L. (V.) braziliensis. Nowadays,there are few reports regarding CPs in L. (V.) braziliensis. Acomplex pattern of CPs, comprised of at least six bandsmigrating in the 20–65-kDa-range in gelatin-SDS-PAGE,has been described in this parasite (Alves et al. 1993). Morerecently, a cpb gene from L. (V.) braziliensis has beencloned and expressed, revealing differences in substrateutilization between L. mexicana and L. braziliensis CPs(Lanfranco et al. 2008). Thus, in the present study, wefurther investigated the CPs from L. (V.) braziliensis byanalyzing, through confocal microscopy, the cellular locationof CPB homologues in the parasites, as well as the mechanismof anchorage to the parasite plasma membrane.

Materials and methods

Chemicals

Bacillus thuringiensis phospholipase C (PLC), bovine serumalbumin (BSA), chromatography columns [concanavalinA-Sepharose 4B (Con A-Sepharose, 10×1.2 cm),diethylaminoethyl-Sephacel (DEAE-Sephacel, 6.0×1.2 cm)],detergents {Tween 20; triton X-100 (TX-100), triton X-114

(TX-114), and 3-[(3-Cholamidopropyl)-dimethylammonium]-1-propanesulfonate (CHAPS)}, fluorescein isothyocianate-labeled goat antirabbit immunoglobulin G (FITC-IgG),horseradish peroxidase-conjugated goat antirabbit IgG(HRP-IgG), molecular mass markers, paraformaldehyde,proteinase substrates [gelatin and pGlu-Phe-Leu-p-nitroanilide (pEFLpNan)], penicillin, proteolytic inhibitor[trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane;(E-64)], p-phenylenediamine, Schneider's insect medium,thiol stimulating agent [dithiothreitol (DTT)], and α-methyl-D-mannopyranoside (α-D-mannose) were purchased fromSigma-Aldrich Chemical Co (St Louis, MO, USA). Fetal calfserum (FCS) was purchased from Cultilab S/A (Brazil).Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) reagents and nitrocellulose membranes werepurchased from BioRad Laboratories Inc. The reagents forchemiluminescence detection were purchased from SantaCruz Biotechnology, Inc (Santa Cruz, CA, USA). TOPRO-3iodide and goat antirabbit immunoglobulin G labeled withAlexa 488 (Alexa 488-IgG) were purchased from MolecularProbes-Invitrogen (Eugene, OR, USA). The anti-cross-reacting determinant (anti-CRD) was provided by Dr.Michael A. J. Ferguson and Dr. Maria Lúcia Guther,University of Dundee, UK. All other reagents were analyticalgrade or superior.

Parasites

Infective promastigotes of L. (V.) braziliensis (MCAN/BR/1998/619) were maintained at 28°C in NNN medium[Novy, McNeal, and Nicolle medium, as described inNicolle (1908)] and subcultured every 4 days, supple-mented with 100 U of penicillin per milliliter. Infectivepromastigotes were obtained from amastigotes isolatedfrom hamster footpad lesions. Promastigote cultures witha density of 5.0×107 cells per milliliter were obtained fromSchneider's insect medium supplemented with 10% FCS.

Triton X-114 extraction and chromatographic procedures

Promastigote forms from stationary-phase (4 days) cultureswere washed three times with phosphate-buffered saline pH7.2 (PBS), and detergent soluble proteins were obtained bythe TX-114 phase separation technique (Bordier 1981).Briefly, 1010 cells were extracted for 40 min on ice withTX-114 2% in TBS (150 mM NaCl, 10 mM Tris, pH 7.4),and soluble proteins were obtained after condensation at37°C, followed by centrifugation at 12,000×g for 15 min.The hydrophobic phase was washed three times with TBSand subjected to a Con A-affinity chromatography procedure.Briefly, the hydrophobic proteins were dialyzed againstequilibrium buffer (20 mM Tris–HCl pH 7.2, 5% glycerol,and 0.5% CHAPS) and then passed through a Con A-

