Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Veterinary Parasitology 141 (2006) 251–259 www.elsevier.com/locate/vetpar

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode Ai Hua Li a,b,1, Sung-Ung Moon a,1, Yun-Kyu Park c, Byoung-Kuk Na d, Myung-Gi Hwang c, Chang-Mi Oh a, Shin-Hyeong Cho a, Yoon Kong e, Tong-Soo Kim a, Pyung-Rim Chung c,* a

Division of Malaria and Parasitic Diseases, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul 122-701, Republic of Korea b State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China c Department of Parasitology, Inha University College of Medicine, Inchon 400-712, Republic of Korea d Department of Parasitology and Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 660-751, Republic of Korea e Department of Molecular Parasitology and Center for Molecular Medicine, Samsung Biomedical Research Institute and Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea Received 20 January 2005; received in revised form 8 May 2006; accepted 15 May 2006

Abstract Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T. solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3–44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9–55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. # 2006 Published by Elsevier B.V. Keywords: Taenia solium metacestode; Cysteine protease; Recombinant protein

1. Introduction

* Corresponding author. Tel.: +82 32 890 0981; fax: +82 32 884 2104. E-mail address: [email protected] (P.-R. Chung). 1 These two authors contributed equally to this work. 0304-4017/$ – see front matter # 2006 Published by Elsevier B.V. doi:10.1016/j.vetpar.2006.05.015

Taenia solium, a pork tapeworm, is a cestode parasite which infects humans as a definitive host. Human infection usually occurs by ingesting raw or inadequately cooked pork infected with cysticerci. The disease has gained attention in several regions including

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Africa, Southern Asia, Eastern Europe and South America due to its reemergence by increasing immigration and frequent travel to endemic areas. T. solium is associated with two distinct infected states in humans: asymptomatic intestinal infection by adult tapeworms (Taeniosis) and symptomatic infection due to the presence of cysticerci (metacestodes) in muscles or organs (cysticercosis). T. solium metacestode (TsM) can persist for years in human tissue. Particularly, the presence of TsM in the central nervous system causes neurocysticercosis, which is one of the most common parasitic diseases in the central nervous system. The main clinical manifestations of the disease are headache, focal and generalized seizure, hydrocephalus and nonspecific neurological deficits according to the number and location of the parasites in the brain (Carpio, 2002). Cysteine proteases of parasites play various essential roles in host-parasite interactions and have gained significant attention as targets for chemotherapy or immunoprophylaxis (Sajid and McKerrow, 2002). A 43 kDa cysteine protease purified from T. crassiceps has been shown to hydrolyze host immunoglobulin G (IgG) (White et al., 1997). Cysteine protease secreted by TsM depleted CD4 lymphocytes in vitro and induced apoptosis in human CD4+ T-cells (Molinari et al., 2000; Tato et al., 2004). Most recently, two cysteine proteases have been purified from TsM and partial biochemical properties of the purified enzymes have been elucidated (Baig et al., 2005; Kim et al., 2005). Both enzymes degraded human immunoglobulin, therefore it has been proposed that these enzymes play key roles in the host-parasite interactions and that they could be employed as targets for chemotherapy. However, elucidation of the precise biological roles of the enzymes has been hampered by difficulties in obtaining a large amount of homogeneous enzymes. In this study, we isolated a cDNA encoding a cathepsin L-like cysteine protease from TsM and characterized partial biochemical properties of the recombinant protein. 2. Materials and methods 2.1. Parasites Taenia solium eggs were isolated from gravid proglottids of the adult worm obtained from a human patient treated with niclosamide, and suspended in cold sterile phosphate buffered saline (PBS, pH 7.2). Three piglets were orally infected with approximately 4  104

