Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis

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Molecular & Biochemical Parasitology 158 (2008) 52–64

Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis夽 Daniel Acosta a , Mart´ın Cancela a,b , Lucia Piacenza c,d , Leda Roche b , Carlos Carmona a , Jos´e F. Tort b,∗ a Unidad de Biolog´ıa Parasitaria, Instituto de Higiene, Facultad de Ciencias, Uruguay Dpto. Gen´etica, Facultad de Medicina, Universidad de la Rep´ublica, Montevideo, Uruguay c Dpto. de Bioqu´ımica, Facultad de Medicina, Universidad de la Rep´ ublica, Montevideo, Uruguay d Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la Rep´ ublica, Montevideo, Uruguay b

Received 2 October 2007; received in revised form 8 November 2007; accepted 14 November 2007 Available online 22 November 2007

Abstract Leucyl aminopeptidases (LAP) from different parasitic organisms are attracting attention as relevant players in parasite biology, and consequently being considered as candidates for drug and vaccine design. In fact, the highest protection level achieved in ruminant immunization by a native antigen was previously reported by us, using a purified LAP as immunogen in a sheep trial against fasciolosis. Here, we report the cloning of a full-length cDNA from adult F. hepatica encoding a member of the M17 family of LAP (FhLAP) and functional expression and characterization of the corresponding enzyme. FhLAP was closely related to Schistosoma LAPs, but interestingly distant from their mammalian host’s homologues, and was expressed in all stages of the parasite life cycle. The recombinant enzyme, functionally expressed in Escherichia coli, showed a marked amidolytic preference against the synthetic aminopeptidase substrate l-leucine-7-amino-4-methylcoumarin (Leu-AMC) and was also active against Cys-AMC and Met-AMC. Both native and recombinant enzyme were stimulated by the addition of divalent cations predominantly Mn2+ , and strongly inhibited by bestatin and cysteine. Physico-chemical properties, localization by immunoelectron microscopy, MALDI-TOF analysis, and cross-reactivity of anti-rFhLAP immune serum demonstrated that the recombinant enzyme was identical to the previously purified gut-associated LAP from adult F. hepatica. Vaccination trials using rFhLAP for rabbit immunization showed a strong IgG response and a highly significant level of protection after experimental infection with F. hepatica metacercariae, confirming that FhLAP is a relevant candidate for vaccine development. © 2007 Elsevier B.V. All rights reserved. Keywords: Leucine aminopeptidase; Metalloprotease; Fasciola hepatica; Trematode; Parasite; Vaccine

1. Introduction

Abbreviations: LAP, leucine aminopeptidase; NEJ, newly excysted juveniles; RACE, rapid amplification of cDNA ends reaction; Trx, thioredoxin; IPTG, isopropyl thio-␣-d-galactoside; Ni-NTA, nickel-nitrilo-tri-acetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DOC, deoxycholic acid; Leu-AMC, l-leucine-7-amino-4-methylcoumarin; ORF, open reading frame; RT, reverse transcription; GSP, gene-specific primer; BSA, bovine serum albumin; GTA, glutaraldehyde. 夽 Note: Nucleotide sequence has been deposited in the GenBank Database under accession number AY64459. ∗ Corresponding author at: Dpto. Gen´ etica, Facultad de Medicina, Universidad de la Rep´ublica, Gral.Flores 2125, CP 11800 Montevideo, Uruguay. Tel.: +598 2 924 95 62; fax: +598 2 924 95 63. E-mail address: [email protected] (J.F. Tort). 0166-6851/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2007.11.011

Proteolysis is a central theme of parasite biology since it has been plausibly demonstrated that peptidases accomplish tasks associated critically with successful parasitism such as invasion, migration, acquisition of nutrients and evasion of inflammatory and immune responses. Although endopeptidases have been extensively studied due to their prominent participation in these processes, exopeptidases have been largely neglected in most parasite models so far [1,2]. Nonetheless, there is a rising interest in aminopeptidases, the exopeptidases that catalyze the removal of amino acids from the unblocked N-termini of peptides and proteins. These peptidases are widely distributed and have been found in many tissues and cells, as either membrane associated or soluble forms. In addition to their role in post-proteasome steps of intracellular proteolysis, aminopeptidases participate

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in specific functions such as antigen trimming for MHC-I presentation, activation or inactivation of biopeptides, removal of N-terminal methionine, and regulation of signal transduction pathways [3–6]. Accumulated evidence from parasitic protozoa and helminths points to a crucial role of leucyl aminopeptidases (LAPs) in parasite biology. Leucyl aminopeptidases members of the M1 or M17 peptidase families constitute a group of diverse and ubiquitous Zn-dependent metallopeptidases, which catalyze the removal of leucine residues from proteins and peptides. LAPs present broader amidolytic activity beyond Leu hydrolysis and participate in either processing/maturation/activation or degradation of substrates [7,8]. The homo hexameric M17 LAPs are being revealed as novel drug targets and vaccine candidates against parasitic infestation. In the intraerythrocytic Plasmodium falciparum malaria parasites, PfLAP participates in the latter stages of haemoglobin catabolism, generating and regulating the pool of free amino acids needed for protein synthesis and osmotic stability maintenance [9–11]. Moreover, parasites overexpressing PfLAP become significantly less susceptible to the broad range aminopeptidase inhibitor bestatin confirming PfLAP as the main target for its anti-malarial effect [11,12]. Accordingly, synthetic broad-spectrum aminopeptidase inhibitors and 1,2-aminoalcohol inhibitors have shown promising anti-Plasmodium activity [11,13]. Furthermore, the aminopeptidase inhibitor arphamenine A has shown activity against the kinetoplastid parasites Trypanosoma brucei and Leishmania species [14,15]. In helminths, LAPs have been poorly characterized although there is evidence that support their participation in vital processes in the vermin life cycle. In the trematode Schistosoma mansoni the enzyme has been localized at the gut, tegument and eggs. Interestingly, the leucyl aminopeptidase inhibitor bestatin is capable of inhibiting miracidial hatching [16–18]. In the gastrointestinal nematode Haemonchus contortus, LAP has been associated to cuticle molting and egg hatching, as both these processes parallel LAP activity, were inhibited by bestatin, and incremented by Zn2+ addition to culture medium [16,19,20]. On the other hand, LAP null mutants of the free-living nematode Caenorhabditis elegans had a slower growth rate and delayed onset of egg-laying, related to its role as digestive enzyme [21]. Fascioliasis is an important freshwater snail-borne helminthiasis caused by the trematode parasites Fasciola hepatica and Fasciola gigantica that produces a chronic liver infection of cattle and sheep, inflicting substantial productive loses on affected animals. While F. hepatica has been recorded in all continents, F. gigantica is restricted to Africa and Asia. In addition, human fascioliasis caused by F. hepatica has been recently recognized as an emerging/re-emerging zoonotic disease in many countries with an estimated prevalence of up to 17 million people infected and 180 million at risk of infection worldwide [22]. Since their initial characterization by Dalton and Heffernan [23], gut-associated cysteine endopeptidases released by adult F. hepatica, later demonstrated to be cathepsin L-like cysteine proteinases and termed FhCL1 and FhCL2, have attracted considerable attention due to their predominant presence in excretory/secretory products of the mammalian stages (revised in [24]). Moreover,

