Experimental Parasitology 153 (2015) 81–90

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Characterization of a gut-associated asparaginyl endopeptidase of Clonorchis sinensis Jung-Mi Kang a,1, Jinyoung Lee a,1, Hye-Lim Ju a, Jung Won Ju b, Jong-Hyun Kim c, Jhang Ho Pak d, Tong-Soo Kim e, Yeonchul Hong f, Woon-Mok Sohn a, Byoung-Kuk Na a,* a Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Republic of Korea b Division of Malaria and Parasitic Diseases, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul 122-701, Republic of Korea c Department of Parasitology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea d Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-736, Republic of Korea e Department of Tropical Medicine, and Inha Research Institute for Medical Sciences, Inha University School of Medicine, Incheon 400-712, Republic of Korea f Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea

H I G H L I G H T S

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G R A P H I C A L

A B S T R A C T

Enzymatic characteristics of CsAEP were similar to those of other helminth parasites. CsAEP was expressed in various developmental stages of C. sinensis. CsAEP was localized in the intestine and intestinal contents of C. sinensis. The antibodies specific for CsAEP were detected beginning 4 weeks post infection.

A R T I C L E

I N F O

Article history: Received 22 October 2014 Received in revised form 12 March 2015 Accepted 20 March 2015 Available online 24 March 2015 Keywords: Clonorchis sinensis Asparaginyl endopeptidase Legumain Intestine

A B S T R A C T

Asparaginyl endopeptidases (AEP: EC 3.4.22.34) are a family of cysteine proteases classified into the MEROPS clan CD, family C13. In this study, we characterized the biochemical and antigenic properties of an AEP of Clonorchis sinensis (CsAEP). The recombinant CsAEP showed hydrolytic activity at pH values ranging from acidic to neutral with optimum activity at pH 6.0. While the recombinant CsAEP was stable at neutral pHs, it was unstable at acidic pHs and resulted in loss of enzymatic activity. The recombinant enzyme was effectively inhibited by iodoacetic acid and N-ethylmaleimide, but not by E-64. The partially purified native CsAEP showed biochemical properties similar to the recombinant enzyme. Native CsAEP is likely to be cleaved into an N-terminal mature enzyme and a C-terminal fragment via autocatalytic activation at acidic pHs. Polyclonal antibody raised against the recombinant CsAEP recognized three forms of CsAEP, proenzyme,

* Corresponding author. Fax: +82 55 772 8109. E-mail address: [email protected] (B.-K. Na). 1 These two authors equally contributed to this study. http://dx.doi.org/10.1016/j.exppara.2015.03.015 0014-4894/© 2015 Elsevier Inc. All rights reserved.

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the N-terminal mature enzyme and the C-terminal fragment, in the worm extract (WE) of C. sinensis. However, only the C-terminal fragment was mainly found in the excretory and secretory (ES) products of the parasite. Strong CsAEP activity was found in the WE, but only a trace level of CsAEP activity was detected in the ES products of the parasite. CsAEP was expressed in various developmental stages of C. sinensis, from metacercariae to adults, and was found to be localized in the intestine of the parasite as well as in intestinal contents. Sera from rats experimentally infected with C. sinensis reacted with CsAEP beginning 4 weeks after infection. These results suggest that CsAEP is a gut-associated enzyme synthesized in the intestine of C. sinensis and subsequently secreted into the intestinal lumen of the parasite. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Helminth parasites express a large set of proteases, which mediate various events critical to the physiology and pathobiology of the parasites (Dalton et al., 2003; Delcroix et al., 2006; Horn et al., 2014; Sajid and McKerrow, 2002). Thus, comprehensive studies have been performed to understand the biochemical and functional properties of helminth parasites’ proteases and to utilize the enzymes as promising targets for the development of vaccines, diagnostic methods, and novel anthelmintic drugs (Abdulla et al., 2007; Dalton et al., 2003; Gonzales Santana et al., 2013; Kang et al., 2013; Loukas et al., 2004; McKerrow et al., 2006; Na et al., 2006; Zawistowska-Deniziak et al., 2013). The Chinese liver fluke, Clonorchis sinensis, is an important human pathogen endemic in Far East Asian countries such as China, Korea, Taiwan, and northern Vietnam, and is estimated to infect about 35 million people residing in those areas (Lun et al., 2005). Infection with the parasite results in clonorchiasis, which is associated with a number of hepatobiliary disorders including cholangitis, obstructive jaundice, cholecystitis, and cholelithiasis. A strong epidemiological correlation between C. sinensis infection and the incidence of cholangiocarcinoma also implicates the parasite as a biological carcinogen (Bouvard et al., 2009). Similar to other helminth parasites, C. sinensis produces a large number of different classes of proteases and some of them have been extensively studied due to their essential roles in the biological processes of the parasite. The enzymes expressed in the gut of C. sinensis in particular have received close scrutiny. Multigene family enzymes of cysteine proteases known as CsCFs or CsCLs are expressed in the intestinal epithelium of C. sinensis, actively secreted into the intestinal lumen of the parasite and participate in host protein hydrolysis (Kang et al., 2010; Li et al., 2012; Na et al., 2008). Two leucine aminopeptidases of C. sinensis (CsLAP1 and CsLAP2) are also synthesized in the intestine of the parasite and may be involved in the final catabolic process of host proteins absorbed from the intestine, though the enzymes are not actively secreted outside the parasite (Kang et al., 2012). Several recent studies have revealed that C. sinensis has a diverse family of proteases (Cho et al., 2006; Yoo et al., 2011; Young et al., 2010), most of which have yet to be characterized. Asparaginyl endopeptidases (AEP) or legumains (EC 3.4.22.34) are a novel family of cysteine proteases classified into the MEROPS clan CD, family C13 (Rawlings et al., 2014). They are ubiquitous in a wide range of organisms including plants, animals, and parasitic organisms, and are known to mediate diverse biological events (Brindley and Dalton, 1996; Delcroix et al., 2006; Hara-Nishimura et al., 1998; Manoury et al., 1998). They specifically hydrolyze the carboxyl end of asparagine residues in peptides and proteins. Helminth AEPs have been identified in several parasitic helminthes including Schistosoma mansoni, Fasciola gigantica, Opisthorchis viverrini, Haemonchus contortus, and Angiostrongylus cantonensis (Adisakwattana et al., 2007; Caffrey et al., 2000; Chang et al., 2014; Laha et al., 2008; Oliver et al., 2006). The AEP of C. sinensis (CsAEP) has been identified previously and its potential as a serodiagnostic antigen for clonorochiasis partially analyzed (Ju et al., 2009), but its biochemical and functional properties are not yet fully understood.