96 Parasitol Res (2009) 106:95–104

Sepharose column previously equilibrated in this buffer. Thecolumn was washed with the equilibrium buffer until nofurther protein was detected, and the retained proteins wereeluted using the same buffer supplemented with 50 mM α-D-mannose. The eluted fraction was concentrated and dialyzedagainst equilibrium buffer by ultra filtration in Centriprep 10filters (Millipore Corporation, Bedforf, USA). Proteins elutedfrom the affinity chromatography were processed by a secondchromatography step on a DEAE-Sephacel column, previ-ously equilibrated. The protein fraction was passed throughthe resin, and unbound proteins were removed by washingwith the equilibrium buffer. The retained proteins were elutedwith a linear gradient from 0 to 0.5MNaCl in the same buffer.Fractions of 1 ml were collected in a flow rate of 0.2 ml/min ina final volume of 120 ml. All chromatography wash andelution steps were accompanied by spectrophotometricmeasurements at 280 nm (Ultrospec 1100 pro; AmershamBiosciences, UK) and were performed in a refrigerated room.Protein concentration was determined by colorimetric assaysas previously described (Lowry et al. 1951), using BSA asstandard.

Electrophoresis assay

SDS-PAGE was performed at room temperature using 12%polyacrylamide gels in Laemmli's buffer (Laemmli 1970).Samples (30 μg) were dissolved in SDS-PAGE samplebuffer (80 mM Tris–HCl pH 6.8, 2% SDS, 12% glycerol,and 0.015% bromophenol blue), supplemented with 5%β-mercaptoethanol, and boiled for 5 min. After electropho-resis, the protein bands were identified by silver staining(Gonçalves et al. 1990).

Zymographic assays

Proteinases were assayed by electrophoresis on 10% SDS-PAGE with 0.1% copolymerized gelatin as substrate(Heussen and Dowdle 1980). Briefly, the gels were loadedper slot with 20 μg of soluble proteins dissolved in SDS-PAGE sample buffer, and following electrophoresis at aconstant voltage of 200 V at 4°C, they were soaked for 1 hat 25°C in 2.5% TX-100. Proteinase activity was detectedby incubating the gels for 16 h at 37°C in 10 mMphosphate buffer pH 7.0, supplemented with 1 mM DTT.Hydrolysis of gelatin was visualized by staining the gelswith 0.2% Coomassie Brilliant Blue R-250 dissolved in40% methanol, 10% acetic acid, and destaining with 10%acetic acid.

Proteinase activity assay using a synthetic substrate

Enzymatic activity of cysteine proteinases was screened,using pEFLpNan as substrate, either in the peak fractions

eluted from DEAE-Sephacel column or in proteins fromthe peak fractions further separated by SDS-PAGE andeluted from the gel. The reaction was initiated by theaddition of each eluted peak sample (50 μl) in 450 μl ofactivation buffer (50 mM sodium phosphate pH 7.0,containing 1 mM DTT and 0.1 mM pEFLpNan). Thereaction was incubated at 37°C for 15 min, andhydrolysis of the peptide substrate was measured at405 nm in a UV 260 recording spectrophotometer(Ultrospec 1100 pro). Alternatively, the eluted peakswere resolved by SDS-PAGE, and the protein bandswere identified after precipitation with 0.3 M ZnCl2solution (Dzandu et al. 1988). Subsequently, gel slices,corresponding to each protein identified, were obtainedwith the aid of a scalpel blade, washed three times (5 minat 4°C) with 50 mM sodium phosphate pH 7.0 containing0.1% CHAPS, and incubated (15 min at 37°C) withactivation buffer into a final volume of 500 µl. For theinhibition tests, the gel slices were previously incubated(5 min at 25°C) with 10 μM E-64. The velocity of thereaction was defined using the formula v=(s−s0)/(t− t0),where v=velocity, (s−s0)=final substrate concentrationsubtracted from the initial substrate concentration, and(t− t0)=final time subtracted from the initial time. Theassays were controlled by verifying the autorelease ofthe chromogenic conjugate at the same time interval. Theenzymatic activity is expressed as micromolar per minuteof pNan and represents the mean value ± standarddeviation of three independent experiments.

