Molecular cloning and expression of the cDNA sequence encoding a novel aspartic protease from Uncinaria stenocephala

Experimental Parasitology 134 (2013) 220–227

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Molecular cloning and expression of the cDNA sequence encoding a novel aspartic protease from Uncinaria stenocephala Katarzyna Wasyl a,⇑, Anna Zawistowska-Deniziak b, Piotr Ba˛ska a,1, Halina We˛drychowicz a,b, Marcin Wis´niewski a a b

Division of Parasitology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Ciszewskiego 8, 02-786 Warsaw, Poland ´ ski Institute of Parasitology Polish Academy of Sciences, Twarda 51/55, 00-818 Warsaw, Poland The Witold Stefan

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Anti-rUs-APR-1 antibodies confirm

the production of the protease in U. stenocephala.  The aspartic protease was detected in various life stages in A. ceylanicum.  An effective vaccine would protect dogs against hookworms not limited to one species.  The vaccine could be constructed from immunogenic epitopes from the key enzymes.

a r t i c l e

i n f o

Article history: Received 9 July 2012 Received in revised form 8 March 2013 Accepted 17 March 2013 Available online 28 March 2013 Keywords: Uncinaria stenocephala Aspartic protease (APR) Hookworm Us-APR-1 Us-apr-1

a b s t r a c t Uncinaria stenocephala belongs to Ancylostomatidae family. Members of this family – hookworms – infect millions of people and animals worldwide. U. stenocephala is most pathogenic in dogs and other Canidae, which are the main hosts, and infection causes anemia or even death. So far no effective hookworm vaccine has been developed that is economically viable. Attempts to identify vaccine antigens have led to a group of aspartic proteases, which play a key role in parasite feeding, migration through host tissues and immune evasion. The cDNA of an aspartic protease from U. stenocephala was cloned using the RACE-PCR method. Computational analysis showed that the cDNA encodes a 447 amino acid protein with a molecular mass of 52 kDa that shows high homology to aspartic proteases from related hookworms. Analysis identified 1 potential N-glycosylation site, 3 potential disulfide bonds and no O-glycosylation sites. The recombinant protein was expressed in Escherichia coli followed by purification and mouse immunization. Using raised anti-Us-APR-12 (Uncinaria stenocephala Aspartic protease-1) serum the presence of Us-APR-1 in the adult stage of U. stenocephala and the expression of homologous protease in L3 and adult stages of A. ceylanicum was confirmed. This analysis is the first phase of work exploring the biological role of Us-APR-1 in parasite-host interactions and raises hope for successful vaccine development against Uncinaria sp. and possibly Ancylostoma sp. Ó 2013 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +48 22 593 60 48. E-mail addresses: [email protected] (K. Wasyl), anna.zawistowska@ twarda.pan.pl (A. Zawistowska-Deniziak), [email protected] (P. Ba˛ska), [email protected] (H. We˛drychowicz), [email protected] (M. Wis´niewski). 1 Present address: Division of Farmacology and Toxicology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Ciszewskiego 8, 02-786 Warsaw, Poland. 2 Us-APR-1 - Uncinaria stenocephala Aspartic protease-1. 0014-4894/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2013.03.013

1. Introduction Uncinaria stenocephala is a parasitic nematode and belongs to the Ancylostomatoidea superfamily (Anderson, 1992). Due to their blood feeding and high prevalence, hookworms constitute a serious medical and veterinary problem. It is believed that approximately 600 million people worldwide are infected with hookworms, mainly in tropical regions (Bethony et al., 2011).