In the present study, we characterized the biochemical and functional properties of a CsAEP. Our results suggest that CsAEP has biochemical properties similar to AEPs of other helminth parasites and is mainly expressed in the intestine of C. sinensis. 2. Materials and methods 2.1. Parasites The metacercariae of C. sinensis were collected from naturally infected Pseudorasbora parva obtained in Korea. Sprague–Dawley rats were infected orally with 100 metacercariae each. The 2-week-old juvenile worms and 4-, 6-, and 9-week-old adult worms were harvested from the livers of rats after experimental infection with the metacercariae, respectively (Kang et al., 2010; Na et al., 2008). The sera of infected rats were also collected at the time of sacrifice. The worms were washed 5 times with cold physiological saline to remove any contamination from the hosts. The collected parasites were used for excretory and secretory (ES) products, stored at −70 °C until use, or used immediately for RNA preparation to synthesize cDNA or to analyze the expression profile. The Gyeongsang National University Animal Care and Use Committee approved all animal studies. 2.2. Cloning of a gene encoding CsAEP The gene sequence encoding CsAEP was obtained in a previous study (Ju et al., 2009). The full length gene for CsAEP was amplified with the primers flanking the open reading frame (5′-ATGCGCCG CTCTTGCCTTCTCATTGCG-3′ and 5′-CTAGGAACAGACTTTATGAAC AGATTG-3′) from the cDNA prepared from C. sinensis adult worms as described previously (Kang et al., 2010; Na et al., 2008). The PCR product was analyzed on 1.2% agarose gel, gel-purified and ligated into the T&A cloning vector (Real Biotech Corporation, Banqiao City, Taiwan). The ligation was transformed into Escherichia coli DH5α competent cells and positive clones were selected by colony PCR for the presence of the appropriate insert. The nucleotide sequence of CsAEP was confirmed by automatic DNA sequencing. Analysis of the primary structure of the deduced amino acid sequences was done with DNASTAR (DNASTAR, Madison, WI, USA), PSORT (http://www.psort.org/) and Signal P (http://www.cbs.dtu.dk/ services/SignalP/). The phylogenetic tree was constructed using the neighbor-joining method with MEGA4 (http://www.megasoftware .net). Bootstrap proportions were used to assess the robustness of the tree with 1000 bootstrap replications. Analysis of physicochemical properties of CsAEP was performed using ProtScale (http://www.expasy.org/tools/protscale.html). 2.3. Expression, purification and refolding of recombinant CsAEP To produce recombinant CsAEP, a fragment without the region encoding the signal peptide was amplified by PCR. The primers used were 5′-GTCGACGCATGGTTAGGCAGTGTCTGCGTC-3′ and 5′-AAGC TTCTAGGAACAGACTTTATGAACAGA-3′, which contain a 5′ Sal I site

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and a 5′ Hind III site, respectively. The purified PCR product was subcloned into the T&A cloning vector (Real Biotech Corporation) and was transformed into E. coli DH5α. The resulting plasmid DNA was digested with the appropriate restriction enzymes, ligated into the pQE-30 expression vector (Qiagen, Hilden, Germany), and then transformed into E. coli M15[pREP4] cells (Qiagen). Expression of recombinant CsAEP was induced by addition of isopropyl-1-thioβ-D-galactopyranoside (IPTG) to 1 mM final concentration for 3 h at 37 °C with shaking at 200 rpm for aeration. The cells were harvested by centrifugation at 10,000 × g for 15 min at 4 °C and suspended in 8 M urea lysis buffer. The recombinant protein was purified by nickel–nitrilotriacetic acid (Ni–NTA) chromatography (Qiagen). The purification and purity of the recombinant CsAEP was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE). Refolding of the purified recombinant CsAEP was carried out as described previously (Kang et al., 2010; Na et al., 2008). The CsAEP was further processed to a fully active enzyme by incubating the refolded protein at pH 5.0 for 30 min at room temperature. The pH was then readjusted to 7.0 with 1 M Tris–HCl (pH 8.5) and the precipitated material was removed by centrifugation. The sample was concentrated with Centricon Plus (cut-off 10 kDa; Millipore).