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their proteolytic activity on natural polypeptide targets shown in vitro [25,26] has led to the consideration of CLs as vaccine candidates and different trials have evaluated their potential either as native or recombinant proteins [27,28]. To further explore F. hepatica digestive enzymes, we characterized a leucine aminopeptidase (LAP, E.C. 3.4.11.1), FhLAP, from the detergent soluble extract of adult worms, based on its activity against different amino acids coupled to 7-amido-4methylcoumarin (AMC) [29]. This metalloenzyme was purified using a combination of gel filtration and bestatin-affinity chromatography. Histochemistry analysis revealed the presence of FhLAP preferentially localized inside the epithelial cells that line the alimentary tract of the adult worm, hence, a participation in the last stages of host protein digestion was proposed. FhLAP showed broad amidolytic activity against fluorogenic substrates at optimum pH 8.0 and its activity was enhanced by the divalent metal cations Zn2+ , Mn2+ and Mg2+ . When used as immunogen, either alone or in combination with native CL1 and CL2, very high level of protection (89 and 76%, respectively) were obtained against challenge infection in ovine fasciolosis [28]. These results, subsequently confirmed in another vaccine trial (unpublished results), represent the highest protection level achieved in ruminants by a natural antigen, therefore positioning FhLAP as one of the leading vaccine candidates in ruminant fasciolosis [30]. In this work we describe the production of a functionally active recombinant hexameric leucine aminopeptidase form (rFhLAP) from F. hepatica, and characterize its expression during the life cycle of the parasite, the biochemical properties, and its localization at the gut epithelial cells of adult liver flukes. Furthermore, we tested the immunogenic potential of the recombinant enzyme in rabbits, showing strong antibody responses and significant protection against experimental infection. These results collectively stress the value of FhLAP as a candidate for vaccination against ruminant fascioliosis. 2. Materials and methods 2.1. Parasites and bacterial strains Mature F. hepatica flukes were obtained from the bile ducts of infected cattle at a local abattoir. Miracidia were obtained from hatching eggs after 15 days incubation at 26 ◦ C. Metacercariae were collected from infected Lymnaea viatrix host snail culture, and newly excysted juveniles (NEJ) were obtained as described elsewhere [31]. Escherichia coli One ShotTM TOP 10 (Invitrogen) was used as a host strain for cloning purposes. 2.2. RNA isolation and cDNA synthesis Total RNA from adult flukes was prepared using the method described by Chomcynsky and Sacchi [32]. Total RNA from miracidia, metacercariae and NEJs were obtained using a micromidi kit (Invitrogen). For cDNA synthesis, 5 ␮g of total RNA from adult or 1 ␮g of RNA from other stages was reversed transcribed using Superscript II kit (Invitrogen) according to the manufacturer’s instructions.

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2.3. Cloning of the FhLAP Based on published cDNAs sequences (GenBank accession numbers U50151: Lycopersicon esculentum; X63444: Arabidopsis thaliana; X15130: E. coli and X77015: Solanum tuberosum), degenerated primers were synthesized against conserved metal binding VGKG, and active site NTDAEGRL regions. The primer sequences were LAPFWD: 5 -TKG TYG GIA ARG GIR TYA YIT WTG AYA-3, and LAPREV: 5 -MCC YTC RGC ATC NGT RTT-3 . By PCR with these primers using Platinum Taq DNA Polymerase (Invitrogen) and adult fluke cDNA as a template a 280 bp fragment was generated.. Cycling conditions were: 94 ◦ C for 1 min; 94 ◦ C for 30 s; 50 ◦ C for 1 min; 72 ◦ C for 2 min; 25 cycles, followed by a 7 min extension at 72 ◦ C. PCR products were gel purified with a QuiaEx extraction kit (Qiagen), sub-cloned in pCR4−TOPOTM (Invitrogen), and sequenced by cycle sequencing with Dye Terminators in a ABI 377 DNA Sequencer (Applied Biosystems). New genespecific primers (GSP) were synthesized based on the sequence obtained (GSP1REV 5 -CCA CAT ACG AAT CTG CAC CAA CAC T-3 , and GSP2FWD 5 -AAG GCG AAC GGA GTG ATG GCT GGA ATG C-3 ), and used in rapid amplification of cDNA ends (RACE)-PCR. For the isolation of the 5 end region, the cDNA was treated with Rnase H, deoxyCytidine-tailed with terminal deoxynucleotidyl-transferase (TdT, Invitrogen), and the cDNA extended at the 5 end in a PCR reaction with GSP1 reverse primer and oligo (dG)12 primers. The 3 -RACE reaction was performed with GSP2 forward primer and oligodT primers. Cycling conditions for both cases were: 94 ◦ C for 1 min; 94 ◦ C for 30 s; 60 ◦ C for 1 min; 72 ◦ C for 2 min; 35 cycles, followed by a 10 min extension at 72 ◦ C. Products of 1.2 kb (5 -RACE) and 0.7 kb (3 -RACE) were obtained, subcloned and sequenced as described above. In order to generate a full-length construct, agarose gel extracted RACE 5 and RACE 3 aliquots were mixed in a 100 ␮l PCR reaction containing no primers, and allowed to extend from the overlapping region. After five cycles, new primers designed from both ends of the FhLAP were added and allowed to amplify for further 25 cycles. These primers included BamHI and SalI (underlined) restriction sites, respectively, to facilitate subcloning in expression vectors. (LAPB(am)FWD 5 -GGG GAT CCG CAA TGG CGG CGT TG-3 and LAPS(al)REV 5 -ACA CGT GTC GAC TGC CCT ATT TGA A-3 ). The expected full-length cDNA product was obtained, subcloned and sequenced as described before. 2.4. Sequence analysis of FhLAP open reading frame Trafficking signatures were predicted by PSORT II [33] or Target P [34] servers. Secondary structure predictions were made using NNPredict [35] and PredictProtein [36] servers. FhLAP nucleotide sequence was used as a query for BLAST searches of databases at the NCBI and EBI servers, and relevant matches retrieved. Sequence alignments were generated with ClustalX package [37]. Secondary structure masks were generated for the alignments based on the structural superposition of E. coli aminopeptidase A (pepA, pdb code 1GYT [38]) and bovine lens