Antiserum

Polyclonal antibody (anti-CPB) was obtained after rabbitimmunization against a peptide derived from the COOH-terminal extension from Leishmania (L.) amazonensiscysteine proteinase, as previously reported by Alves et al.2001, 2005.

Agglutination assay

For the agglutination assays, the promastigotes (1.0×106

cells per milliliter) were harvested by centrifugation(1,500×g, 4oC, 5 min), washed three times with PBS,and incubated (45 min at 25°C) with anti-CPB dilutedfrom 1:10 to 1:1,000 in PBS. The assays were performedin 96-well amicroplates in a final volume of 100 μl/well.The agglutination was evaluated by comparison withparasites treated with rabbit preimmune serum observedin a Zeiss Axiovert light inverted microscope. Theimages were digitally recorded using a cooled CCDcamera (Color ViewXS, Analysis GmBH, Germany)and analyzed with AnalySIS system software (AnalySIS,Germany).

Parasitol Res (2009) 106:95–104 97

Confocal microscopy

The parasites (1×107 cells) were harvested by centrifugation(1,500×g, 4oC, 5 min), washed with PBS and fixed with 4%paraformaldehyde in PBS (pH 7.2) for 30 min, and allowedto settle on glass coverslips treated with 0.01% poly-L-lysinefor 30 min. Subsequently, the cells were incubated for 1 hwith a blocking solution (2.5% BSA, 10% FCS, 1%skimmed milk diluted in PBS) to inhibit unspecific labeling.The cells were further incubated with anti-CPB, diluted1:500 in PBS supplemented with 1% BSA, washed withPBS for three times for 5 min each, and incubated for anadditional hour with goat antirabbit immunoglobulin G (IgG)labeled with Alexa 488, diluted 1:750 in a solution of 1 µMTOPRO-3 iodide in PBS. These cells were washed threetimes with PBS, and the coverslips were mounted in slideswith p-phenylenediamine in buffered glycerin. A negativecontrol was prepared by substituting the incubation step withanti-CBP for incubation with rabbit preimmune serum. Thematerial was analyzed and the images captured in a CarlZeiss LSM 510 META confocal microscope.

Parasite treatment with phospholipase C

Promastigotes (1.0×107 cells) were harvested by centrifu-gation (1,500×g, 4°C, 5 min), fixed at 4°C in 0.4%paraformaldehyde in PBS, followed by extensive washingin the same buffer. The parasites were then incubated (2 h at37°C) with 0.1 U/μl of phospholipase (PLC) in PBS(100 μl) supplemented with 25% glycerol. Control cellswere subjected to the same experimental conditions, exceptfor the presence of PLC. The cells maintained theirmorphological integrity as verified by microscopic obser-vation. Supernatants from the reaction mixtures werecollected by centrifugation (8,000×g at 4°C for 2 min),filtered in a 0.22-μm membrane, and used in the immuno-blotting assays (d’Avila-Levy et al. 2005).

Flow cytometry assay

Untreated and PLC-treated promastigotes (1.0×107 cells)were incubated (2 h at 25°C) with anti-CPB (1:1,000) inPBS and then incubated for an additional hour with FITC-IgG (1:200) in PBS. The cells were washed three times inPBS (1,500×g, 4°C, 5 min) and analyzed by flowcytometry in FACSCalibur (BD Bioscience, USA). Non-treated cells and those treated either with the rabbitpreimmune antiserum or the secondary antibody alonewere assayed in parallel as controls. Each experimentalpopulation was then mapped using a two-parameterhistogram of forward-angle light scatter vs side scatter.The mapped population (n=50,000) was then analyzed forlog green fluorescence using a single-parameter histogram.