the reaction was stopped by washing the membrane with distilled water. The CsAEP activity in the ES products and the WE were assayed using the same method as described earlier. Native CsAEP was partially purified from the WE by two steps of fast protein liquid chromatography (FPLC) using an Äcta FPLC system (GE Biosciences, Pittsburgh, PA, USA). The WE (10 mg) was loaded onto a HiTrap DEAE column (GE Biosciences; gel volume 1 ml, flow rate 1 ml/min) equilibrated with 50 mM Tris–HCl (pH 7.5) and fractions (each 1.0 ml) were collected. The column was washed with 10 volumes of the same buffer and bound proteins were eluted with a continuous gradient of 1 M NaCl. CsAEP activity in each collected fraction was assayed with the same method described earlier and fractions with CsAEP activity were pooled, dialyzed against 20 mM Tris–HCl (pH 8.0) containing 0.15 M NaCl, and concentrated with Centricon Plus (cut-off 10 kDa; Millipore). The sample was then applied to a Superdex 200 HR 10/30 column (GE Sciences), which was equilibrated and eluted with 20 mM Tris–HCl (pH 8.0) containing 0.15 M NaCl at a flow rate of 0.5 ml/min, and fractions (each 1.0 ml) were collected. CsAEP activity was assayed and the fractions with CsAEP activity were pooled and concentrated. The partially purified CsAEP was confirmed by 12% SDS–PAGE and immunoblot using anti-CsAEP.

2.4. Enzyme activity assay

2.7. Biochemical properties of native and recombinant CsAEP

CsAEP activity was assayed by cleavage of the fluorescent substrate benzyloxycarbonyl-L-alanyl-L-alanyl-L-asparagine 4-methylcoumaryl-7-amide (Z-Ala-Ala-Asn-MCA; Peptide International, Louisville, KY, USA). In brief, 10 μl of the enzyme solution (1 μg) was added to 190 μl of sodium phosphate buffer (pH 6.0) containing 5 μM Z-Ala-Ala-Asn-MCA, and the amount of released fluorescence was measured with excitation and emission wavelengths of 355 nm and 460 nm over 20 min at room temperature with a Fluoroskan Ascent FL (Thermo, Vantaa, Finland).

The optimal pH for maximum activity of CsAEP was assessed in sodium acetate (pH 4.0–5.5), sodium phosphate (pH 6.0–6.5), Tris– HCl (pH 7.0–8.5) and glycine–NaOH (pH 9.0). Native or recombinant CsAEP (50 nM) was added to each pH buffer supplemented with 5 μM Z-Ala-Ala-Asn-MCA and the enzyme activity was measured as described earlier. For each pH, the appropriate blank was separately measured as the control. The pH stability of CsAEP was also examined between pH 5.0 and 8.0 by incubating the enzyme at 37 °C in the appropriate buffers. The effects of protease inhibitors on CsAEP activity were analyzed by preincubation of the enzyme with different concentrations of each inhibitor, for 30 min at room temperature, followed by measuring the residual enzyme activity using Z-AlaAla-Asn-MCA as a substrate. Results were expressed as % residual activity with respect to the control, which was not treated with any inhibitors and was regarded as having 100% activity. The following inhibitors were used in this study: PMSF, pepstatin A, E-64, iodoacetic acid (IAA), N-ethylmaleimide (NEM), ethylenediaminetetraacetic acid (EDTA), and amastatin. All of the inhibitors used in this study were purchased from Sigma or Peptide International. All experiments were carried out in triplicate, and the mean and standard deviation (SD) were calculated. To analyze the glycosylation state of CsAEP, the WE was incubated with endoglycosidase H (Endo H; New England BioLabs, Ipswich, MA, USA) at 37 °C for 2 h and the sample was analyzed by SDS–PAGE followed by immunoblot with anti-CsAEP.

2.5. Production of polyclonal antibody for CsAEP Polyclonal antibody against recombinant CsAEP (anti-CsAEP) was produced by immunizing rats with the purified recombinant CsAEP. The protein (100 μg) was mixed with Freund’s adjuvant (Sigma, St. Louis, MO, USA) and intraperitoneally injected into rats three times at 2-week intervals. Two weeks after the final booster, the rats were sacrificed and the sera were collected. The immunoglobulin G (IgG) fraction was further isolated from the sera with a Protein G-Sepharose column (Amersham Biosciences, Piscataway, NJ, USA). The specificity of the antibody was confirmed by immunoblot analysis. 2.6. Identification and partial purification of native CsAEP Native CsAEP in the ES products and the soluble worm extract (WE) of adult C. sinensis was examined by immunoblot analysis using the anti-CsAEP. The ES products were prepared with the same method described previously (Kang et al., 2010; Na et al., 2008). The WE were prepared by homogenizing the freshly collected worms in physiological saline containing phenylmethylsulphonyl fluoride (PMSF: final concentration 1 mM) and trans-epoxy-succinyl-Lleucylamido(4-guanidino)butane (E-64: final concentration 10 μM) and centrifuging at 15,000 × g for 15 min at 4 °C. The supernatant was collected and used as the WE. The ES products and the WE were separated by 12% SDS–PAGE and transferred to nitrocellulose membrane (Millipore). The membrane was blocked with PBST supplemented with 3% skim milk for 1 h at room temperature. The membrane was then incubated with anti-CsAEP diluted 1:1000 in PBST at room temperature for 2 h. After several washes with PBST, the membrane was incubated with 1:1000 diluted horseradish peroxidase (HRP)-conjugated anti-rat IgG (Sigma). The immunoreactive bands were visualized with 4-chloro-1-naphthol (Sigma) and