LAP (1 LAP [39]). The alignments were manually corrected using GeneDoc [40]. Conserved regions were used to generate neighbor joining unrooted trees. 2.5. Expression and purification of soluble recombinant FhLAP The full-length cDNA product was cloned in frame in BamHI and BglII sites of linearized pThio HisC E. coli expression vector (Invitrogen) fused downstream of the E. coli thioredoxin (Trx) gene, and transformed into E. coli Top 10 cells. One liter culture of transformed E. coli cells was grown at 37 ◦ C to an OD600 of 0.4, then switched to 28 ◦ C, induced with Isopropyl thio-␤-d-galactoside (IPTG) (1.0 mM final concentration), and harvested 3 h later by centrifugation for 15 min at 4000 × g at 4 ◦ C. The pellet was resuspended in 100 ml of 0.05 M Tris–HCl buffer (pH 8.5) containing 0.1 M NaCl and 0.005 M Imidazole. Cells were sonicated for 1 min with 30 s burst cycles and after three cycles of freeze-thawing at 37 ◦ C, the lysate was centrifuged at 9000 × g for 30 min at 4 ◦ C, and applied to a ProBondTM (Invitrogen), nickel-nitrilo tri-acetic acid (Ni-NTA) charged agarose column equilibrated in 0.05 M Tris–HCl buffer (pH 8.5) containing 0.1 M NaCl. Bound proteins were washed with equilibration buffer and stepwise elution was carried out in the same buffer containing 0.01, 0.02 and 0.05 M imidazole. LAP activity was monitored with l-Leu coupled to 7-amino4-methylcoumarin (AMC). In some instances, the Trx tag was removed by cleavage with enterokinase (Sigma). Sample purity was evaluated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels stained with Coomassie Blue. 2.6. Purification of the native FhLAP (nFhLAP) Native FhLAP was purified according to a previously described protocol [29]. Briefly, washed adults flukes were incubated in a solution consisting of 10 ml of 1% deoxycholic acid (DOC) in 0.15 M glycine buffer (pH 9.0) 0.5 M NaCl for 60 min at room temperature, 30 min at 37 ◦ C, and then 30 min at 4 ◦ C. The material was centrifuged at 20,000 × g for 60 min, and the supernatant applied onto Superdex 200 HR 16/50 column (Pharmacia) equilibrated with 0.05 M Tris–HCl, pH 8.5. Samples were eluted with the same buffer, and fractions containing activity against Leu-AMC were applied to bestatin-affinity column equilibrated in Tris–HCl pH 8.5. The column was washed with 10 bed volumes of the equilibration buffer at a flow rate of 0.3 ml/min. Stepwise elution was carried out with 0.05 M Tris–HCl buffer, pH 8.5, containing 0.1, 0.25, 0.5 and 1.0 M NaCl. Fractions exhibiting amidolytic activity were pooled, dialyzed against 0.1 M glycine pH 8.0, 0.02% sodium azide, and stored at 4 ◦ C until used. 2.7. Enzymatic analysis Substrate specificity was determined using different amino acids coupled to AMC. Assays were carried out in 96-well black plates using a final volume of 200 ␮l in glycine buffer 0.1 M pH

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8.5 containing 1 mM MnCl2 in the presence of the fluorogenic substrates (50 ␮M). The amount of AMC released was measured in a fluorescence microplate reader (Fluostar Galaxy) at λex = 360 nm and λem = 430 nm. One unit of enzyme activity was defined as the amount that catalyzes the release of 1 nmol of AMC per minute at 37 ◦ C. The KM and Vmax for rFhLAP were estimated using substrate concentrations ranging from 5 to 800 ␮M and data analyzed by the Hanes-Woolf Plot ([S]/v vs. [S]). Activity was measured under the same conditions as described above and expressed as the mean of three different experiments. The kcat was determined from kcat = Vmax /[E]0 where [E]0 represents the concentration of rFhLAP monomer (by mass, assuming 100% activity). The [E]0 for rFhLAP was determined using a molecular mass of 68,102 kDa which takes into account the added mass of the Trx tag. The effects of different peptidase inhibitors were assayed by pre-incubating the enzyme (2 ␮g) with different concentrations of the inhibitors for 10 min at 37 ◦ C. After the incubation period remaining enzyme activity was assayed as above using l-Leu-AMC (50 ␮M) as substrate. Results are expressed as % of or relative activity respect to control conditions taken as 100%. The Ki values were determined for bestatin and Cys using l-Leu-AMC (0–200 ␮M) as substrate in the presence of fixed inhibitor concentrations (0–100 nM). Data obtained from the reciprocal plot (slope at different inhibitor concentrations) were replotted vs. the inhibitor concentration. The rFhLAP pH profile was assayed in 0.1 M glycine buffer ranging from pH 5.0 to 10.0. The effect of ionic strength was measured with increasing concentrations of NaCl (0–2 M) in glycine buffer at pH 8.5. The thermal stability of rFhLAP was measured by incubating the enzyme during 10 min at the respective reaction temperature and then placed on ice prior to the enzymatic assay as described above. The influence of different metals ions was determined by assaying rFhLAP activity after pre-incubation with various concentrations of divalent cations added as chloride or sulphate salts. 2.8. Preparation of polyclonal antiserum and immunoblot analysis Polyclonal antiserum to nFhLAP was obtained by immunizing a Corriedale sheep twice, at 4-week intervals (100 ␮g). For the production of anti-rFhLAP antiserum, a New Zealand rabbit was immunized subcutaneously four times at 3-week intervals, with purified rFhLAP (50 ␮g). Both proteins were formulated in Freund’s Complete or Incomplete adjuvant. Serum was obtained 10 days after the final immunization. For immunoblotting purified native and recombinant enzymes were electrophoretically separated by reducing 12.5% SDS-PAGE gels and transferred onto nitrocellulose membranes (BioRad) with a standard protocol. The membranes were probed with sheep anti-nFhLAP (1:2000) or rabbit anti-rFhLAP (1:5000). The appropriate HRP-conjugated secondary antibodies produced in goat (Sigma) were used and detection was carried out with 4-chloro-1-naphthol and H2 O2 as substrates. Protein concentration was measured using the BCA protein assay kit (Pierce).