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Among parasites of carnivorous animals the most important are Ancylostoma caninum, A. ceylanicum, A. tubaeformae, A. braziliense and U. stenocephala. U. stenocephala is commonly found in temperate climates, predominantly in central and northern Europe (Eckert et al., 2005; ESCCAP, 2007) and its eggs can survive in soil several weeks at temperatures below zero (Bajer et al., 2011). Hookworm infections can cause severe damage in their hosts from diarrhea to anemia and even death. While attached to the intestine they ingest blood for nutrition. Human hookworms (depending on species) may cause average blood loss 0.04 or 0.2 ml blood/worm/day (Pawlowski et al., 1991). Heavy infections lead to hookworm disease including a reduction in hemoglobin level, iron deficiency anemia, protein malnutrition, or even death (Bajer et al., 2011; Hotez et al., 2004). Dogs and other Canidae are the main definitive hosts for U. stenocephala. Cats are nonspecific hosts, where the parasite cannot fully develop (Ramisz and Martynowicz, 1963). Cases of human infections, causing ‘creeping eruption’, have also been reported, including in children from South America (Pezzani et al., 1996), in 22% of prisoners in Madrid (Alonso-Sanz et al., 1995), among population of the village in north of Iran (Ghadirian, 2007) and a probable case in an adult in France (Tamminga et al., 2009). Tamminga et al. (2009) point to rising temperature and changing climate as a cause for the increasing incidence of the zoonotic infections in northern regions, which can lead to expansion of the global distribution of more infective species or increase the current prevalence of zoonotic hookworms such as U. stenocephala. The problem of uncinariosis has been neglected, though some prevalence data has been reported to date. U. stenocephala was found to be the most prevalent parasite of dogs (33.28%) in Córdoba, Spain (Martínez-Moreno et al., 2007), while hookworm infections were present in 6.9% of dogs in Switzerland (Sager et al., 2006), 11.4% in Belgium (Vanparijs et al., 1991) and between 9.7% and 14.4% in the USA. (Kirkpatrick, 1988; Nolan and Smith, 1995). Borkovcová (2003) reported a very low Ancylostoma sp./Uncinaria sp. prevalence (0.6%) in Moravia, the Czech Republic, as did Epe et al. (2004) (1.4%) in Germany. The common hookworm in dogs in Canada is U. stenocephala (Kennedy, 2001) but there are no accurate records available in regards to its prevalence. In Tirana (Albania) 72 from 111 dogs studied were infected with U. stenocephala (Xhaxhiu et al., 2011). Very frequent infections with this parasite were found in Poland, with 2.9% of pet dogs in Poznan´, 5–10% in Wrocław, 10.1–47.1% of stray dogs in Warsaw and 75% of country dogs around Wrocław infected with U. stenocephala (Okulewicz et al., 1994; Górski et al., 1996; Mizgajska and Luty, 1998). Some archival data point to a very high prevalence of uncinariosis (70%) in the Niger Delta (Arene, 1984) and (26%) Australia (Blake and Overend, 1982). Generally hookworm infections are more likely to occur in stray and shelter dogs than in household dogs (Grandemange et al., 2007), and in rural dogs compared to urban dogs, the latter probably because of food consisting of uncooked remains of farm animals (Dubná et al., 2007). Apart from dogs, U. stenocephala also infects foxes. A prevalence of 58.2% was reported in red foxes in Guadalajara, Spain (CriadoFornelio et al., 2000), with prevalence rates of 26% in Western Poland (Balicka-Ramisz et al., 2003), 45.5% in Wolin Island, Poland (Mizgajska-Wiktor and Jarosz, 2010), 40.4% in Belarus (Shimalov and Shimalov, 2003), 41.3% in Great Britain (Smith et al., 2003), 68.6% in Denmark (Saeed et al., 2006) and 78.2% in Geneva, Switzerland (Reperant et al., 2007). Wild foxes can easily contribute to the spread of hookworm infection in other animals such as dogs because of the coprophagic behaviour of the latter. Due to reinfection of hosts, transition into dormant larvae stage, developing parasite drug resistance and ease of transmission among animals, the need to develop an effective vaccine has