2.8. Inhibitory effect of C. sinensis endogenous cysteine protease inhibitors, CsStefins, on CsAEP Recombinant CsStefin-1 and CsStefin-2 were prepared as described previously (Kang et al., 2011, 2014). Native and recombinant CsAEP (each 1 μg) was mixed with different concentrations (1 and 10 μg) of recombinant CsStefin-1 or CsStefin-2 and incubated at room temperature for 30 min. After incubation, the residual activity of CsAEP was assayed as described earlier. CsAEP, which was inactivated by NEM, was used as a negative control. Assays were carried out in triplicate, and the mean and SD were calculated. 2.9. Expression profile of CsAEP in different developmental stages Expression profile of CsAEP in different developmental stages of C. sinensis, metacercariae, 2-week-old juveniles, and 4-, 6-, and 9-week-old adults, was analyzed by semi-quantitative reverse

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transcription PCR (RT-PCR) and immunoblot analysis. Total RNAs were isolated from C. sinensis worms of each different developmental stages using TriZol reagent (Invitrogen, Carlsbad, CA, USA) and were treated with RNase-free DNase (GIBCO BRL, Rockvile, MD, USA) to remove any contaminating DNA. Reverse transcription and the subsequent amplification of CsAEP transcripts were performed with the same amounts of total RNA (1 μg each) and specific primers using the ProtoScript® II RT-PCR Kit (Invitrogen) according to the manufacturer’s instruction. The CsAEP specific primers were 5′-ATGCGCCGCTCT TGCCTTCTCATTGCG-3′ and 5′-CTAGGAACAGACTTTATGAACAGATTG-3′. The C. sinensis actin gene (EU109284) was also amplified for an internal control as described previously (Kang et al., 2010; Na et al., 2008). The amplified PCR products were analyzed on 1.2% agarose gel. Each developmental stage of C. sinensis worms was homogenized in physiological saline and collected as described earlier. The protein concentration in each WE was determined by a Bradford assay (Bio-Rad, USA). The same amount of WE (10 μg) from worms of each developmental stage were separated on 12% SDS–PAGE and transferred to nitrocellulose membrane (Millipore). The membrane was immunoblotted with anti-CsAEP using the same method described earlier. 2.10. Localization of CsAEP To determine the localization of CsAEP in C. sinensis, immunofluorescence assay (IFA) was performed. The freshly prepared C. sinensis adult worms (6-week old) were washed with PBS (20 mM, pH 7.4) and fixed in 4% paraformaldehyde. The worms were dehydrated with a graded ethanol series, embedded in paraffin blocks and stored in a desiccator until use. Sections (4 μm thickness) were mounted on slide glasses, deparaffinized, rehydrated and rinsed with PBS. The slides were incubated with 1:200 diluted anti-CsAEP at room temperature for 2 h and washed with PBS several times. Mouse anti-CsCF-6 was also applied as a standard antibody. After incubation with fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG (Sigma) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated antimouse IgG (Sigma) diluted 1:200 for 2 h, the slides were washed with PBS several times and observed using a confocal laser scanning microscope FV-1000 (Olympus, Japan).

and 194. Three potential N-glycosylation motifs (Asn-Xaa-Ser/Thr sequences) were found at residues 67NIS69, 83NNS85, and 172NKT174, with high potentials at 67NIS69 and 172NKT174. The conserved N-terminal WAVLAV motif and four cysteine residues at the C-terminal were also identified in the CsAEP (Fig. 1). Multiple sequence alignment of the deduced amino acid sequence of CsAEP with those of AEPs from other helminth parasites and mammals revealed that the N-terminal portion was well conserved between the sequences, while the C-terminal portion was less conserved. Analysis of the physico-chemical properties of CsAEP suggested that the putative C-terminal processing site was located at 242N in the N-terminal region of the SHV motif (Fig. 1). CsAEP showed highest sequence identity (76.9%) with the AEP of O. viverrini. The overall sequence identity between CsAEP and AEPs of other trematode parasites was between 39.1 and 76.9%. Phylogenetic analysis revealed that CsAEP was clustered into a clade closely related with AEPs from other trematode parasites, supporting the notion that they are orthologs that descend from a common ancestor (Fig. 2). 3.2. Production of recombinant CsAEP and anti-CsAEP Recombinant CsAEP was expressed in E. coli as an insoluble form. The recombinant protein was purified by Ni–NTA affinity chromatography and its purity was confirmed by SDS–PAGE. The approximate molecular mass of the recombinant protein was 50 kDa, consistent with the estimated molecular mass of the deduced amino acid sequence of CsAEP (Fig. 3a). The recombinant CsAEP was correctly refolded into soluble protein, but only a small portion of the protein (less than 5%) was successfully converted into a soluble form. The refolded sample was further processed under acidic conditions to fully activate the enzyme. SDS–PAGE analysis of the sample showed two proteins with approximate sizes of 30 kDa and 20 kDa (Fig. 3b). Enzyme assay of the sample against Z-Ala-Ala-Asn-MCA showed hydrolytic activity. To produce polyclonal antibody specific to CsAEP, rats were immunized with the recombinant protein and the polyclonal antibody was purified from the sera of immunized rats. The anti-CsAEP specifically reacted with the recombinant CsAEP in immunoblot (Fig. 3c). 3.3. Detection and partial purification of native CsAEP