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2.9. Mass spectrometry Native and recombinant FhLAPs were analysed by mass spectrometry in a Voyager DE-PRO MALDI-TOF equipment (Applied Biosystems) at the Unit of Analytical Biochemistry (IIBCE/Faculty of Sciences, Montevideo, Uruguay). Procedures followed to prepare and measure tryptic digests for peptide mass mapping from protein gel band samples were essentially as previously described [41]. Peptide masses of tryptic peptides obtained from the nFhLAP were compared to theoretical masses of a virtual digestion of the expected protein sequence deduced from DNA sequence of the cloned gene (0.1 Da accuracy). 2.10. Immunoelectron microscopy F. hepatica adult worms were rinsed in PBS, allowed to release their gut contents, and fixed for 1 h in 2% double-distilled glutaraldehyde (GTA) in 0.1 M sodium cacodylate buffer (pH 7.2) containing 3% sucrose at 4 ◦ C. Following thorough washing in buffer, specimens were dehydrated through graded ethanol propylene oxide, infiltrated and embedded in LRWhite resin. Ultrathin sections (40–70 nm) were cut on a RMC MT-X ultramicrotome, collected on 200-mesh nickel grids and dried at room temperature. For immunogold labeling, sections were etched with 10% hydrogen peroxide diluted in H2 O for 5 min and rinsed with 20 mM Tris–HCl buffer (pH 8.2) containing 0.1% bovine serum albumin (BSA) and Tween 20 (1:40 dilution). Grids were incubated in normal rabbit serum (1:20 dilution) for 20 min and then transferred to primary antibody diluted to 1:10.000 with 0.1% BSA/Tris–HCl buffer for 12–18 h. Grids were then washed in BSA/Tris–HCl and transferred to a 20 ␮l drop of goat antirabbit IgG conjugated to 10 nm gold particles (Sigma) for 1 h. Following another buffer wash, grids were lightly fixed with 2% double-distilled GTA for 3 min, and double stained with uranyl acetate (5 min) and lead citrate (3 min), examined in a JEM 1010 electron microscopy (JEOL), operating at 80 kV. Images were captured with a digital camera (4742-95 model, Hamamatsu Photogenics). To verify the specificity of labeling, controls grids in which the primary antibody was omitted were included. 2.11. Immunization protocol Fourteen Californian rabbits (average weight 1.5 kg) were separated into two groups and housed individually. All vaccines were prepared by mixing 100 ␮g of purified recombinant protein in Freund’s Complete Adjuvant for the first immunization (priming; week 0) or Freund’s incomplete adjuvant for the second immunization (booster; week 4) and administered intramuscularly. The control group received PBS with the corresponding adjuvant. Six weeks after the immunization (week 6), the animals were orally challenged with a gelatin capsule containing 50 metacercariae. Blood was collected from all animals prior to the first immunization and weekly until the time of necropsy. The serum was obtained and then stored at −80 ◦ C. During the trial, one animal from each group died. Rabbits were humanely

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slaughtered 20 weeks after the first immunization. Flukes in the main bile ducts and gall bladder were removed. The liver was then cut into 1-cm-thick pieces, which were subsequently soaked in water at 37 ◦ C for 30 min, squeezed, and forced through a 300␮m-mesh sieve; the retained material was analyzed for immature or damaged flukes. For comparison between the two groups the Student’s t test was performed. A p value <0.05 was considered significant.

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Antibody responses were analyzed by ELISA. The wells of polystyrene microplates (Nunc, Roskilde, Denmark) were coated with a 2 ␮g/ml solution of purified rFhLAP in PBS and incubated overnight at 4 ◦ C. The plates were blocked with 5% dry milk in PBS for 1 h at 37 ◦ C, and washed three times in washing solution (0.05% Tween 20 in PBS). Sera were diluted 1:200 in dilution buffer (2.5% dry milk and 0.05% Tween 20 in PBS) and added to the wells (100 ␮l per well). After a 1-h incubation at 37 ◦ C, the plates were washed as described above, and bound antibody was detected with HRP-conjugated anti-rabbit IgG (Sigma) (1:7000 in dilution buffer) with the substrates o-phenylenediamine and H2 O2 (Sigma). The enzyme reaction was stopped after 15 min by addition of 10% HCl (50 ␮l per well) and the optical densities at 492 nm (OD492 ) were determined. Antibody concentrations, expressed in terms of arbitrary units and OD492 values, corresponding to dilutions of the reference serum were correlated by linear-regression analysis. ELISA data (OD492 values) for every sample were converted to antibody concentrations equivalent to this reference for analytical consistency.

of 1569 base pairs predicted to encode a 523 amino acids residue polypeptide with a calculated molecular mass of 56,401 Da, and a predicted pI of 7.26. The stop codon is followed by 156 bp 3 UTR region. No intracellular or transmembrane trafficking signatures were predicted by the PSORT II [33] or Target P [34] servers, indicating a cytoplasmic localization. No potential glycosylation or phosphorylation sites were found for this sequence. A comparison of the deduced amino acid sequence of FhLAP cDNA insert with sequences in GenBank data bases indicated that encoded a protein with characteristic features of members of the MEROPS M17 family of cytosolic leucine aminopeptidases [42]. The enzyme is more closely related to LAP enzymes isolated from S. mansoni and Schistosoma japonicum with overall identities of 63 and 65%, and similarities of 76 and 79%, respectively. The carboxy-terminal domain (residues 184–512) matches the InterPro IPR000819 domain shared by all M17 aminopeptidases, with 75% identity and 88% similarity to the more closely related members of the family. The more conserved regions are those containing the metal coordinating residues (Lys269, Asp274, Asp293, Asp352, Glu354) and the positively charged Lys281 and Arg356 residues in the active site. However, the N-terminal portion of FhLAP (amino acids 1–110) does not match to IPR008283 M17 N-terminal domain, although it is predicted to have the characteristic five-stranded beta sheet surrounded by four alpha helices (Fig. 1A). A phylogenetic comparison with homologous enzymes from different species demonstrate that the all metazoan LAPs constitute a well defined group diverse from similar enzymes from bacteria, plants and unicellular eukaryotes. FhLAP and other flatworm orthologs constitute a well-defined cluster distant to the vertebrate enzymes (Fig. 1B).

3. Results

3.2. Expression of FhLAP in during the life cycle

3.1. Isolation of full-length cDNA encoding FhLAP

cDNA obtained from miracidia, metacercariae, NEJs and adult flukes was assayed in PCR reactions using GSP primers. An amplification product of the expected 170 bp size was found in all the life cycle stages examined (Fig. 2).

2.12. Antibody responses analysis

Using degenerate PCR primers designed on the conserved active site regions of M17 LAPs, a 280 bp fragment was obtained from adult fluke cDNA. Specific primers generated from this fragment were used to generate overlapping 5 and 3 fragments by RACE from adult fluke cDNA, and the full-length clone constructed by extending the overlapped products. The full-length cDNA had a short 5 untraslated region (UTR) (18 bp) that was followed by a large open reading frame (ORF)

3.3. Expression and purification of soluble recombinant F. hepatica LAP (rFhLAP) The coding region corresponding to FhLAP was subcloned downstream of the E. coli Trx gene, in the pThio-HisC E. coli