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emerged. It was previously reported that aspartic proteases, which host-specifically digest hemoglobin, serum proteins and skin macromolecules, are promising antigens for a hookworm vaccine (Hotez et al., 2010; Loukas, 2005; Williamson et al., 2002, 2003a,b, 2004). These studies were conducted in relation to human and canine infections with Ancylostoma sp. (A. duodenale and A. caninum) and Necator americanus. This report is focused on an aspartic protease from the related species U. stenocephala, a representative parasite from a non-tropical climate. 2. Materials and methods 2.1. Obtaining the infective stage L3 larvae Fecal samples were collected from a dog shelter in Celestynów near Warsaw and were examined for the presence of the hookworm U. stenocephala using the flotation method (Henriksen and Christensen, 1992). From every positive sample a coproculture was set up using the Baermann technique. 2.2. Isolation of total RNA from infective U. stenocephala stage L3 larvae Approximately 10,000 L3 larvae were suspended in 1 ml of Trizol (Invitrogen). The larvae were then homogenized using an electric homogenizer (GLAS-COLÒ Variable Speed Laboratory Motor). Total RNA was then obtained using the Total RNA isolation kit (A&A Biotechnology) according to the manufacturer’s instructions. 2.3. Cloning and generating pBS and pET constructs The first step was the synthesis of single stranded cDNA as a result of reverse transcription. Then the addition of a homopolymeric tail to the 30 terminus of the DNA molecule was conducted using terminal deoxynucleotidyl transferase. Amplification of 50 and 30 ends of cDNA was performed by RACE-PCR as previously described (Ba˛ska et al., 2013; Mieszczanek et al., 2000; Wis´niewski et al., 2004). The primer Acaspr1L (50 -GAACTCCAGCGCAGAATTTCACAGTGATT) and Acasp1R (50 -GGGCCTGGCCAACATCGAAGAC) were designed in correspondence to the A. caninum aspartic protease cDNA sequence (GenBank ID: U34888.1). PCR amplification with both gene specific primers was performed using 1 lg of cDNA template, 0.4 lM of each primer, 2 mM MgCl2 (Fermentas), 0.2 lM dNTPs (Fermentas), 1 Taq buffer (Fermentas), 1 U Taq Polymerase (Fermentas), 0.5 U Pfu Polymerase (Fermentas) in a final volume of 50 lL. The cycling conditions were 94 °C for 3 min, followed by 35 cycles of 94 °C for 20 s, 56 °C for 30 s, 72 °C for 1 min, and final elongation of 72 °C for 5 min. The results was analyzed using 1% agarose gel electrophoresis, the PCR products were isolated from the gel using the Gel-Out kit (A&A Biotechnology) and confirmed by sequencing at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences. 2.4. 30 and 50 RACE-PCR cDNA was synthesized by reverse transcription, then a homopolymeric tail was added to the 30 terminus of the DNA molecule using terminal deoxynucleotidyl transferase. Amplification of 50 and 30 ends of cDNA was performed by RACE-PCR as previously described (Mieszczanek et al., 2000; Wis´niewski et al., 2004). with the primers Acaspr1L (50 -GAACTCCAGCGCAGAATTTCACAGTGATT) and Acasp1R (50 -GGGCCTGGCCAACATCGAAGAC) designed in correspondence to the A. caninum aspartic protease cDNA sequence