eggs each. At 4 weeks after infection, the developed T. solium metacestode (TsM) was harvested by dissection. After several washings with PBS, the collected parasites were stored at 70 8C until use or were immediately used for RNA isolation. 2.2. Cloning of TsM cysteine protease (TsCL-1) Total RNA was isolated from 100 mg of frozen worms by using Trizol reagent (GIBCO BRL, Grand Island, NY, USA) following the manufacturer’s instruction. First-strand cDNA was synthesized from the total RNA by using oligo dT30-CDC (50 -AAGCAGTGGTATCAACGCAGAGTACT(30)VN-30 ) and a SMARTTM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). Degenerate primers for polymerase chain reaction (PCR) were designed based on the highly conserved regions of amino acids from cysteine proteases of other helminth parasites. The sequence of the forward primer was 50 -AAGAANCAGGGCCARTGCGGNTCNTGTTG-30 and that of reverse primer was 50 -RCAACTGTTCTTNACYAACCARTA-3. The amplification reaction was done with a thermal cycling profile of 94 8C (4 min), 35 cycles at 94 8C (1 min), 50 8C (1 min), and 72 8C (1.5 min), followed by a final extension at 72 8C (10 min). The amplified gene fragment was purified from the gel with a Nucleospin Gel Extract Kit (Macherey-Nagel GmbH, Germany) and ligated into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA). After being transformed into competent Escherichia coli TOP10 cells (Invitrogen), nucleotide sequence of the cloned gene was determined by using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystem, UK) and an ABI automated DNA sequencer. From this sequence data, a pair of specific primers (GSPf: 50 CAAACGCACCCTCCAACGCTCCTG-30 and GSPr: 50 -TGCTCGAGTAAATTCCTCAACCACGG-30 ) were made and were used for rapid amplification of cDNA ends (RACE)-PCR to obtain the 30 - and 50 -ends of the gene using a SMARTTM RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instruction. The amplified products were purified from the gel, cloned into pCR2.1-TOPO vector (Invitrogen), and sequenced. To eliminate the possibility that the cloned TsCL-1 may originate from pig, the host animal used for TsM collection, RT-PCR analysis using mRNAs from TsM and normal pig tissue was performed by using TsCL-1 specific primers, 50 -CCGGAGAGTAGAGTAGCTGGAAGGCGT-30 and 50 -GTTCCCCTCTAGAATATCACCAATATC-30 . The amplified product was analyzed by agarose gel electrophoresis.

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2.3. Molecular analysis of TsCL-1 The predicted amino acid sequence of the putative full-length clone was analyzed for similarity to other known sequences using the BLAST program within the NCBI server. Multiple alignment of amino acid sequences was conducted using the MegAlign program in the DNASTAR (Madison, WI, USA). Phylogenetic analysis of the aligned sequences was done with distance methods by using the MEGA program version 3.1. A Bootstrap consensus tree was constructed by using the neighbor-joining (NJ) method. The stability of the trees was estimated by bootstrap analysis for 1000 replications using the same program. The detection of signal peptide was done with the Signal P 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/). The nucleotide sequence of TsCL-1 has been deposited in the GenBank database with the Accession number AY515271. 2.4. Expression of recombinant TsCL-1 (rTsCL-1) in E. coli To produce rTsCL-1, a portion of TsCL-1, which did not contain the 17 amino acid-length signal peptide, was amplified by PCR using primers 50 -AGTCGTTCATATGGAAACCTCGGCACTTC-30 and 50 -GAACA GTCGACTTAAACGTATGGGAAATC-30 . Restriction enzyme sites, NdeI and SalI, were incorporated into primer sequences for cloning. The PCR product was purified, ligated into pCR2.1-TOPO vector, and transformed into competent E. coli TOP10 cells (Invitrogen). After purification, plasmid DNA was digested with NdeI and SalI and ligated into pET-28a expression vector (Novagen, Madison, WI, USA) predigested with the same enzymes. The resulting plasmid was transformed into competent E. coli BL-21 (DE3) cells (Novagen) and spread onto Luria–Bertani agar plates containing 30 mg/ml of kanamycin. The expression of the recombinant protein was induced by adding isopropyl-1-thio-b-D-galactopyranoside (IPTG) to a final concentration of 1 mM and the expressed protein was purified with a nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen, Valencia, CA, USA) and Ultrogel AcA202 gel filtration chromatography under native conditions containing protease inhibitor (Roche, Germany). 2.5. Expression of rTsCL-1 in Pichia pastoris For the expression of TsCL-1 in a yeast system, the open reading frame of TsCL-1 was amplified with the