Fig. 1. Phylogenetic relationships of Fasciola hepatica LAP protein. (A) Alignment of leucyl aminopeptidase amino acid sequences. Conserved residues are indicated in gray. Conserved metal coordinating and active site residues are indicated by black and white arrowheads, respectively. Secondary structures (either crystallographic or deduced) are indicated by arrows: beta strands or circles: alpha helixes. The conserved carboxy-terminal domain is indicated by a line under the secondary structures. (B) Neighbor joining unrooted tree of the conserved C-terminal domain of LAPs. Sequence names indicate species, Uniprot or Ensembl accessions and common names. Species included are Escherichia coli (Uniprot accession P11648), Arabidopsis thaliana (Q8RX72, P30184 and Q944P7), Lycopersicum esculetum (tomato, Q42876, and Q10712), Solanum tuberosum (potato, P31427), Plasmodium falciparum (Q8IL11), Plasmodium yoelii (Q7RNC2), Leishmania donovani (Q95V75), Leishmania amazonensis (Q95V76), Leishmania major (Q9U0Z6), Schizosaccharomyces pombe (YA55 SCHPO), Encephalitozoon cuniculi (Q8SQZ7), F. hepatica (AY064459), Schistosoma mansoni (P91803), Schistosoma japonicum (Q9GQ37), Haemonchus contortus (HCC01102), Ascaris suum (ASC01167), Caenorhabditis elegans (P34629-LAP1 and Q27245-LAP2), Anopheles gambiae (Q7QLA3, Q7Q3D0 and Q7QFS4), Drosophila melanogaster (Q9V7Q5, Q9V6S5, Q9V7Q8, Q961W5, Q9V6T4, Q9VSM6, Q9VFQ9 and Q9VG16), Fugu rubipes (Fugu fish, Ensembl accessions SINFRUP00000144215, SINFRUP00000160378 and SINFRUP00000129217), Danio renio (zebrafish, ENSDARP00000019981, ENSDARP00000016686, Q6NWE8), Mus musculus (mouse, Q6NSR8 and Q99P44), Bos taurus (cattle, P00727), Homo sapiens (Q9HAI5 and Q6P0L6).

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enzyme with immunized sheep anti-nFhLAP sera, resulted in several peptides that fully coincide with the sequence obtained for the rFhLAP (data not shown). In addition, rabbit antibodies generated against the recombinant protein, and anti-nFhLAP sheep antibodies, both recognized rFhLAP (Fig. 3B). Transmission immunoelectron microscopy confirmed its localization at the cytosol of the intestinal epithelial cells. Interestingly, a strong reactivity was detected at the apical lamella (Fig. 4). Fig. 2. Analysis of LAP expression in different stages of F. hepatica. cDNAs from adult (AD), miracidium (MI), newly excysted juvenile (NEJ) and metacercariae (MC) were obtained as described in Section 2 and used as template for PCR reactions using GSP2 forward and GSP1 reverse primers. The 170 bp LAP fragment obtained is indicated. The faster migrating bands correspond to primer adducts.

expression vector, and rFhLAP was purified from induced culture supernatants by a single step affinity chromatography on Ni-NTA agarose with a yield of approximately 3.5 mg/l of culture supernatant. The purified protein was resolved as a single band with apparent molecular mass of 67 kDa on SDS-PAGE (Fig. 3A). This value is in good agreement with the value estimated from the deduced amino acid sequence of the rFhLAP plus the 11.7 kDa of E. coli Trx which contain His tag. Positive rFhLAP protein identification was done by MALDITOF mass spectrometry, with eleven masses assigned to expected peptides covering 32% of the sequence and comprising 166 amino acids out of 523. On the other hand, trypsin digestion of the main immunoreactive bands detected in the purified native

3.4. Biochemical characterization of rFhLAP Purified recombinant and native FhLAPs were tested for enzymatic activity using different synthetic fluorogenic substrates. As shown in Table 1 the pattern of amidolytic activity is very similar, being Leu, Cys, Arg, Thr and Met the preferred substrates. The highest amidolytic activity in both cases was against l-Leu-AMC being 1.6-fold higher for rFhLAP. The high affinity displayed by rFhLAP for l-Leu-AMC, lCys-AMC and l-Met is reflected in both the low KM and high Kcat resulting in an overall catalytic efficiency (Kcat /KM ) of 6745, 3740 and 3045 M−1 s−1 , respectively. Substrates lArg-AMC, l-Ala-AMC, l-Phe-AMC and l-Thr-AMC were hydrolyzed considerable less efficiently with values of Kcat /KM below 1000 M−1 s−1 (Table 2). The inhibitor profile of rFhLAP is shown in Table 3. The reducing agent DTT (5 mM) and the chelating agent 1-10-ophenantroline (1 mM) inhibited rFhLAP activity by more than

Fig. 3. Recombinant expression of FhLAP. (A) Purification of the rFhLAP-thioredoxin fusion protein. The soluble E. coli cell fraction was centrifuged, sonicated as described in Section 2 and loaded onto a ProBond, nickel-charged agarose column (Ni-NTA) equilibrated in 0.05 M Tris–HCl buffer (pH 8.5) containing 0.1 M NaCl. Bound proteins were washed out with equilibration buffer and the stepwise elution was carried out in the same buffer containing 0.01, 0.02 and 0.05 M imidazole. Fractions were analyzed by 10% SDS-PAGE under reducing conditions: Non induced soluble E. coli cell fraction (lane 1), soluble E. coli cell fraction after 3 h of IPTG induction (lane 2), stepwise elution 0.01 M (lane 3), 0.02 M (lane 4) and 0.05 M imidazole (lane 5). The 68.1 kDa fusion protein is indicated by the arrow. (B) Western blot analysis of IgG response against FheLAP in sheep and rabbit. nFhLAP (lanes 1 and 2) and rFhLAP (lanes 3 and 4) separated by SDS-PAGE (10%) under reducing conditions and electrotransferred onto nitrocellulose filters, were probed with sheep anti-nFhLAP antibodies (lanes 1 and 3) or rabbit anti-rFhLAP antibodies (lanes 2 and 4).

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Fig. 4. Detection of FhLAP in adult F. hepatica gut by immunoelectron microscopy. (A) Localization of FhLAP in the intestinal secretory cell. The characteristic secretory vesicles and microvilli forming the surface lamellae of the intestinal secretory cells of F. hepatica are observed. (B) Localization of FhLAP at the microvilli and cytoplasm of the epithelial cell (C) Control without primary antibody in a similar view as B. Scale bar A: 2 ␮m, B and C: 500 nm. Table 1 Hydrolytic activity towards aminoacyl-AMC substrates by nFhLAP and rFhLAP Substrate

nFhLAPa

l-Leu-AMC l-Cys-AMC l-Arg-AMC l-Thr-AMC l-Met-AMC l-Phe-AMC l-Ala-AMC l-Ile-AMC l-Tyr-AMC l-Pro-AMC l-Ser-AMC l-Gly-AMC l-Val-AMC l-Glu-AMC l-Asp-AMC