2.11. Antigenic properties of CsAEP

3. Results

The native CsAEP in the ES products and WE of C. sinensis adult worms was identified by immunoblot analysis using anti-CsAEP. The anti-CsAEP recognized three proteins in the WE with approximate molecular masses of 58, 36, and 22 kDa, respectively (Fig. 4a). Meanwhile, only two proteins were identified in the ES product; a weakly reactive 36 kDa protein and a 22 kDa protein with a stronger reaction to the anti-CsAEP (Fig. 4a). Treatment of WE with Endo H resulted in reduction of the sizes of the protein species identified by anti-CsAEP (Fig. 4a), suggesting the CsAEP is a glycoprotein. We also assayed the CsAEP activity in the ES product and the WE of the parasite. High levels of CsAEP activity were detected in the WE, but only a trace amount of CsAEP activity was observed in the ES product (Fig. 4b). The native CsAEP was partially purified from WE and confirmed by SDS–PAGE followed by immunoblot. SDS–PAGE analysis of the partially purified native CsAEP showed a major protein band with an approximate molecular mass of 36 kDa which reacted strongly with the anti-CsAEP (Fig. 4c).

3.1. Molecular characterization of CsAEP

3.4. Biochemical properties of native and recombinant CsAEPs

The gene encoding CsAEP consists of 1299 bp which encodes a protein of 432 amino acid residues with a predicted molecular mass of 49.5 kDa. Primary sequence analysis of CsAEP revealed that it had a typical N-terminal signal peptide sequence. The catalytic histidine and cysteine residues are located at amino acid positions 153

The biochemical properties of native and recombinant CsAEPs were characterized. Both enzymes showed a hydrolytic activity for Z-Ala-Ala-Asn-MCA at pH values ranging from acidic to neutral with optimum activity at pH 6.0 (Fig. 5a). Both enzymes were stable at neutral pHs, but acidic pHs under pH 6.0 resulted in instability and

Production of antibodies specific for CsAEP in rats experimentally infected with C. sinensis was analyzed. Five rats were infected with 100 C. sinensis metacercariae, and the sera from each rat were collected at 0, 2, 4, 6, 8, and 12 weeks post-infection. Purified recombinant CsAEP was separated by 12% SDS–PAGE and transferred onto nitrocellulose membrane (Millipore). The membrane was cut into strips and blocked with PBST supplemented with 3% skim milk for 1 h. The strips were then incubated with a 1:200 dilution of sera from each rat collected at the different timepoints, respectively, at room temperature for 3 h. After several washes with PBST, the strips were incubated with 1:1000 diluted peroxidase-conjugated antirat IgG (Sigma). The immuno-reactive bands were visualized with 4-chloro-1-naphthol (Sigma), and the reaction was stopped by washing the strips with distilled water.

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Fig. 1. Multiple sequence alignment of deduced amino acid sequence of CsAEP with those of AEPs from other organisms. The dashes represent gaps introduced to maximize alignment. The predicted signal peptide region of CsAEP is underlined. The sequences of the N-terminal WAVLVA motif and the SHV motif, which are well conserved in AEPs, are marked with a bold and a dotted line on the sequences, respectively. The four conserved cysteine residues of the C-terminal peptide are labeled with open reverse triangles. Asterisks indicate the conserved active site residues. The putative N-glycosylation sites are marked with closed circles. The possible C-terminal processing site of CsAEP, which is essential for fully activating the enzyme, is indicated by an arrow. Percentage of sequence identity is represented as shading. C. sinensis (GAA42795); Opisthorchis viverrini (ABD64147); Fasciola hepatica (CAC85636); Fasciola gigantica (ABQ02437); Schistosoma mansoni (AAA29895); Schistosoma japonicum (AAR30508); Haemonchus contortus (CAJ45481); Brugia malayi (EDP29073); human (AAH03061); mouse (CAA04439).