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(GenBank ID). Briefly, amplification of the 30 end of U. stenocephala aspartic protease cDNA was undertaken with primers Acasp1L and pTBam (50 -CGCCACGCGTGGATCCGTTTTTTTTTTTTTTTTT), and amplification of the 50 end of the cDNA was performed using primers Acasp1R and pG (50 -CGAGGAATTCGGGGGGGGGG). PCR amplifications were performed using 1 lg of cDNA template, 0.4 lM of each primer, 2 mM MgCl2 (Fermentas), 0.2 lM dNTPs (Fermentas), 1 Taq buffer (Fermentas), 1 U Taq Polymerase (Fermentas) and 0.5 U Pfu Polymerase (Fermentas) in a final volume of 50 ll. The cycling conditions were 94 °C for 3 min, followed by 30 cycles of 94 °C for 20 s, 56 °C for 30 s, 72 °C for 1.5 min, and final elongation of 72 °C for 5 min. Results were analyzed by 1% agarose gel electrophoresis, and the bands of predicted molecular weight were extracted from the gel and purified using the Gel-Out kit (A&A Biotechnology). A blunt ending reaction was conducted using Pfu Polymerase. 2.5. pBS/Us-apr-1 constructs Vector pBluescript II SK(+) (pBS, Stratagene) was prepared by Eco RV (Fermentas) digestion. Both 30 and 50 ends of aspartic protease cDNA were separately ligated into pBS vectors using 4 U of T4 DNA ligase (Fermentas) at 22 °C for 2 h and then 16 °C for 16 h. Bacterial competent cells (Escherichia coli strain DH5a) were transformed with the resulting constructs. Recombinant plasmids containing inserts of appropriate size were confirmed by Xba I and Hind III digestion and by sequencing at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences. Based on the results of sequencing reactions, new primers were designed allowing amplification of the complete U. stenocephala aspartic protease (Us-apr-1) cDNA sequence: Usap1L (50 -GATGGTTCGTCTCGTATTGTTCC) containing an ATG start codon, and Usap1R (50 -GGATGATGTATTCACTCTCTAA) containing a TAA stop codon. Amplification of the complete Us-apr-1 cDNA sequence was conducted as described above using the newly designed primers. Subsequent purification, ligation into pBS vector, transformation of bacterial competent cells, and analysis and sequencing of clones was performed as previously described. 2.6. Bioinformatics analysis Comparison of the nucleotide sequences of aspartic proteases from related hookworms in the GenBank database was carried out using Basic Local Alignment Search Tool (Blast) available at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi. The amino acid sequence was obtained using Translate tool available at http:// .web.expasy.org/translate/. Nucleotide and amino acid alignments comprising the novel U. stenocephala sequence and homologous sequences from related hookworms A. ceylanicum, A. caninum and N. americanus were constructed using ClustalW (accessible at http://www.ebi.ac.uk/Tools/msa/clustalw2/). The signal peptide was determined with the SignalP 3.0 server available at http:// www.cbs.dtu.dk/services/SignalP/. Characteristic domains were identified using InterProScan Sequence Search accessible at http://www.ebi.ac.uk/Tools/pfa/iprscan/. NetOGlyc and NetNGlyc were used to determine N and O-glycosylation sites (www.cbs.dtu.dk/services). The probable three-dimensional structure of U. stenocephala aspartic protease was obtained with the use of 3D Jigsaw Comparative Modelling 2.0 (available at http:// www.bmm.cancerresearchuk.org/~3djigsaw). The structure was then visualized using PyMOL (available at http://www.pymol.org). 2.7. Protein expression and purification The gene was subcloned prior to expression. The complete cDNA encoding the protein was amplified by PCR and then ligated