Immunoblotting

The proteins (20 μg), separated by SDS-PAGE as previouslydescribed, were transferred to 0.2-µm nitrocellulose mem-branes according to Towbin et al. (1979). Nonspecific bindingsites were blocked with 5% skimmed milk in PBS containing0.5% Tween 20 (2 h at 25°C). The blots were washed threetimes with PBS containing 0.05% Tween 20 (PBST) andincubated (1 h at 25°C) with anti-CPB (1:400) or anti-CRD(1:400) in PBST. After six washings with PBST, the blotswere incubated (1 h at 25°C) with HRP-IgG (1:25,000).Finally, the nitrocellulose membranes were washed six timeswith PBST, and the immune complexes were revealed bychemiluminescence.

Results

In the present report, we used a simple strategy forobtaining in L. (V.) braziliensis a fraction rich in hydro-phobic proteinases containing mannose residues. It con-sisted of an association of the TX-114 extraction coupledwith the affinity and anion exchange chromatographies.The hydrophobic proteins, obtained from TX-114 solubili-zation of 1010 promastigotes, were submitted to a Con A-Sepharose affinity chromatography, and the eluted fractionwas subjected to an anion-exchange DEAE-Sephacel column.

The chromatography results indicated that from a total of3.7±0.4 mg of hydrophobic proteins extracted from theinfective strain, about 0.43±0.1 mg (11.62%) bound to aCon A-Sepharose affinity column and were eluted withα-D-mannose. This fraction was applied on a DEAE-Sephacel column, revealing that 95% (0.40±0.03 mg) ofthe proteins remained adsorbed to the solid phase afterthe washing step, indicating a predominance of positivecharges in the proteins, which were recovered only afterelution with a NaCl gradient. The peak that displayed thehighest proteolytic activity in this column was gatheredand further characterized (Fig. 1). SDS-PAGE andgelatin-SDS-PAGE analyses of the chromatographic pro-cedures revealed a considerable enrichment of proteinases(Fig. 2a, b).

The zymographic analysis of the fraction obtained afterthe affinity chromatography revealed three bands ofapproximately 63, 43, and 40 kDa with proteolytic activityat neutral pH (Fig. 2b, lane 1), which were weakly inhibitedby E-64 (data not shown). When analyzing the fractionsfrom the ion-exchange column, we observed only twomajor proteolytic digestion haloes at neutral pH that werecoincident with the major protein bands observed in thispooled fraction (Fig. 2a, b, lane 2). The eluted fraction fromthe ion-exchange column was tested for crossreactivity withan anti-CPB antiserum generated against a COOH-terminal

98 Parasitol Res (2009) 106:95–104

extension from L. (L.) amazonensis cysteine proteinase(Alves et al. 2001, 2005). Our data revealed a strongrecognition of the proteins migrating at 63 and 43 kDa(Fig. 2c). Lysates of L. (L.) amazonensis were stronglyreactive with the anti-CPB antiserum (data not shown). Theproteolytic active fraction from the DEAE-Sephacel columnwas further separated in SDS-PAGE, and the 63- and43-kDa bands were sliced from the gel and assayed withpEFLpNan. Both enzymes were capable of degrading thissubstrate and were partially inhibited by E-64 (64% and53% of inhibition, respectively; Table 1).

Direct agglutination assay of live promastigotes withanti-CPB was performed to identify CPB homologues andto assess if these molecules may be associated with theparasite surface. The anti-CPB antiserum induced cellularagglutination at 1:50 dilution (Fig. 3a), while control cells,incubated with the preimmune rabbit serum showed noagglutination (Fig. 3b). The location of CPB homologueswas also assessed by confocal laser scanning microscopyusing the anti-CPB serum. The reactive protein wasvisualized in the flagellar pocket and on the parasite surface(Fig. 4a, b). No labeling was detected in control preparationsincubated with rabbit preimmune serum (Fig. 4c).