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primers 50 -AGTCGTTGAATTCGAAACCTCGGCACTTC-30 and 50 -GAACAGGTACCTTAAACGTATGGGAAATC30 . Restriction enzyme sites, EcoRI and KpnI, were incorporated into 50 of the primer for cloning. The amplified PCR product was purified and subcloned into the pCR2.1 vector, and transformed into E. coli TOP10 cells. A positive transformant was selected and the plasmid was purified and digested with EcoRI and KpnI. The resulting insert was purified and ligated into the pPICZaA vector (Invitrogen). E. coli TOP10 cells were then transformed with the construct. Bacteria were plated on agar plates containing low-salt Luria broth (1% tryptone, 0.5% NaCl and 0.5% yeast extract) and Zeocin (25 mg/ml). Positive clones were selected by PCR and sequenced to confirm the reading frame of the insert. Recombinant plasmid was linearized and transformed into Pichia pastoris strain KM71H (MutS phenotype) by using an EasySelect kit (Invitrogen). Transformed cells were selected on YPDS (1% yeast extract, 2% peptone, 2% dextrose and 1 M sorbitol) plates supplemented with Zeocin (100 mg/ml) and grown for several days at 30 8C. Positive clones containing the insert were selected and grown at 30 8C in 10 ml of BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 1% glycerol, 0.00004% biotin and 0.1 M potassium phosphate; pH 6.0) in 50 ml tubes with vigorous shaking. Cells were harvested by centrifugation, resuspended in 2 ml of BMMY medium (BMGY medium with 0.5% methanol substituted for glycerol), and cultured for 6 days. During the induction period, methanol was added every 24 h to maintain the final concentration of 0.5% (vol/vol). Large-scale culture was carried out for 5 days after induction. The cells were pelleted by centrifugation and the resulting supernatants were harvested and concentrated by 75% ammonium sulfate precipitation. The resulting pellet was dissolved in distilled water and equilibrated with phosphate-buffered saline (PBS) (pH 7.4) by using a PD-10 column (Amersham Biosciences, Uppsala, Sweden). The expressed rTsCL-1 was purified by Ni-NTA agarose and gel filtration chromatography by using Ultrogel AcA202 and Superdex 75 (Amersham Biosciences), and was concentrated by lyophilization. The purity of the purified rTsCL-1 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Blue staining. For substrate gel analysis, rTsCL-1 was mixed with SDSPAGE sample buffer without 2-mercaptoethanol and electrophoresed onto an SDS-polyacrylamide gel copolymerized with 0.1% gelatin. The gel was washed twice with 2.5% Triton X-100 at room temperature for 30 min, incubated overnight at 37 8C in 100 mM sodium phosphate (pH 6.5) containing 10 mM DTT, stained with

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Fig. 1. Multiple sequence alignment of the deduced amino acid sequence of TsCL-1 with those of other cathepsin L cysteine proteases. The deduced amino acid sequences were aligned using the MegAlign program in DNASTAR. Gaps were introduced to maximize the alignment. The conserved ERFNIN, GNFD and GCNGG motifs are indicated on top of the alignment. An arrowhead indicates the putative mature domain processing site of TsCL1. Asterisks represent the residues which are essential for active site formation. C. sinensis (AFF40479); F. hepatica (AAF76330); P. westermani (AAB93494); S. erinacei (BAA09821); Human (AAH12612); Mouse (NP_034114); Pig (BBA07140); Rat (CAA68691); Dog (AJ279008).