into pBS vector by incorporating Bam HI and Xho I restriction sites:pETBUL, 50 -AGGATCCAGCATCCATCGGAGGAC (Bam HI restr. site) and pETUR, 50 -TCTCGAGTTAGAGAGTGAATACAT (Xho I restr. site). The PCR conditions were as follows: 94 °C for 3 min followed by 40 cycles of 94 °C for 20 s, 55 °C for 30 s, 72 °C for 1.5 min. Final elongation was held at 72 °C for 10 min. Gel electrophoresis, DNA extraction and purification, ligation into pBS vector and transformation of E. coli DH5a competent cells was conducted as previously described. The isolation of the recombinant plasmid was performed using the Plasmid Mini isolation kit (A&A Biotechnology) according to the manufacturer’s instructions. The plasmid underwent restriction digestion using Bam HI and Xho I enzymes (Fermentas). The digestion product of approximately 1400 bp was extracted from an agarose gel after electrophoresis and was purified and ligated into the pET expression vector (Novagen) previously digested with Bam HI and Xho I. Transformation of E. coli Rosetta™ competent cells (Novagen) with the Us-apr1/pET construct was conducted similarly as described above. Recombinant protein expression was performed in E. coli Rosetta™ with IPTG as expression inductor and with addition of glucose (0.5%) and kanamycin (60 lL/mL). The protein was purified on a His-Select™ Cartridge column (Sigma Aldrich) under denaturing condition (8 M urea) according to the manufacturer’s instructions. 2.8. Immunisation of a mouse with recombinant U. stenocephala aspartic protease Antiserum against recombinant Us-APR-1 (rUs-APR-1) was generated by immunizing a C3H mouse with a total of 150 lg of recombinant protease mixed at a ratio of 2:1 with ImjectÒ Alum (Pierce) as adjuvant. Injections were administered in 4 doses at 2 weekly intervals: the first dose of 50 lg was injected intramuscularly, a second equal dose was injected subcutaneously, and the last two doses each containing 25 lg of the protein were administered both intramuscularly and subcutaneously. After collection, blood was incubated at 37 °C for 2 h then 4 °C overnight. The following day the blood was centrifuged (2500g, 10 min) and the serum was collected and stored at 70 °C for further analysis. All experiments were approved by the Third Local Ethical Committee of Warsaw University of Life Sciences – SGGW. The study was conducted according to the institution’s guidelines for animal husbandry. 2.9. Analysis of the specificity of obtained polyclonal antibodies to rUsAPR-1 Four samples of purified rUs-APR-1 were separated on a 12.5% polyacrylamide gel during SDS-PAGE, prior to transfer to a nitrocellulose membrane. Blocking of the membrane occurred in 5% skim milk in phosphate buffered saline (PBS) for 1 h at RT. The membrane was then cut into 4 pieces (one for each protein sample) and each strip was incubated with the mouse anti-rUs-APR-1 serum at the following dilutions: 1:100, 1:200, 1:500 and 1:1000 (in 5% skim milk in PBS) for 1 h at RT under gentle agitation. After washing the membrane strips with PBS (3  10 min) the conjugated secondary antibodies (goat Anti-Mouse IgG (Fc specific)–Peroxidase antibody, Sigma Aldrich) diluted 1:5000 in 5% skim milk in PBS were added and incubated for 1 h at RT. After washing with PBS (3  10 min) the peroxidase substrate was added and detection by photographic film was conducted. Purified rUs-APR-1 incubated with serum obtained from a non-immunized mouse (diluted 1:500) was used as a negative control.

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2.10. Analysis of the specificity of polyclonal anti-recombinant A. ceylanicum aspartic protease (rAce-APR-1) antibodies to rUs-APR-1 An elution fraction of purified rUs-APR-1 was separated in a 12.5% polyacrylamide gel, followed by transfer to a nitrocellulose membrane and blocking with 5% skim milk in PBS for 1 h at RT. The primary antibodies, anti-rAce-APR-1, were produced in rabbit during other experiment. Briefly, a New Zealand white rabbit was immunized subcutaneously at two week intervals with purified protein produced in E. coli. The IgG fraction was isolated from the whole serum using a Protein G affinity column (Amersham Pharmacia Biotech). The membrane was then incubated with rabbit anti-rAce-APR-1 serum diluted 1:3000 in 5% skim milk in PBS for 1 h at RT under gentle agitation. After washing the membrane with PBS (3  10 min) the secondary antibodies were added (goat AntiRabbit IgG (whole molecule)–Peroxidase antibody, Sigma Aldrich) diluted 1:5000 in 5% skim milk in PBS for 1 h at RT. After washing with PBS (3  10 min) the substrate for peroxidase was added and detection by photographic film was conducted. Purified rUs-APR-1 incubated with serum obtained from a non-immunized rabbit (diluted 1:3000) was used as a negative control. 2.11. Analysis of the expression of Us-apr-1 in adult U. stenocephala In order to analyze aspartic protease expression in vivo, homogenate from the adult stage of U. stenocephala was prepared: 10 adult hookworms obtained post mortem from the dogs’ small intestine at Veterinary Clinic at Warsaw University of Life Sciences were ideintified as U. stenocephala based on microscopic examination of the buccal cavity. Worms were homogenized in 200 lL ice-cold PBS and centrifuged (15,000g, 15 min). Homogenate protein concentration was determined using the BCA Protein Kit Assay (Pierce) according to manufacturer’s instructions. Approximately 70 lg of the homogenate was separated in a 12.5% polyacrylamide gel during SDS–PAGE and then transferred