134.4 95.3 75.2 69.4 65.7 14.1 8.3 9.3 5.4 3.2 1.9 0.5 0.6 0.2 0.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

rFhLAPa

0.5 0.8 0.3 0.5 1.0 0.3 0.1 0.2 0.1 0.2 0.2 0.1 0.1 0.0 0.01

214.5 83.1 29.1 35.5 25.3 6.8 7.5 6.2 6.4 5.6 2.1 1.9 2.5 0.1 1.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.5 1.2 0.8 1.1 0.5 0.2 0.1 0.3 0.5 0.1 0.1 0.1 0.3 0.0 0.2

Activity expressed as nmol min−1 mg−1 . Data reflect the mean relative activity ± S.D. (n = 3). a

80%. The inhibition observed with PMSF the aspartic protease inhibitor pepstatin, the serine proteases inhibitors leupeptin and TLCK was about 60%, while aprotinin A and E-64 decreased the enzymatic activity by 50%. The strong inhibitory effect exerted by Cys on the native enzyme [29] confirmed here for the recombinant FhLAP, prompt us to investigate its Ki and compare it

Table 2 Kinetic parameters of rFhLAP on different aminoacyl-AMC substrates Substrate

Kcat × 103 s−1

l-Leu-AMC l-Cys-AMC l-Met-AMC l-Ala-AMC l-Arg-AMC l-Phe-AMC l-Thr-AMC

246.0 288.1 283.2 192.0 59.9 33.8 16.2

KM (␮M) 38 77 93 231 78 66 60

Kcat /KM (M−1 s−1 ) 6475 3740 3045 832 768 512 270

with bestatin, the standard family inhibitor. We found that both Ki are in the nM range, ∼1 nM for bestatin (Fig. 5A) and ∼25 nM for Cys. The pH profile, temperature stability and influence of the ionic strength over the amidolytic activity of rFhLAP were assayed using l-Leu-AMC as substrate. Optimal activity was observed at pH 8–8.5, exhibiting a rapid decline under mildly acidic (pH ≤ 7) and basic (pH ≥ 9) conditions (Fig. 5B). The recombinant enzyme is active at a wide range of temperatures, with two peaks, at 37 and 50 ◦ C (Fig. 5C). Preheating rFhLAP at 50 ◦ C produced the maximum specific activity while at higher temperatures (≥60 ◦ C) the enzyme resulted inactivated. No appreciable loss of activity was observed when kept frozen at −80 ◦ C over a period of 3 months, being resistant to several cycles of freezing and thawing (not shown).

Table 3 The effect of different inhibitors on rFhLAP hydrolysis of l-Leu-AMC Inhibitor

Concentration (mM)

DTT 1,10-o-Phenantroline l-Cysteine Bestatin PMSF Pepstatin A Leupeptin TLCK Aprotinin E64 Control

5 1

a

Activity expressed as nmol min−1 mg−1 .

1 1 0.01 0.01 0.005 0.005

Specific Activitya 2.33 ± 0.10 4.46 ± 0.37

2.78 2.97 3.72 3.29 6.70 4.90 11.38

± ± ± ± ± ± ±

0.14 0.01 0.37 0.37 2.14 1.56 0.68

Relative activity (%) or Ki 20.5 18.8 Ki = 25 ± 3 nM Ki = 1 ± 0.5 nM 24.5 26.1 32.7 28.9 58.9 43 100

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Fig. 5. Biochemical characterization of the recombinant FhLAP. (A) Inhibition of rFhLAP with bestatin. rFhLAP (2 ␮g) was pre-incubated in the presence of bestatin (0–100 nM) for 15 min at 37 ◦ C and activity assayed with l-Leu-AMC (50 ␮M). Inset. After pre-incubation of the enzyme (2 ␮g) with bestatin (0–5 nM) activity was assayed in the presence of l-Leu-AMC (0–200 ␮M). Slopes from the reciprocal plot were obtained and replotted vs. bestatin concentration. (B) pH profile of rFhLAP. Activity towards l-Leu-AMC assayed in the presence of 1 mM Mn2+ . The results are expressed as percentage of the maximum activity obtained at 8.5. (C) Thermal stability. Activity assays towards l-Leu-AMC after incubation of the enzyme at the indicated temperatures for 10 min. (D) Effect of the ionic strength. Activity assays in the presence of increasing concentrations of NaCl in glycine 0.1 M, 1 mM Mn2+ buffer.

The influence of ionic strength on rFhLAP was determined using a standard assay mix containing different NaCl concentrations. A sharp increase in enzymatic activity was observed up to 1 M. At higher concentrations amidolytic activity showed only a slight increase up to 2 M (Fig. 5D). The effects of divalent metal ions on rFhLAP enzymatic activity are shown in Table 4. Basal (no metal addition, control condition) rFhLAP activity was progressively activated by MnCl2 (1 ␮M to 10 mM) with an optimum at 1 mM, yielding a 43-fold increase. CoCl2 at 1 mM increased the specific activity 18-fold and at 10 mM, even though there is still an increment in

respect to control conditions, is only 3.5-fold. MgCl2 showed an optimal concentration at 10 mM that increased specific activity by 12-fold. ZnCl2 modestly affects the enzymatic activity and CaCl2 inhibited the enzymatic activity in all the range tested. No relevant effect was seen with NiCl2 , FeSO4 and CuSO4 (not shown). 3.5. Immunization trial In order to analyze the immunoprophylatic potential of the recombinant enzyme, a pilot immunization trial in rabbits was

Table 4 Effect of divalent metal ions on rFhLAP hydrolysis of l-Leu-AMCa Metal

MnCl2 MgCl2 ZnCl2 CoCl2 CaCl2 Control a

Concentration (mM) 0.001

0.01

4.59 ± 0.51 1.03 ± 0.35 1.00 ± 0.20 0.89 ± 0.25 0.89 ± 0.12 1.00

2.12 1.58 1.58 1.13 0.74

0.1 ± ± ± ± ±

1.75 0.30 0.01 0.50 0.05

41.28 3.86 1.53 10.89 0.59

Data expressed as mean relative activity ± S.D. to control incubated without metal.

1 ± ± ± ± ±

0.15 0.40 0.50 5.15 0.10

43.06 6.53 2.37 18.01 0.49

10 ± ± ± ± ±

2.90 0.30 0.01 0.55 0.05

36.83 ± 0.30 11.78 ± 1.00 <0.001 3.56 ± 0.30 0.44 ± 0.05

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Table 5 Worm recovery in rabbits vaccinated with rFhLAP Groups

Control LAP a

Flukes recovered Mean ± S.D.