Flow cytometric analysis provided measurements for therelative expression of L. (V.) braziliensis surface CPB-likemolecules (Fig. 5a). The percentage of positively labeledpromastigotes was substantially higher in promastigotesincubated with anti-CPB (73%) than in those incubatedwith rabbit preimmune serum (0.9%; Fig. 5a). Collectively,our results suggest a surface location of the CPB homologuesin this parasite. Since in trypanosomatids, a great number ofsurface proteins are attached to the plasma membrane via aglycosylphosphatidylinositol (GPI)-anchor (Ferguson 1999),we treated the parasites with PLC and then measured thelevels of surface CPB-like molecules. The results showed aconsiderable reduction in the binding of anti-CPB antibodiesin the PLC-treated parasites when compared to the non-treated ones (Fig. 5a). Further evidence that these moleculesare GPI-anchored can be obtained by probing for thepresence of inositol 1,2 cyclic monophosphate epitope onthe released polypeptide with the anti-CRD antibody (Zamzeet al. 1988). Therefore, the supernatant obtained from PLC-treated L. (V.) braziliensis cells was probed with anti-CRD oranti-CPB antibodies, revealing that proteins migrating at 63and 43 kDa were recognized by both antisera (Fig. 5b, lanes1–2). Moreover, our results showed an additional protein

0.7 0.5

0 20 40 60 80 100 1200.0

0.1

0.2

0.3

0.4

0.5

0.6

OD

280

nm

NaC

l (M

)

0

×10-

2µM

/min

of p

Nan

50

100

150

200

0

Fig. 1 Fractioning of hydrophobic proteins from L. (V.) braziliensisby DEAE-Sephacel column. Hydrophobic proteins from a TX-114extraction were previously enriched of mannose containing polypep-tides by a Con A-Sepharose chromatography and then fractioned intoa DEAE-Sephacel column. The retained proteins were eluted with alinear gradient (0 to 0.5 M) of NaCl, and the fractions were collectedin a flow rate of 0.2 ml/min. The elution steps were accompanied bymeasuring proteins at 280 nm (filled circle) and proteinases at 405 nmthrough the hydrolysis of pEFLpNan (empty circle)

6343

29

634340

6343

1 2 1 2 2

a b c

Fig. 2 Analysis of the chromatographic fractions. a SDS-PAGE and bzymography assay in gelatin-SDS-PAGE of proteins obtained after theCon A-affinity (lane 1) and DEAE-anion exchange chromatographies(lane 2); c immunoblotting assay revealed with anti-CPB antiserum ofthe proteins obtained after DEAE-anion exchange chromatography(lane 2). The numbers in the left indicate the apparent molecular mass(in kilodaltons) of the proteins. These results are representative of twoindependent experiments

Table 1 Enzymatic activity of the 63- and 43-kDa immobilized bandsfrom Leishmania (V.) braziliensis

Enzymatic activity (μM/min pNan)

63kDa 43kDa

Control 2.2 ± 0.3 3.0 ± 0.2

E-64 1.03 ± 0.1 1.08 ± 0.1

The proteins obtained after a TX-114 extraction coupled with affinityand anion exchange chromatographies were separated by SDS-PAGE;the 63- and 4-kDa proteins were sliced from the gel, and theenzymatic activity was assayed by the chromogenic substratepEFLpNan (0.1 mM) in 50 mM sodium phosphate pH 7.0, containingDTT (1 mM) in the absence (control) or presence of E-64 (10 μM).The hydrolysis product (pNan) was measured at 405 nm, and theenzymatic activity was expressed in micromolar per minute of pNan.The values represent mean ± standard deviation of three independentexperiments, which were performed in triplicate.

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band, not recognized by the anti-CPB antiserum, migratingat approximately 120 kDa that was recognized by the anti-CRD antibody (Fig. 5b, lane 1). These results suggest thatthe polypeptides migrating at 43, 63, and 120 kDa are GPI-anchored to the cell surface. Control assays performed withthe supernatant from fixed control cells showed no reactionwith the anti-CPB or anti-CRD antisera (Fig. 5b, lane 3).