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Fig. 2. Phylogenetic analysis of TsCL-1. The unrooted neighbor-joining method was used to construct the phylogenetic tree using the MEGA 3.1 program. The numbers at nodes are the proportions of 1000 bootstrap resamplings that support the topology shown. The scale bars indicate a distance of 0.1.

Coomassie Blue and then destained to identify proteolytic activity as clear bands on the gel. The protein concentration was determined by the Bradford method with BSA as the standard. 2.6. Characterization of rTsCL-1 The proteolytic enzyme activity was assayed as the hydrolysis of the fluorogenic dipeptide substrate, benzyloxycarbonyl-Leu-Arg-7-amino-4-methyl coumarin (Z-LR-AMC; Peptides International, Louisville, KY, USA). The purified rTsCL-1 was added to sodium phosphate buffer (320 ml, pH 6.5) containing 5 mM ZLR-AMC and 10 mM DTT, and the fluorescence levels were quantified at 355 nm (excitation) and 460 nm (emission) over a 20 min period, using a SpectraMAX Gemini fluorometer (Molecular Devices, Sunnyvale, CA, USA). The optimal pH was assessed in sodium acetate (pH 4.5–5.5), sodium phosphate (pH 6.0–6.5) and Tris–HCl (pH 7.0–9.0). The rTsCL-1 was added to each buffer supplemented with 5 mM Z-LR-AMC and 10 mM DTT. Relative activity was measured as

described above. For each pH step, the appropriate blanks were separately measured as control groups. The effects of reducing agents were examined under various concentrations of DTT, and pH stability was examined at pH 5.0, 6.5 and 8.0 by incubating the purified enzyme at 37 8C in the appropriate buffers. Active site titration was done using a specific inhibitor, E-64. The hydrolysis of the peptide substrate in the presence of a constant amount of rTsCL-1 (25 nM) and varying substrate concentrations was assessed in 100 mM sodium phosphate (pH 6.5) containing 10 mM DTT. The kinetic parameters of rTsCL-1 for Z-LR-AMC were determined at room temperature using varying concentrations of the substrate in 100 mM sodium phosphate (pH 6.5) containing 10 mM DTT. To determine the hydrolytic activity of rTsCL-1 against macromolecular proteins, human immunoglobulin G (IgG), bovine serum albumin (BSA) and collagen were incubated with the purified rTsCL-1 (20 nM) for 3 h at 37 8C. The reactions were stopped by adding an equal volume of reducing sample buffer, and were analyzed by SDS-PAGE.

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2.7. Western blot analysis Purified rTsCL-1, which expressed in E. coli, was separated by 12% SDS-PAGE and was transferred to a polyvinylidene difluoride membrane. The membrane was cut into strips, blocked with 5% non-fat milk and probed with 1:200 diluted pooled sera from patients with helminth infections for 1 h at room temperature. The primary antibody consisted of pooled sera from 10 patients infected with Clonorchis sinensis, Paragonimus westermani, T. solium, Spirometra erinacei and/or Fasciola hepatica, or from 10 healthy peoples. The membranes were washed three times with PBS containing 0.05% Tween-20 (PBST) and further were incubated in 1:1000 diluted horseradish peroxidaseconjugated anti-human rabbit IgG (Amersham Biosciences, Uppsala, Sweden). Immunoreactive bands were visualized with 3,30 -diaminobenzidine tetrahydrochloride in the presence of 0.05% hydrogen peroxide. 3. Results 3.1. Molecular analysis of TsCL-1 The putative sequence of TsCL-1 was obtained by sequential PCR procedures of PCR with degenerate primers and RACE-PCR. The identified TsCL-1 sequence had 1216 bp with an open reading frame (ORF) of 1020 bp and deduced amino acids of 339. The 50 -UTR prior to the ATG start codon was 22 bp long, whereas the 30 -UTR after the TGA stop codon consisted of 174 bp, but lacked the consensus polyadenylation signal sequence AATAAA. The most probable cleavage site between the signal sequence and the pro-domain was determined to be between Asn17 and Val18. A theoretical cleavage site between the pro-domain and the mature domain was determined to be between Gly123 and Leu124 by multiple sequence alignment with several well-known cathepsin L-like proteases. ERFNAQ, GNFD and GCNGG motifs, which are conserved in the cathepsin-L cysteine protease family, were identified in both the pro- and mature domains. The amino acid residues Q142, C148, H286, N306 and W308, which are crucial for active site formation of cysteine proteases, were also conserved in the mature domain of TsCL-1. Two putative N-glycosylation sites at Asn223 and Asn241 were also found. Multiple sequence alignments of the entire sequences of TsCL-1 revealed a low degree of similarity of 27.3–44.6 to other cathepsin L-like cysteine proteases of other helminth parasites and mammals; however, the similarity