Flukes/animal

9 ± 1.78 2 ± 1.67a

10; 12; 8; 8; 7; 9 4; 0; 3; 3; 2; 0

Infection units recovered (%)

Reduction (%)

18 4

– 78

p > 0.0001.

performed. Rabbits were immunized with the recombinant protein and challenged at 6 weeks with 50 metacercariae. Parasite burden after 20 weeks show a marked difference between the control and immunized group with a significant reduction of 78% (Table 5). Specific IgG antibodies against rFhLAP were found in immunized animals shortly after the first immunization, and increased steadily up to a plateau established at 11 weeks. A minimal increase in antibody titres was detected in the control group between weeks 3 and 5 (Fig. 6). 4. Discussion Peptidases have been one of the main focuses of research in molecular parasitology since they are involved in key processes related to parasite invasion and survival, being suitable targets for immunological or pharmacological intervention. The ubiquitous M17 LAPs are becoming exciting novel drug targets and vaccine candidates in parasitic infestation, since specific inhibitors have shown to affect key stages of parasite development [11,13,14]. In this report we extend the work on a previously purified and characterized LAP, a gut-associated digestive enzyme isolated from adult liver flukes, proved to confer high levels of protection against fasciolosis in ruminants. The sequence obtained from adult liver fluke cDNA corresponds to a gene encoding a protein with all the characteristics of LAPs that are collectively grouped in the family M17 of

Fig. 6. Antibody responses against rFhLAP in rabbits. Rabbits were immunized (I) with rFhLAP or PBS (control group), received a booster (B) after 4 weeks and were challenged (C) with F. hepatica at week six. Titers of anti-LAP specific IgGs in serum samples taken during the course of the experimental immunization were measured. Open symbols are the measurements for each animal of the control group and filled symbols correspond to the treated group.

the MEROPS peptidase database. A distinctive feature that distinguishes M17 from M1 LAPs is the absence of the metallopeptidase signature HEXXH in the catalytic domain. Additionally, M17 LAPs are homo-hexameric peptidases and bind two metal cations whilst M1 LAPs are not hexameric and bind a single cation [8]. The FhLAP is predicted to be cytoplasmic and folded in two domains present in the family, a C-terminal domain that bears the active site, and a smaller, more variable N-terminal domain that seems to be important in the multimerization mechanism. The residues that by homology might be related to metal coordination and the catalytic function are well conserved, indicating that this might be an active enzyme. The comparison of the sequence obtained to other members of the M17 family showed that general conservation is restricted to the C-terminal domain while the N-terminal portion of the protein is more divergent and similar to a restricted set of sequences that include other worms. The phylogenetic analysis based on the conserved regions around the active site indicated an early divergence of eukaryotic LAPs from their bacterial ancestors, with well-defined clades of enzymes from animal and plant origin and long deep branches for the enzymes from unicellular eukaryotes. Within metazoans, LAPs are further divided into diverse branches, one constituted by the vertebrate aminopeptidases, related to the insect enzymes, and other cluster constituted by two branches, one with enzymes from vertebrates, insects and nematodes, and a second one, which includes the trematode enzymes, and homologues from nematodes and fishes but not from mammals. It is noteworthy that the flatworm enzymes are included in a group devoid of enzymes from their hosts and vice versa, strengthening the possibilities of these catalysts as candidates for specific drug design or their use as vaccines. Furthermore, the phylogenetic distribution of M17 LAPs is consistent with the existence of at least three different LAP genes early in metazoan evolution, with selective losses in diverse lineages. A fully active recombinant form of the FhLAP was produced in the pThioHisC inducible bacterial system as Trx fusion protein and affinity purified. The molecular mass of the recombinant protein corresponds to the monomeric unit plus the Trx-His tag, suggesting that no posttranslational modifications occur, in accordance to predictive methods. Tryptic fragments of nFhLAP analyzed by MALDI-TOF match the recombinant enzyme, and antibodies raised against nFhLAP strongly reacted to purified rFhLAP. Furthermore, the amidolytic preferences of the expressed enzyme are similar to the enzyme purified from adult flukes (Table 1). Collectively, these data indicate that the recombinant enzyme is in fact the LAP previously characterized and assayed as immunogen.

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Consistent with other M17 LAPs, nFhLAP and rFhLAP showed optimal amidolytic activity against Leu and exhibited a broad pattern of substrate specificity, cleaving other non polar aliphatic (Thr), sulfur-containing (Met), polar (Cys) and basic (Arg) amino acids with KM in the nanomolar range. Very low activity towards Phe, Ala, Tyr, Pro, Ser, Gly, Val, Ile and Asp was observed. A comparative study of M17 LAPs from animal (pig), plant (tomato) and bacteria (E. coli) attempted to deduce some amidolytic preferences for the three kingdoms [43]. It established that while Leu and Met are the best N-terminal residues for the three species, Arg is more efficiently cleaved by porcine and plant LAPs than E. coli PepA. Asp, Ser and Gly are uniformly the worst substrates. More recently, physico-chemical data have been obtained from functional recombinant parasite M17 LAPs. In an extensive examination of substrate preferences on amino acid residues, LAPs from three Leishmania species exhibited a more restricted cleaving pattern, with significant activity on exposed Leu, Cys and Met residues, very poor activity against Ala, Ile and Tyr, and no effect on the rest of the 20 amino acids tested [15]. Recombinant LAPs from P. falciparum and Schistosoma species S. mansoni and S. japonicum were cloned and functionally produced in insect cells. P. falciparum enzyme showed very high catalytic efficiency against Leu compared with Phe and Pro, with no amidolytic activity against Ala, Ile, Gly, Asp, Glu or Arg [10]. On the other hand, Schistosoma LAPs studied with a set of four fluorogenic substrates, displayed a marked preference for Leu, and very low amidolytic activity against Tyr, Ala, and Pro [18]. The catalytic efficiency (kcat/ KM ) for substrates acted upon by rFhLAP are about one order of magnitude lower than those found for LAPs from bovine, tomato and E. coli. Although the phylogenetic analysis suggested the existence of different LAPs in metazoans that might represent diverse catalytic properties, the data available is still not enough to validate or reject this hypothesis. All M17 LAPs isolated so far, showed a marked preference for Leu, and varied amidolytic activity towards other residues. Nevertheless, a systematic comparative analysis of enzyme specificity, using LAPs from diverse organisms representing the different clusters with a broad spectrum of substrates, is needed to give an insight into this interesting issue. Divalent cations are essential for metallopeptidase activity and several studies demonstrated that M17 LAPs activity is enhanced by their addition, and suppressed by metal chelating agents. Crystallographic data from bovine LAP show that two coordination sites occupied by Zn atoms exist per monomeric unit. The 3D-structures of the enzyme-inhibitor complexes demonstrates that Zn488 (bovine LAP numbering) is coordinated by the side chain of the conserved residues Asp255, Asp332 and Glu334, while Zn489 is coordinated to Lys250, Asp255, Asp273 and Glu334 [39]. These metal binding sites show different affinities and kinetics toward divalent metal ions. Zn488 occupies the so-named readily exchangeable site or site 1 that can be substituted by Mn2+ , Co2+ and Mg2 ions, whereas Zn489 is located in the so-called tight-binding site or site 2 that allows Zn2+ replacement only when both sites are unoccupied [44]. Not surprisingly, we found a several-fold increase in rFhLAP activity by the addition of Mn2+ , Co2+ and