Discussion

Since leishmaniasis treatments are not always effective andpose serious side effects, finding alternative ways forcontrolling this disease is in demand. In this context, parasiteproteinases represent an interesting chemotherapeutic targetdue to the possibility of selective blockage of key functionsperformed by these molecules in the parasite life cycle and inhost–parasite interactions (Sajid andMcKerrow 2002). Out ofthe four proteinases classes (serine-, cysteine-, aspartic- and

metallo-), CPs have a crucial role in Leishmania spp.survival and pathogenesis (Mottram et al. 2004). AlthoughCPs have been identified and studied for more than 30 yearsin Leishmania spp. (Zuckerman 1975), there is still limitedinformation, especially in L. (V.) braziliensis, where enzymaticevidences of the CPs are scarce. Most of the CPscharacterized in Leishmania spp. appears to be intracellularhydrophilic molecules with strict megasomal localization(Pral et al. 2003; Mottram et al. 2004). Here, we have shownthat an infective strain of L. (V.) braziliensis possessesproteinases with hydrophobic properties, which are rich inmannose residues and seems to be associated with theparasite surface. Additionally, we have shown that enrichmentmethods are crucial for detection of these enzymes, since CPscannot be detected in L. (V.) braziliensis crude extracts(Cuervo et al. 2006).

Proteinase activity in whole promastigotes extract waspreviously investigated in five different strains of L. (V.)braziliensis, and only metalloproteinases were identified(Cuervo et al. 2006). Our group reported the presence ofCPs by zymographic assays only after TX-114 extraction(Alves et al. 1993). The metalloproteinase gp63 is the mostabundant surface protein in Leishmania spp. (Yao et al.2003). It is reasonable to assume that this enzyme impairsthe detection of CPs in L. (V.) braziliensis crude extracts.Conversely, in parasites where CPs are the most abundantenzymes, the detection of metalloproteinases could only beachieved by adding E-64 before parasite protein extraction(Branquinha et al. 1995).

The enzyme described herein possesses catalytic propertiesof CPs, which are characterized by their active site composedof cysteine, histidine, and asparagine, as they were capable ofhydrolyzing the pEFLpNan substrate. This substrate isspecific for proteinases with a thiol group at the active center,such as papain (Carica papaya), ficin (Ficus carica), orbromelin (Ananas comosus; Filippova et al. 1984). Thehydrolysis of substrates containing phenylalanine residues atposition P2 is compatible with the specificity of proteinaseswith a thiol group. Furthermore, our data indicated that theCPs are rich in mannose residues, a characteristic previouslyobserved in CPs from L. mexicana (Robertson and Coombs1990).

The electrophoresis assays, revealed by silver staining,indicated a progressive simplification of the protein profileas samples were subjected to chromatographic procedures.In addition, proteins migrating at 63 and 43 kDa wererecognized by the anti-CPB antiserum, which was raisedagainst CPB from L. amazonensis, a member of L. (L.)mexicana complex. So, it is possible that these proteinsmay be related to CPs from other species of Leishmania,such as the 45-kDa precursor form, the 40-kDa intermedi-ary product, the 27-kDa mature enzyme, and a 15-kDafragment from Leishmania (L.) pifanoi (Duboise et al.

b

a

Fig. 3 Reactivity of anti-CPB antiserum on the surface of intactL. (V.) braziliensis promastigotes. The antiserum reactivity wasevidenced by direct agglutination (a), and control was performedwith rabbit preimmune serum (b). The bar indicates 10 µm

100 Parasitol Res (2009) 106:95–104

1994); the 65-, 56-, 30-, and 27-kDa proteinases fromnoninfective promastigotes of L. (L.) major (Alves et al.1993) and, also, a 43-kDa cysteine proteinase previouslydescribed in other L. (V.) braziliensis strain (Alves et al.1993). Recently, a CPB proteinase was cloned andsequenced in L. (V.) braziliensis, bands migrating at 37,28, 27, and 24 kDa were identified and correspond to the

pro form and to the active mature forms of the enzyme(Lanfranco et al. 2008).