increased to 33.8–55.0 when compared to mature domains (Fig. 1). Phylogenetic analysis was also performed to clarify the phylogenetic relationship of TsCL-1 with cathepsin L-like cysteine proteases from other organisms. The topology of the phylogenetic tree demonstrated that TsCL-1 shared a more inclusive clade with cathepsin L-like cysteine proteases of trematode and cestode parasites, such as S. erinacei and F. hepatica than with those from nematodes and mammals (Fig. 2). RT-PCR analysis using TsCL-1 specific primers showed that the amplified PCR product was detected in mRNA sample from TsM, but not the sample from pig tissue (data not shown). 3.2. Expression and characterization of rTsCL-1 The rTsCL-1 expressed in E. coli was purified and analyzed by SDS-PAGE. The molecular weight of the purified rTsCL-1 was approximately 40 kDa, which corresponded well to the predicted size from the primary sequence of the gene (Fig. 3A). However, the rTsCL-1 expressed in E. coli did not show proteolytic enzyme activity. Therefore, we tried to express TsCL-1 in a eukaryotic expression system, P. pastoris. The rTsCL-1 expressed in P. pastoris was found predominantly in the culture supernatant. Its molecular weight was about 28 kDa on SDS-PAGE and it showed clear

Fig. 3. Expression and purification of rTsCL-1. (A) Expression and purification in E. coli. Lane M, molecular mass standards (masses shown on the left side); lane 1, E. coli lysate without IPTG induction; lane 2, E. coli lysate with IPTG induction; lane 3, unbound fraction of Ni-NTA affinity chromatography; lane 4, purified rTsCL-1 by Ni-NTA affinity chromatography; lane 5, further purified rTsCL-1 by gel filtration chromatography. (B) Expression and purification in P. pastoris. The expressed rTsCL-1 was purified by Ni-NTA and gel filtration chromatographies. Left, SDS-PAGE analysis of the purified rTsCL-1; right, Zymography analysis of the purified rTsCL-1.

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Fig. 4. Characterization of rTsCL-1 expressed in P. pastoris. (A) Optimum pH. Enzyme activities were assayed in 100 mM sodium acetate (pH 4.5– 5.5), sodium phosphate (pH 6.0–6.5) and Tris–HCl (pH 7.0–9.0), in the presence of 1 mM DTT. (B) Enzyme stability. The rTsCL-1 was incubated at 37 8C in different pH buffers, after which residual activity was detected in 100 mM sodium phosphate (pH 6.5) containing 1 mM DTT at the indicated time points. The entire assay was carried out employing a Z-LR-AMC substrate. pH 5.0 (&); pH 6.5 (*); pH 8.0 (*). (C) Degradation of macromolecular proteins. Each protein was incubated at 37 8C for 3 h with or without rTsCL-1 and the reaction mixtures were analyzed by SDSPAGE. Lane 1, human IgG without rTsCL-1; lane 2, human IgG with rTsCL-1; lane 3, BSA without rTsCL-1; lane 4, BSA with rTsCL-1; lane 5, collagen without rTsCL-1; lane 6, collagen with rTsCL-1. (D) Western blot analysis of rTsCL-1 against the pooled sera from patients with helminth parasite infections or normal controls. Cy, cysticercosis; Sp, sparganosis; Fh, fascioliasis; Pw, paragonimiasis; Cs, clonorchiasis; NC, normal control.