rapid loss of enzymatic activities (Fig. 5b). The native and recombinant CsAEPs were not inhibited by amastatin, PMSF, E-64, EDTA or pepstatin A. However, they were effectively inhibited by both IAA and NEM (Fig. 5c). We also analyzed inhibitory effects on the activity of CsAEP by CsStefins, which are endogenous cysteine protease inhibitors of C. sinensis. Both native and recombinant CsAEPs were not affected by CsStefins (Fig. 5d). 3.5. Expression pattern and localization of CsAEP The expression profile of CsAEP in different developmental stages of C. sinensis was analyzed by semi-quantitative RT-PCR. CsAEP was expressed through all examined developmental stages of the parasite, which included metacercariae, juveniles and adult worms. The transcription level of the gene increased gradually with the maturation of the parasite from metacercariae to adults (Fig. 6a). To further investigate the developmental expression of CsAEP, we also analyzed its expression by immunoblot using the anti-CsAEP. The anti-CsAEP recognized relevant proteins in the WE, consistent with the results of semi-quantitative RT-PCR (Fig. 6b). IFA was used to determine the localization of CsAEP in the parasite. Strong fluorescence signals were detected in the epithelial cells lining the intestine and also in the intestinal contents of the parasite (Fig. 6c). These results collectively demonstrate that CsAEP is synthesized in the

epithelial cells lining the parasite intestine and is subsequently secreted into the intestinal lumen of the parasite. 3.6. Antigenic property of CsAEP The antigenic property of CsAEP was analyzed by immunoblot. CsAEP showed antigenicity against the sera from rats experimentally infected with C. sinensis. Specific antibodies for CsAEP were detected beginning 4 weeks after experimental infection (Fig. 7). 4. Discussion Gut-associated proteases of helminth parasites have been recognized as promising targets for novel anthelmintic drugs (Delcroix et al., 2006; McKerrow et al., 2006; Na et al., 2006; Sajid and McKerrow, 2002). They are involved in a wide range of essential biological functions in parasites, such as nutrient uptake, host tissue invasion, and host immune evasion. Similar to other trematode parasites, gut-associated proteases have been extensively studied in C. sinensis due to their essential roles in the biological processes of the parasite. The enzymes studied most extensively have been CsCFs or CsCLs, multigene family enzymes abundantly expressed in the intestinal epithelium and actively secreted outside of the parasite (Kang et al., 2010; Li et al., 2009; Na et al., 2008). They play primary

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Fig. 2. Phylogenetic analysis of CsAEP and other related enzymes. The tree was built with the neighbor-joining method using the MEGA 4 program. Numbers on the branches indicate bootstrap proportions (1000 replicates).

proteins absorbed from the intestine (Kang et al., 2012). Helminth parasites express diverse classes of proteases in their intestine and the enzymes construct a complex proteolytic cascade or network (Delcroix et al., 2006). Identification of proteases expressed in the intestine of C. sinensis and understanding the role of each enzyme can therefore provide clear insight into the functional network of the enzymes in the parasite’s intestine. In this study, we characterized the biochemical and antigenic properties of CsAEP, a clan CD cysteine protease that is functionally expressed in the intestine of C. sinensis. Sequence and phylogenetic analyses of CsAEP revealed that the enzyme is a typical AEP belonging to the clan CD, family C13. The characteristic active site forming amino acid residues (histidine and cysteine) and several motifs, which are well conserved in the AEP family of enzymes, were present in CsAEP. It also displayed a high level of sequence identity to homologous enzymes from other trematode parasites. CsAEP

roles in the nutrient uptake of the parasite, however the fact that they are actively released into outside of the parasite suggests possible extracorporeal roles of the enzymes (Kang et al., 2010; Li et al., 2012). Strong antigenic responses of the enzymes against sera from experimentally infected animals and/or patients with C. sinensis infection also render them as promising candidates for the development of serodiagnostic method for clonorchiasis (Lv et al., 2012; Na et al., 2002). Cathepsin B of C. sinensis (CsCB) is reported to be expressed in the intestine and eventually secreted into outside of the parasite (Chen et al., 2011). We also discovered that C. sinensis express a family of CsCB which are encoded by at least 5 different genes (GenBank accession numbers: EF071861, EF102086, EF102087, EF207785 and EF443056). Some of those enzymes are synthesized in the intestine of the parasite (our unpublished data). Two CsLAPs are also synthesized in the intestine epithelial cells of the parasite and may be involved in the final catabolic process of host

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Fig. 3. Productions of recombinant CsAEP and anti-CsAEP. (a) Expression and purification of recombinant CsAEP. Recombinant CsAEP was produced as an insoluble protein in E. coli. Proteins were analyzed by SDS–PAGE and stained with Coomassie blue. Lane 1, Escherichia coli lysate control; lane 2, IPTG-induced E. coli lysate; lane 3, purified recombinant CsAEP using Ni-NTA affinity chromatography. (b) Refolding and activation of CsAEP. The purified recombinant CsAEP was refolded and the refolded protein was activated under acidic condition. Proteins were analyzed by SDS–PAGE and stained with Coomassie blue. Lane 1, refolded CsAEP; lane 2, activated CsAEP under acidic conditions. (c) Production of anti-CsAEP. Rats were immunized recombinant CsAEP, the IgG portion was purified from the sera of immunized rats and immunoblotted against recombinant CsAEP. Lane 1, pre-immunized normal rat IgG; lane 2, anti-CsAEP.