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to a nitrocellulose membrane. Blocking of the membrane occurred in 5% skim milk in PBS for 1 h at RT. Next, anti-rUs-APR-1 primary antibodies were added (1:500) and incubated with the membrane for 1 h at RT in 1 mL of 5% skim milk in PBS. After washing with PBS (3  10 min) secondary antibodies were added and the assay proceeded as described above. A negative control was prepared with the same homogenate sample incubated with antibodies obtained from a non-immunized mouse (diluted 1:500). 2.12. Analysis of the specificity of obtained polyclonal antibodies to an aspartic protease from the related hookworm A. ceylanicum (Ace-APR1) The A. ceylanicum strain was cultivated in Syrian hamsters under standard conditions according to local regulations, with all experiments approved by the Third Local Ethical Committee of Warsaw University of Life Sciences. The study was conducted according to the institution’s guidelines for animal husbandry. Approximately 50 adult A. ceylanicum were picked from the gut of euthanized Syrian hamsters. Worms were incubated for 2 h in phosphate buffered saline (PBS) at 37 °C to remove host tissue. Larvae L3 from A. ceylanicum were cultured from feces from infected hamsters using Baermann technique. Adult A. ceylanicum and larvae L3 were homogenised separately in 200 lL ice-cold PBS and centrifuged (15,000g, 15 min). Concentration of proteins in the homogenates were determined using BCA Protein Kit Assay (PIERCE). SDS–PAGE was conducted with a 12.5% polyacrylamide gel with the following samples: 5 lg of adult A. ceylanicum homogenate, 40 lL excretory/secretory (ES) solution from A. ceylanicum adults, 5 lg A. ceylanicum L3 larva homogenate and 40 lL of ES solution from activated A. ceylanicum L3 larva. Blocking, washing, incubation with antibodies and detection were as described in the section above. All the negative controls were prepared with the same homogenate and solution samples incubated with antibodies obtained from a non-immunized mouse (diluted 1:500).

Fig. 1. Probable three-dimensional structure of the U. stenocephala aspartic protein 1. Model for Us-APR-1 was built based on the known structure of human cathepsin D. The two active site Aspartate residues, at positions 98 and 285 (mature protein) are shown. The arrow indicates the propeptide consisting of two a-helices, which blocks the active site of the enzyme.

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from activated L3 larva, L3 larva homogenate, and the adult A. ceylanicum homogenate and ES fraction (Fig. 5).