Mg2+ . In addition, no influence was detected by incubation with Ni2+ , Fe2+ , and Cu2+ , while Ca2+ produced an inhibitory effect. The effect of Zn2+ is variable, being a modest activator at low concentrations (1 mM) and acting as inhibitor at higher concentrations (10 mM). Similar results were obtained with the native enzyme purified from adult worms [29]. Since Zn was the only ion detected in nFhLAP by atomic absorption spectroscopy at a 1.86:1 Zn-LAP subunit ratio (not shown), it is reasonable to assume that the activation observed is due to the replacement of Zn atoms at site 1. Bestatin is an inhibitor that slowly attains equilibrium with the enzyme to form a tightly bound complex. The biphasic reaction progress curves observed for the hydrolysis of l-Leu-AMC in the presence of the inhibitor suggest a slow binding inhibition mechanism [45]. The high inhibition profile shown by thiol compounds can be explained by a chelating effect of these compounds. The pH optimum of 8.0–8.5 for rFhLAP is similar to other cytosolic M17 LAPs which are between 8.5 and 9.5 [3,5,6]. An outstanding physico-chemical feature of the rFhLAP is its thermal stability. Activity increases with rising temperature and reaches it maximum when it is incubated at 50 ◦ C for 10 min previous to substrate addition. Similar thermal stability has been observed for LAPs from Solanum tuberosum [46], Haemaphysalis longicorins [47] and Leishmania spp. [15]. In sharp contrast to other members of M17 LAPs from bacteria [48] and protozoa [15] which are inhibited by 0.1 M salt concentration, amidolytic activity on Leu-AMC was positively influenced by ionic strength showing optimal activity at 1–2 M NaCl. It remains to be determined whether these characteristics of the enzyme are of physiological relevance. The complex life-cycle of F. hepatica involves a definitive herbivorous mammal host that harbors the adult worm at the bile ducts, and freshwater Lymnaeid snails as intermediate host in which the parasite reproduce asexually. Ruminants and humans are infected by ingestion of the metacercariae encysted in vegetation. Inside the duodenum the juvenile (NEJ) breaks free and rapidly penetrate throughout the gut wall, then migrates through the liver and finds its way to the bile ducts where it matures into an adult fluke 8–10 weeks post-infection. The hermaphrodite worms release embrionated eggs that pass in stools and after 2 weeks a miracidium, the ciliated free- living stage, actively swims and invades the susceptible snail. We analyzed the expression pattern of FhLAP in adult worms, metacercariae, eggs, and the invading stages NEJs and miracidia, and found transcripts in all stages assayed. Similarly, we searched the S. mansoni EST database [49] and found that ESTs that might correspond to the orthologous gene were detected in diverse stages from eggs to adults. Using chromogenic substrates, leucine amidolytic activity was predominantly detected by histochemistry at the cells lining the alimentary tract of adult flukes [29]. Here, we examined the intracellular localization of FhLAP in gut cells by immunoelectron microscopy and found very strong cytoplasmic reactivity particularly at the apical lamella, situating LAP close to the lumen where the proteolytic events degrading host proteins takes place in adult trematoda. Although LAP activity is consistently

D. Acosta et al. / Molecular & Biochemical Parasitology 158 (2008) 52–64

very low in cathepsin L-rich ES products, it has been recently shown that FhLAP is not only detected in ES products from adult flukes by 2D gels coupled to MALDI-TOF analysis, but also that it was specifically recognized by sera from human patients from the Peruvian Altiplano (Marcilla, unpublished). In this sense, its presence in lumen contents could be explained by continuous “leakage” from the apical part of gut cells as they undergo cyclical remodelling between absorptive and secretory phenotypes [50]. If this is the case, the lack of activity observed in vitro might be related to the strong inhibitory effect that incubation in ES products exert on FhLAP activity (not shown) caused by either degradation by cathepsin Ls, mild acidic lumen microenvironment, or a combination of both factors. In blood-feeding helminths host blood proteins are the main source of amino acids required for development and reproduction of invading and adult stages. The cooperative participation of clan CA and CD cysteine proteases and clan AA aspartic proteases in a digestive network or cascade that degrades haemoglobin (Hb) has been proposed in hookworms [51] and more recently in S. mansoni [52]. In this trematode parasite, the model proposes the initial attack to the Hb monomer by cathepsin D or cathepsins B1 and L1 generating proteolytic fragments that are further cleavage into peptides by the action of cathepsins L1 and B1. In the last steps, absorbable peptides or amino acids are released by the action of cathepsin B1 and the exopeptidases cathepsin D and LAP. However, this key catabolic process is completely unknown in F. hepatica where cathepsins L1 and L2 are the only proteolytic enzymes shown to be actively secreted by intestinal cells inside the gut lumen in adult worms [24]. Additionally, a dipeptidyl peptidase was isolated in ES products from liver fluke mammalian stages, and it was postulated that it functions in degrading peptides and producing dipeptides that can thus diffuse into absorptive intestinal cells [53] as demonstrated for mammal jejunal enterocytes [54]. In this hypothetical scenario, FhLAP most significant contribution in the digestive process would be the cytosolic hydrolysis of absorbed Hb-derived dipeptides. The analysis of bovine Hb amino acid sequence shows that Leu, Cys, Met and Arg, FhLAP most preferred substrates, correspond to 26% of Hb globins. However, preferences on absorbed di- or tri-peptides could not be exactly predicted based on amidolytic activities on fluorogenic substrates as P 1 or P 2 are important in determining LAP substrate affinity [43]. Native FhLAP induced strong IgG response in vaccinated sheep that inhibited enzymatic activity. This property was proposed as the basis for the very high level (89%) of protection obtained [28]. The molecular characterization reported here confirms the lack of O- or N-glycosylation sites highlighting the potential of the recombinant form as a plausible vaccine. Finally, the immunization of rabbits whit rFhLAP resulted in an important protection similar to the one obtained previously in sheep with the native antigen [28]. In addition the induced humoral response increased early and persisted during the study in a similar fashion than in the previous trial. The results in two different model organisms confirm that FhLAP is a relevant candidate for vaccination against fasciolosis in diverse animal models.

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Acknowledgements This work was supported by grants from Fondo Clemente Estable and Comisi´on Sectorial de Investigaci´on Cient´ıfica, UdelaR, Uruguay. Daniel Acosta was recipient of an MSc scholarship from the Programa de Maestr´ıas en Biotecnolog´ıa, Facultad de Ciencias, Udelar. We thank Rosario Duran and Carlos Cerve˜nansky at the Institut Pasteur Montevideo/IIBCE/Facultad de Ciencias for performing the MALDI-TOF work, and Gabriela Casanova and Alvaro Duran at the Unidad de Microscopia Electr´onica, Facultad de Ciencias, for immuno electron microscopy.

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