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Fig. 4. Detection and partial purification of native CsAEP. (a) Detection of native CsAEP in the ES products and WE of C. sinensis adult worms. The ES products (20 μg) and WE (20 μg) of C. sinensis were separated on 12% SDS–PAGE, transferred onto nitrocellulose membrane, then probed with anti-CsAEP. Endoglycosidase H-treated WE (Endo H) was also analyzed by immunoblot with anti-CsAEP to determine the glycosylation state of CsAEP. (b) Enzyme assay. The activity of CsAEP in the ES products (20 μg) and WE (20 μg) of C. sinensis was assayed using Z-Ala-Ala-Asn-MCA as a substrate. 4W, 4-week-old adult worms; 6W, 6-week-old adult worms. (c) Partial purification of native CsAEP. The partially purified native CsAEP (10 μg) was analyzed by 12% SDS–PAGE followed by Coomassie blue staining (SDS–PAGE) or immunoblot with anti-CsAEP (IB).

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pH 6.0 and are unstable at acidic pHs. Both enzymes were effectively inhibited by IAA and NEM but not by E-64 or by inhibitors for other classes of proteases. Mammalian AEPs can be inhibited by some cystatins, cystatin C, cystatin E/M, and ovocystatin

Residual activity (%)

shared similar biochemical properties with AEPs from other organisms including helminth parasites (Adisakwattana et al., 2007; Chang et al., 2014; Chen et al., 1997; Laha et al., 2008; Oliver et al., 2006). Both native and recombinant CsAEPs have an optimum activity at

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Fig. 5. Biochemical properties of native and recombinant CsAEP. (a) Optimal pH. Enzyme activity was assayed in various pH buffers ranging from pH 4.0 to 9.0. Maximal activity for each enzyme was shown as 100%. Native CsAEP (○) and recombinant CsAEP (●). (b) pH stability. Recombinant or native CsAEP was incubated in buffers at various pH at 37°C for the indicated time and residual enzyme activity was assayed. pH 5.0 (●), pH 6.0 (○), pH 7.0 (■), pH 8.0 (□), pH 9.0 (▲). (c) Effects of protease inhibitors. Native (white bar) and recombinant (grey bar) CsAEP (each 1 μg) were preincubated with each inhibitor in 50 mM Tris–HCl (pH 7.0) at room temperature for 30 min. Following the addition of substrate, the residual enzyme activity in each sample was measured in 50 mM sodium acetate (pH 6.0) using Z-Ala-Ala-Asn-MCA as a substrate. Results are expressed as % relative activity compared to the control. The control was not treated with any inhibitors and was considered as having 100% relative activity. PMSF, phenylmethylsulphonyl fluoride; E-64, trans-epoxy-succinyl- L -leucylamido(4-guanidino)butane; IAA, iodoacetic acid; NEM, N-ethylmaleimide; EDTA, ethylenediaminetetraacetic acid. (d) Effect of CsStefins on the activity of CsAEP. Native (white bar) and recombinant (grey bar) CsAEP (each 1 μg) were preincubated with either CsStefin-1 or CsStefin-2 (1 or 10 μg) in 50 mM Tris–HCl (pH 7.0) at room temperature for 30 min. After incubation, the residual enzyme activity in each sample was assayed in 50 mM sodium acetate (pH 6.0) using Z-Ala-Ala-Asn-MCA as a substrate. Results are expressed as % activity with respect to the control, which was not treated with any CsStefins. All the assays were carried out in triplicate and the mean and standard deviation (SD) were calculated.

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a

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IN Control Fig. 6. Expression pattern and localization of CsAEP. (a) Semi-quantitative RT-PCR. Transcriptional profile of CsAEP gene at various developmental stages of C. sinensis. Semiquantitative RT-PCR was performed as described in materials and methods. The reaction was conducted in the presence (+) or absence (–) of reverse transcriptase (RTase) to verify DNA contamination. C. sinensis actin gene was amplified as an internal control. PCR products were analyzed on 1.2% agarose gel with ethidium bromide staining. M, metacercariae; 2W, 2-week-old juvenile worms; 4W, 4-week-old adult worms; 6W, 6-week-old adult worms; 9W, 9-week-old adult worms. (b) Immunoblot analysis. The soluble lysate of each developmental stage of C. sinensis were separated on 12% SDS–PAGE, transferred onto nitrocellulose membrane, and then immunoblotted with anti-CsAEP. 2W, 2-week-old juvenile worms; 4W, 4-week-old adult worms; 6W, 6-week-old adult worms; 9W, 9-week-old adult worms. (c) Immunofluorescence assay. Sections of C. sinensis adult worms were probed with rat anti-CsAEP and mouse anti-CsCF-6 followed by FITC-conjugated anti-rat IgG and TRITC-conjugated anti-mouse IgG. The slide was observed with a confocal laser scanning microscope (×10 magnification). IN, intestine; U, uterus. Scale bar indicated 100 μm. Control, non-immunized mouse sera.