proteolytic activity on zymography gel (Fig. 3B). The rTsCL-1 expressed in P. pastoris showed typical properties of cysteine proteases. It showed optimal activity at pH 6.5 (Fig. 4A), and the activity was increased by reducing agents such as DTT but was significantly inhibited by E-64 (data not shown). The enzyme was stable at pH 6.5 and its residual activity after 24 h incubation at 37 8C was 34.2%. Meanwhile, it was relatively unstable at pH 5.0 and 8.0 (Fig. 4B). The rTsCL-1 degraded human IgG and BSA, but not collagen (Fig. 4C). The kcat/Km for Z-LR-AMC was calculated to be 5.8  104 s1 M1. The rTsCL-1 showed strong antigenic reactivity against the serum samples from patients with cysticercosis and sparganosis, and showed cross reaction against the sera from patients infected with F. hepatica; however, weak or no cross-reactivity was found against the serum samples from patients with clonorchiasis or

paragonimiasis and normal controls in Western blot analyses (Fig. 4D). 4. Discussion Parasite cysteine proteases are involved in various physiological processes important to development and survival of the parasites and therefore have been considered as potential targets for chemotherapy or vaccine development. Cysteine proteases of protozoa, nematode and trematode parasites have been extensively studied and they have been proposed to be involved in various essential roles including the maintenance of cellular physiology of the parasite itself and in the host-parasite interactions by mediating several pathophysiological events, including host’s immune evasion, nutrient uptake and parasite invasion (Que and Reed, 2000; Dalton et al., 2003; Guiliano

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et al., 2004; Kang et al., 2004; Lustigman et al., 2004; Dvorak et al., 2005; Na et al., 2006). However, only limited biochemical and immunological studies on the cysteine proteases of cestode parasites have been done. In this study, we isolated a gene (TsCL-1) encoding for cathepsin L-like cysteine protease from the cestode parasite, T. solium. Although TsCL-1 showed a low sequence similarity, 27.3–44.6%, with cathepsin L-like cysteine proteases of other helminth parasites, it had well conserved amino acid residues, Q, C, H, N, and W, which are required for active site formation of cysteine proteases and typical ERFNIN, GNFD and GCNGG motifs which found in cathepsin L-like cysteine proteases. However, the sequence similarity was increased to 33.8–55.0% when aligned with mature domains of the enzymes. These results collectively suggested that the gene identified in this study was classified into a cathepsin L-like cysteine protease. Low sequence similarity of TsCL-1 with pig cathepsin L and RT-PCR analysis also suggested that the gene was originated from T. solium, not from the pig, which was the host animal used to obtain the parasite. Although the bacterially expressed rTsCL-1 did not show enzyme activity, the rTsCL-1 expressed in P. pastoris revealed typical biochemical properties of cysteine proteases. It degraded human IgG and BSA, but not collagen. Recently, partial biochemical properties of two cysteine proteases purified from TsM have been characterized (Baig et al., 2005; Kim et al., 2005). Both enzymes showed similar biochemical properties including substrate specificity and IgG degradation, but they differ significantly from each other in molecular weight, 29 and 48 kDa. As most of helminth parasites have multiple cysteine proteases (Dalton et al., 2003; Guiliano et al., 2004; Dvorak et al., 2005; Lee et al., 2006), it seems that T. solium also has multiple cysteine proteases. Considering the estimated molecular weight of the mature domain of TsCL-1, it can be postulated that TsCL-1 would encode the 29 kDa enzyme rather than the 48 kDa enzyme; however, further detailed studies are needed to clarify this. Moreover, comprehensive studies to determine the precise physiological role of TsCL-1 also are required. The rTsCL-1 revealed antigenicity against the sera from patients with cysticercosis and sparganosis, and showed cross reactions with the sera from patients infected with F. hepatica. Meanwhile, weak or no crossreactivity was found with the serum samples from patients with clonorchiasis or paragonimiasis and normal controls in Western blot analyses. These results may be due to the similarity of the sequences of the enzymes. In deed, the sequence of the mature domain of