3. Results The 30 RACE-PCR resulted in a product of approximately 1100 bp, and 50 RACE-PCR resulted in a 1200 bp product. Newly designed primers: Usap1L and Usap1R allowed the amplification of the whole 1400 bp U. stenocephala aspartic protease cDNA. The sequence was deposited in GenBank database (GenBank ID: FJ147198). Subcloning of the cDNA into expression vector pET 28a (+) using primers: pETBUL and pETUR was confirmed by the restriction analysis with Bam HI and Xho I enzymes. Comparison of nucleotide sequences of aspartic proteases cDNAs from related hookworms using Blast showed 88% identity to A. ceylanicum cathepsin D-like aspartic protease, 87% to A. duodenale aspartic protease, 86% to A. caninum aspartic protease and 80% to N. americanus necepsin II. The amino acid sequence was obtained using Translate tool and deposited in Genetic sequence database at the NCBI (GenBank ID: ACI02330). The SignalP 3.0 Server was utilized to identify the signal peptide, which spanned residues 1–16, with the sequence: M V R L V L F L A L C T L A V A. InterProScan identified a composition of the active site of the aspartic protease and approximate structure of propeptide A. This propeptide consisted of two a-helices, which blocks the active site of the enzyme. The propeptide is joined with the catalytic site by hydrogen bonds, which stabilise its conformation and probably take part in transformation of proenzyme into the mature enzyme (Sielecki et al., 1991). The rUs-APR-1 has a predicted molecular size of 4789 kDa similarly to other aspartic proteases from related hookworms previously reported. The novel aspartic protease exhibited two catalytic DTG active-site motifs which are characteristic for the aspartic protease family. These two motifs consist of Asp acid residue in 114 and 301 positions which build the active site of the enzyme. The flanking regions of the catalytic site are also conserved: VIFDTGSSNLWVPS and AIADTGTSLIAG (Tang and Wong, 1987; PROSITE). The amino acid sequence has high identity to Ace-APR-1 from A. ceylanicum (94,9%), Ad-APR-1 from A. duodenale (94,6%), Na-APR-2 from N. americanus (89%) and bores only 88,4% identity to Ac-APR-1 from A. caninum although it is also a canine hookworm. That suggests that cross immunity could be low because of the differences in the sequence. Visualisation of the probable three-dimensional model of U. stenocephala aspartic protease is shown in the Fig. 1. NetNGlyc and NetOGlyc determined one possible N-glycosylation site and no O-glycosylation sites (Table 1). Fig. 2 shows the alignment of 5 aspartic proteases from related hookworms with indication of the major features. Analysis of the specificity of the anti-rAce-APR-1 polyclonal antibodies to rUs-APR-1 was performed using Western Blot. Fig. 3 shows detection of the recombinant protease. The obtained serum recognized recombinant Us-APR-1 when used at optimum dilution of 1:500. The aspartic protease U. stenocephala was detected by anti-rUs-APR-1 serum in U. stenocephala homogenate (Fig. 4). The anti-rUs-APR-1 antibodies also detected the aspartic protease in the related hookworm A. ceylanicum, in the ES fraction

4. Discussion The novel aspartic protease cDNA from U. stenocephala was cloned using RACE-PCR technique and product of 1344 bp was translated to 447 amino acid protein. The gene, named Us-apr-1 (Uncinaria stenocephala aspartic protease-1), encoded protein Us-APR-1. Computational analysis showed post translational modifications (N-glycosylation and disulfide bonds). Post translational modification seem to be essential for appropriate folding of hookworm aspartic proteases since Na-APR-1 expressed in yeast was catalytically active (Ranjit et al., 2009) and Us-APR-1 expressed in E. coli was insoluble in the neutral pH buffer in non denaturing conditions (without addition of 8 M urea). We identified the presence of Us-APR-1 in the adult parasite homogenate what is consistent with results of Ranjit et al. (2009) who localized the Na-APR-1 in brush border of the intestine of adult worms. The serum cross reacted with homogenate and ES fraction from various life stages from A. ceylanicum but this is rather quality not quantity method because determination of the concentration of the protein was impossible since the presence of FBS in ES fraction of ssL3 larvae what generate high background. Suprisingly our result might indicate that aspartic protease is present in ES fraction of ssL3 and adult A. ceylanicum. This result might be caused due to presence of worm gut tissue fragments in the parasite waste products. Western blot analysis showed difference between protein expression in adult and L3 A. ceylanicum. Higher protein expression in adult worms would be consistent with parasite physiology. L3 larvae in opposite to adult worms do not feed on mammal hemoglobin so the enzyme responsible for hemoglobin digestion seems to be unnecessary. Aspartic proteases are important component of hemoglobin cleaving pathway not only for hookworm (Ranjit et al., 2009) but also contribute to hemoglobin cleavage during malaria (Francis et al., 1997). Recently Subramanian et al. (2009) suggested that hemoglobin hydrolysis by Plasmodium falciparum is not a highly ordered process but rather that cysteine proteases rapidly cleave hemoglobin at multiple sites to facilitate rapid hydrolysis of this substrate. Hookworms utilize rather ordered network of proteases to digest hemoglobin since Na-CP-3 (Necator americanus Cysteine Protease 3) and Na-MEP-1 (N. americanus Metalloprotease 1) could not cleave hemoglobin but were able to digest hemoglobin fragments that were produced after Na-APR-1 digestion (Ranjit et al., 2009). Aspartic proteases of both N. americanus and A. caninum were localized in gut of adult parasite. But antibodies of serum raised against aspartic protease (APR) attached also to esophagus, excretory gland and amphidial glands and some reproductive organs (Williamson et al., 2002). We produced the serum against Ace-APR-1 and Us-APR-1. The cross reactivity was observed. Although Williamson et al. (2002) showed that hookworm aspartic hemoglobinases cleave hemoglobin from specific host with higher