(Alvarez-Fernandez et al., 1999; Chen et al., 1997; Smith et al., 2012). In this study, we analyzed the inhibitory effect of CsStefins on CsAEP. CsStefins are gut-associated endogenous cysteine proteases inhibitors of C. sinensis that belong to the family I stefins (Kang et al., 2011, 2014). Two CsStefins, CsStefin-1 and CsStefin-2, are synthesized at the intestine epithelium of C. sinensis and are involved in regulating the processing and activity of CsCFs (Kang et al., 2011, 2014). CsAEP activity was not influenced by either CsStefins, which suggested that CsAEP activity is not regulated by CsStefins. Indeed, it has also been reported that mammalian AEPs are not significantly influenced by type 1 cystatins (cystatins A and B) or by the low molecular weight inhibitor kininogen (Alvarez-Fernandez et al., 1999). AEPs are synthesized as proproteins and autocatalyzed to the mature form through cleavage of N- and/or C-terminal peptides (Caffrey et al., 2000; Chen et al., 2000; Halfon et al., 1998; Hiraiwa et al., 1999; Li et al., 2003). This process results in the formation of two fragments, an active N-terminal enzyme fragment and a

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12345 12345 12345 12345 12345 12345

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Fig. 7. Antigenic property of CsAEP. Time-course antibody production specific for CsAEP in rats experimentally infected with C. sinensis was analyzed. Five rats were infected with 100 C. sinensis metacercariae and the sera from each rat were collected at 0 (0W), 2 (2W), 4 (4W), 6 (6W), 8 (8W), and 12 (12W) weeks post-infection. The purified recombinant CsAEP was separated on 12% SDS–PAGE, transferred onto nitrocellulose membrane, cut into strips, and then probed with sera from five rats.

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C-terminal peptide fragment. The internal cleavage sites of only a few AEPs are known currently, but it is likely that AEPs undergo autocatalytic processing at different positions due to sequence dissimilarities (Adisakwattana et al., 2007). Comparison with known or predicted cleavage sites in other AEPs suggests that the cleavage positions possess hydrophilic and flexible characteristics and the cleavages occur at an asparagine residue located between the SHV motif and the first cysteine residue of four conserved cysteine residues at the C-terminal (Adisakwattana et al., 2007; Chen et al., 2000; Kuroyanagi et al., 2002). The approximate molecular mass of the CsAEP proenzyme was predicted to be 49.5 kDa when calculated from its deduced amino acid sequence and this coincided well with the molecular mass of recombinant CsAEP. Immunoblot analysis of the WE of C. sinensis using anti-CsAEP revealed three proteins with approximate molecular masses of 58 kDa, 36 kDa, and 22 kDa, respectively, that specifically reacted with anti-CsAEP, suggesting the three proteins originated from CsAEP. Interestingly, CsAEP do not have an asparagine residue between SHV motif and four cysteine residues conserved in the C-terminal portion. From analysis of the physico-chemical properties of the CsAEP sequence and the expected molecular masses of the three reactive proteins detected in immunoblot analysis, the putative C-terminal autocatalytic cleavage site of CsAEP was predicted to be at 242N, in the N-terminal region of the SHV motif. Consequently, the approximate molecular mass of the processed N-terminal active enzyme of CsAEP was expected to be 28 kDa, while the removed C-terminal fragment was expected to be 22 kDa. This conclusion was also supported by the activation process of the refolded recombinant CsAEP. Activation of the refolded recombinant CsAEP (about 50 kDa) in acidic pH resulted in two fragments with approximate molecular masses of 28 and 22 kDa. Taken together, these results indicate that the 58 kDa protein recognized by anti-CsAEP in immunoblot corresponds to the CsAEP proprotein, while the 36 kDa and 22 kDa proteins identified in the immunoblot assay correspond to the N-terminal and C-terminal parts of the cleaved CsAEP, respectively. However, the molecular masses of the proenzyme and the N-terminal portion of native CsAEP identified were higher than predicted by ~8 kDa. CsAEP has three potential N-glycosylation sites in its N-terminal end which could explain the discrepancy in molecular weights between the sizes of predicted and observed proteins in the immunoblot assay. N-glycosylation at any of the potential sites on the N-terminal fragment may increase the molecular mass of the native CsAEP. Treatment of Endo H resulted in size reduction of native CsAEP, supporting glycosylation state of CsAEP. Further in-depth analysis to determine the exact C-terminal cleavage of CsAEP is therefore necessary. CsAEP is expressed in various developmental stages of C. sinensis, from metacercariae to adults, and is localized in the intestine of the parasite and in intestinal contents. These results indicate that CsAEP is a protein synthesized in the intestine of C. sinensis and secreted into the intestinal lumen of the parasite, then subsequently released outside the parasite as part of the ES products. However, in our immunoblot analysis, CsAEP is not clearly detected in the ES products, though the enzyme is obviously present in the WE of the parasite. The 22 kDa C-terminal portion of CsAEP was identified in both the ES products and the WE, but the 36 kDa N-terminal mature enzyme and the 58 kDa proenzyme detected in the WE were not observed in the ES products. CsAEP activity assay of the ES products also suggested that only trace amounts of active CsAEP were present. It has been reported that the AEP of O. viverrini (Ov-AEP1) was identified in the ES products of the parasite (Laha et al., 2008). Meanwhile, two AEPs of F. gigantica (FgLGMN-1 and FgLGMN-2) were not identified in the ES products of the parasite (Adisakwattana et al., 2007). Similar to other trematode parasites, the pH of the intestinal lumen of C. sinensis was estimated to be acidic (around pH 5.0–6.0) (Kang et al., 2010). Since CsAEP was unstable and rapidly

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lost its activity at acidic pH (