TsCL-1 showed higher sequence similarity to the cysteine proteases of S. erinacei (55.0%) and F. hepatica (51.7%) than to those of C. sinensis (33.8%) and P. westermnai (35.9%). In conclusion, we have identified TsCL-1, a cathepsin L-like cysteine protease of T. solium, and characterized biochemical properties of the recombinant enzyme. The precise biological role of TsCL-1 is not clear, but its ability to degrade human IgG suggests its possible roles in host–parasite interactions. Further detailed biochemical and immunological studies of TsCL-1 are underway to clarify the biological role of the enzyme. Acknowledgments This work was supported by grants from the National Institute of Health, Ministry of Health and Welfare, Republic of Korea (NIH-348-6111-158) and International Cooperation Programme of KOSEF (F01-2001200023-0). References Baig, S., Damian, R.T., Molinari, J.L., Tato, P., Morales-Montor, J., Welch, M., Talhouk, J., Hashmeys, R., White Jr., A.C., 2005. Purification and characterization of a metacestode cysteine proteinase from Taenia solium involved in the breakdown of human IgG. Parasitology 131, 411–416. Carpio, A., 2002. Neurocysticercosis: an update. Lancet Infect. Dis. 2, 751–762. Dalton, J.P., Neill, S.O., Stack, C., Collins, P., Walshe, A., Sekiya, M., Doyle, S., Mulcahy, G., Hoyle, D., Khaznadji, E., Moire, N., Brennan, G., Mousley, A., Kreshchenko, N., Maule, A.G., Donnelly, S.M., 2003. Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines. Int. J. Parasitol. 33, 1173–1181. Dvorak, J., Delcroix, M., Rossi, A., Vopalensky, V., Pospisek, M., Sedinova, M., Mikes, L., Sajid, M., Sali, A., McKerrow, J.H., Horak, P., Caffrey, C.R., 2005. Multiple cathepsin B isoforms in schistosomula of Trichobilharzia regenti: identification, characterisation and putative role in migration and nutrition. Int. J. Parasitol. 35, 895–910. Guiliano, D.B., Hong, X., McKerrow, J.H., Blaxter, M.L., Oksov, Y., Liu, J., Ghedin, E., Lustigman, S., 2004. A gene family of cathepsin L-like proteases of filarial nematodes are associated with larval molting and cuticle and eggshell remodeling. Mol. Biochem. Parasitol. 136, 227–242. Kang, T.H., Yun, D.H., Lee, E.H., Chung, Y.B., Bae, Y.A., Chung, J.Y., Kang, I., Kim, J., Cho, S.Y., Kong, Y., 2004. A cathepsin F of adult Clonorchis sinensis and its phylogenetic conservation in trematodes. Parasitology 128, 195–207. Kim, J.Y., Yang, H.J., Kim, K.S., Chung, Y.B., 2005. Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes. Korean J. Parasitol. 43, 157–160. Lee, E.G., Na, B.K., Bae, Y.A., Kim, S.H., Je, E.Y., Ju, J.W., Cho, S.H., Kim, T.S., Kang, S.Y., Cho, S.Y., Kong, Y., 2006. Identification of immunodominant excretory-secretory cysteine proteases of adult

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