Although the partial inhibition of the 63- and 43-kDaproteinases by E-64 together with their capacity to degradethe pEFLpNan substrate suggest that these proteins may beCPs, the lack of total inhibition by E-64 raises someconcerns. The E-64 reagent is a specific irreversible

control

100 101 102 103 104

0

128

anti-CPB

PLC/anti-CPB

a

150

63

43

1 2 3

b

Fig. 5 Anchoring of CPB from L. (V.) braziliensis to the parasitesurface. Flow cytometric analysis (a) showing the anti-CPB binding tothe cellular surface of promastigotes. Paraformaldehyde-fixed cellswere treated (PLC/anti-CPB) or not (control and anti-CPB) for 2 h at37°C with phospholipase C and subsequently incubated in the absence(control) or in the presence (anti-CPB) of anti-CPB as described in theMaterials and Methods, and then analyzed by flow cytometry. Western

blotting (b) showing the reactivity of polypeptides released by PLCwith anti-CRD (lane 1) or anti-CPB (lane 2) antibodies. Thesupernatant from fixed cells untreated with PLC was probed witheither anti-CPB or anti-CRD for control (lane 3). For simplicity, onlythe lane revealed with anti-CPB is shown, since the result for anti-CRD was similar. The numbers in the left indicate the apparentmolecular mass (in kilodaltons) of the reactive polypeptides

Fig. 4 Confocal scanning imagesof L. (V.) braziliensis.Promastigote forms wereincubated in the presence ofanti-CPB (1:500) andsubsequently in the presence ofAlexa 488-IgG. The punctuallabeling suggests membranelocalization (thin arrow), andthere is also a labeling in theflagellar pocket (thick arrow).a and b Anti-CBP and TOPRO 3labeling, c negative control withrabbit preimmnune serum.d, e, and f Overlay of a, b, andc with differential interferencecontrast microscopy. The scalebar indicates 20 μm(a, c, d, and f) or 10 μm(b and e)

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inhibitor of several CPs, such as cathepsin K, cathepsin B,cathepsin H, actinidin, calpain among others (Lecaille et al.2002). However, some orthologous CPs are slowly inacti-vated, or are insensitive to E-64, such as bromelin (Harrachet al. 1998) and falcipain-1 (Goh et al. 2005), respectively.Thus, it is possible that some CPs expressed by thepromastigote forms of L. (V.) braziliensis may differ fromthe CPs strongly inhibited by E-64.

The composition of the cell surface is of primaryimportance in the cellular response to environmental stimuliand for specific recognition between parasites and hostcells. In protozoa surface, CPs are involved in nutrition orescape from host defense mechanisms. These proteinasescan also modulate macrophage activity by acting directlyon the host cell surface (Olivier et al. 2005). In this context,we investigated if the CPB homologues may have a surfacelocalization in L. (V.) braziliensis promastigotes.

The surface location of the CPB-like molecules in thepromastigote forms of L. (V.) braziliensis was assayed bydirect agglutination assays and confocal microscopy usingthe anti-CPB antiserum. Our data revealed that CPBhomologues can be detected on the surface membrane andalso in the flagellar pocket of this parasite. The wellcharacterized CPBs from the L. (L.) mexicana complex areabundant in the amastigote stage of the life cycle thatoccurs in the mammalian host. In this stage, CPBs arelocated in large lysosomes called megasomes (Pupkis et al.1986), whereas in metacyclic promastigotes (the infectivestage in the sandfly vector), they are thought to be presentin low amounts in an unusual lysosomal compartmentdesignated the multivesicular tubule (Mullins and Bonifacino2001). However, CPB homologues were already described inthe cellular surface of L. amazonensis (Alves et al. 2005; deAraújo Soares et al. 2003). Cellular trafficking of leishmanialCPB in metacyclic promastigotes is unusual, being primarilytargeted to the flagellar pocket (Brooks et al. 2000), withtargeting signals residing in the prodomain. Onward transferfrom the flagellar pocket to the lysosomes appears to requireremoval of the prodomain, presumably with activation of theenzyme (McConville et al. 2002). The localization of CPBon the cellular surface of the parasite is not frequentlydescribed because the known route for the cellular traffic ofthese enzymes does not predict their addressing to theparasite surface. Since CPB homologues were detected by anantibody raised against the C-terminal region of CPB, whichpresumably would be removed in the flagellar pocket, wehypothesize that L. (V.) braziliensis has CPB isoforms withspecial location on the promastigote membrane surface at anunprocessed form.