Table 1 Human and canine parasites – similar proteases. Organism

U. stenocephala A. ceylanicum A. duodenale A. caninum N. americanus a

GenBank accession number

Amino acid identity (%)

Amino acid similarity (%)

ACI02330 AAO22152 ACI04532 AAB06575 CAC00543

– 94.9% 94.6% 88.4% 89.0%

– 97.5% 97.3% 91.6% 94.0%

Only partial signal peptide sequence is known.

Mature protein AA

Mw (Da)

pI

431 430 430 433 430

47886.60 47964.76 47945.63 48405.06 47867.60

7.02 7.52 7.50 7.56 6.41

Signal pepitide(AA)

O-Glycosylation sites

N-Glycosylation sites

16 16 16 >9a 16

0 0 0 0 0

1 1 1 1 1

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Fig. 2. Alignment of five aspartic proteases from related hookworms. Ace-APR-1 – from Ancylostoma ceylanicum, Ad-APR-1 – from A. duodenale, Acasp1 – from A. caninum, UsAPR-1 – U. stenocephala, NcpII – from Necator americanus. The sequences of signal peptide and propeptide are indicated in frame with solid and dotted line respectively. NGlycosylation site is indicated in bolt frame and marked with square (j). The conserved cysteine residues are in bold frames and marked with triangle (N). The conserved flanking regions of the catalytic site are in bold frames and Asp residues building active site are marked with an arrow (;). The alignment was prepared using ClustalW (http:// www.ebi.ac.uk/Tools/msa/clustalw2/). The identical amino acid residues are marked with asterisk (⁄).

efficiency, the mentioned cross reactivity could be very helpful in creating a one vaccine for dogs based on recombinant aspartic protease which would develop a cross-immunity to many related

hookworms e.g. A. caninum, A. ceylanicum, A. braziliense. This kind of research showed promising results since dogs vaccination with mutated Na-APR-1 (strictly human parasite) resulted in

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significantly reduced parasite egg burdens and weight loss among canines after A. caninum infection (Pearson et al., 2009). All these studies could evolve better protection against more than one species of hookworm. Although the vaccine based on one antigen might be insufficient in raising protective response against hookworm infection whereas the vaccine comprised of enzymes crucial for parasite survival might be the efficient tool for control of hookworm infection. Acknowledgments The authors are grateful to Luke Norbury for providing language help and writing assistance. References Fig. 3. Analysis of the specificity of the anti-rAce-APR-1 polyclonal antibodies to rUs-APR-1. rUs-APR-1 probed with (1) rabbit anti-rAce-APR-1 antibodies, and (2) antibodies from a non-immunized rabbit (negative control).

Fig. 4. Analysis of the expression of Us-apr-1 in adult U. stenocephala. Native aspartic protease from homogenized adult stage U. stenocephala detected by (1) mouse anti-rUs-APR-1 antibodies, and (2) antibodies from a non-immunized mouse (negative control).

Fig. 5. Analysis of the specificity of anti-rUs-APR-1 antibodies to aspartic protease from related hookworm Ancylostoma ceylanicum. ES solution from activated A. ceylanicum larva L3 (1 and 2), A. ceylanicum larva L3 homogenate (3 and 4), A. ceylanicum adult ES (5 and 6) and adult A. ceylanicum homogenate (7 and 8) probed with antibodies from a non-immunized mouse diluted 1:500 (1, 3, 5 and 7, negative controls) and mouse anti-rUs-APR-1 sera (2, 4, 6 and 8).

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