Generally, the plasma membranes of the divergenteukaryotic parasites, such as Leishmania and Trypanosoma,are highly specialized, with a thick coat of glycoconjugatesand glycoproteins playing a central role in virulence.

Unusually, the majority of these surface macromoleculesare attached to the plasma membrane via a GPI anchor(Ferguson 1999). The composition of this surface coat isexquisitely regulated during the course of leishmaniasisdisease (Ilgoutz and McConville 2001). Through flowcytometry assay, we demonstrated that at least some CPBisoforms are GPI-anchored proteins since the treatment ofpromastigotes with PLC reduces the number of CPBpositive cells. Moreover, the 63 and 45 kDa CPB-likemolecules were released by this treatment, and proteinswith the same molecular mass were recognized by the anti-CRD antibody (Ferguson 1999; d'Avila-Levy et al. 2005).Taken together, these results suggest that these moleculesmay be GPI-anchored to the parasite surface.

The most intensively studied GPI-anchored molecules intrypanosomatids are the variant surface glycoprotein ofbloodstream-form T. brucei (Pays and Nolan 1998) and themetalloprotease gp63 (Frommel et al. 1990), and theglycoconjugates, lipophosphoglycan and the glycoinositolphospholipids (Alexander et al. 1999) from Leishmaniaspp. The surface localization of the CPB homologues andthe suggestion that this enzyme is a peripheral proteincovalently attached to a membrane lipid, such as GPI,provides an alternative to transmembrane domains foranchoring these proteins to the parasite surface, as occursin eukaryotes (Lalanne et al. 2004).

Parasites usually undergo considerable changes duringtheir life cycle. This is of special interest in the case of theparasitic protozoa of the genus Leishmania, which isexposed to different environments within the vertebrateand invertebrate hosts. Our findings may reveal severalimportant biological functions of thiol-containing proteinasesfrom promastigotes that possess activity in neutral pH, whichis in accordance with the pH in the insect vector. In theenvironment of the sandfly gut, a pH near neutrality may befavorable for the catalysis of some substrates necessary to thepromastigotes nutrition. Also, it is known that enzymesthat hydrolyze the pEFLpNan substrate can inhibit thebiosynthesis of proinflammatory prostaglandins (Taussig1980) and reduce clotting efficiency by affecting fibrinogen(Livio et al. 1978). Moreover, our results indicated that theproteinases are capable of hydrolyzing different peptidebonds, since they were capable of degrading substrateshaving multiple (gelatin) or defined peptide bonds(pEFLpNan). The immunological similarities detected inCPs from promastigotes of L. amazonensis and L. (V.)braziliensis could constitute an example of convergentadaptation by which distinct microorganisms of differenttaxonomic groups expose analogous structures that permittheir fitness in a common environment and against acommon selective pressure. Further studies on the role ofthis enzyme in the parasite are necessary in order to betterreveal its precise biological function.

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Acknowledgments This study was supported by the Brazilianagencies: MCT/CNPq (Conselho Nacional de Desenvolvimento Cien-tífico e Tecnológico), FAPERJ (Fundação de Amparo à Pesquisa doEstado do Rio de Janeiro), CAPES (Coordenação de Aperfeiçoamentode Pessoal de Nível Superior), PAPES/CNPq (Programa Estratégicode Apioo à Pesquisa em Saúde/CNPq) and FIOCRUZ (FundaçãoOswaldo Cruz). C.M.D.L and C.R.A. are CNPq research fellows.

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