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Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis
Veterinary Parasitology 190 (2012) 510–518
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis Jong Nam Park a,b,1 , Sang Kyun Park a,1 , Min Kyoung Cho a , Mi-Kyung Park a , Shin Ae Kang a , Dong-Hee Kim c , Hak Sun Yu a,∗ a b c
Department of Parasitology, School of Medicine, Pusan National University, Yangsan 626-870, South Korea Park Jong Nam Internal Medicine Clinic, Busan 604-030, South Korea Department of Nursing, College of Nursing, Pusan National University, 626-870, South Korea
a r t i c l e
i n f o
Article history: Received 23 March 2012 Received in revised form 18 June 2012 Accepted 25 June 2012
Keywords: Trichinella spiralis Aspartic protease Ts-Asp Muscle stage larva Excretory–secretory protein
a b s t r a c t In a previous study, we identified an aspartic protease gene (Ts-Asp) from the Trichinella spiralis muscle stage larva cDNA library. The gene sequence of Ts-Asp was 1281 bp long and was found to encode a protein consisting of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95. The deduced Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, a common characteristic of aspartic proteases. In addition, the deduced amino acid sequence of Ts-Asp was found to possess significant homology (above 50%) with aspartic proteases from nematode parasites. Results of phylogenetic analysis indicated a close relationship of Ts-Asp with cathepsin D aspartic proteases. For production of recombinant Ts-Asp (rTs-Asp), the pGEX4T expression system was used. Like other proteases, the purified rTs-Asp was able to digest collagen matrix in vitro. Abundant expression of Ts-Asp was observed in muscle stage larva. Ts-Asp was detected in ES proteins, and was able to elicit the production of specific antibodies. It is the first report of molecular characterization of aspartic protease isolated from T. spiralis. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Infection by the parasitic nematode T. spiralis occurs upon consumption of meat contaminated with infective, first-stage larvae. The parasite is released from muscle by digestive enzymes in the stomach of the host (Capo and Despommier, 1996). Larvae of T. spiralis invade the epithelium of the small intestine, where they undergo four molts, maturation, mating, and reproduction (Despommier, 1993). Newborn larvae travel via the bloodstream, eventually entering skeletal muscle, where
∗ Corresponding author at: Department of Parasitology, School of Medicine, Pusan National University, Beomeo-ri, Mulgeum-eup, Yangsansi, Gyeongsangnam-do 626-870, South Korea. Tel.: +82 51 510 8022; fax: +82 51 980 0872. E-mail address: [email protected] (H.S. Yu). 1 Jong Nam Park and Sang Kyun Park equally contributed to this study. 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.06.029
each larva invades a single, terminally differentiated muscle cell (Despommier, 1975). Over a period of 20 days, the parasite modifies the infected myotube by reentry into the cell cycle, remodeling of the cytoplasmic matrix, synthesis of a collagen capsule, and formation of a capillary net around the altered cell (Despommier, 1993). It is remarkable how T. spiralis make a new architecture in the host tissue, by morphological remodeling. The formation of nurse cell is thought to be orchestrated by molecules (excretory–secretory proteins; ES proteins) elaborated by the parasite (Despommier, 1993). Proteinases secreted by parasitic organisms may be involved in a wide variety of adaptive functions such as tissue penetration, larval migration, immune evasion, retardation of blood coagulation, digestion, molting, and degradation of cellular matrix (Nagano et al., 2009). Recently, several authors have reported on proteinases in ES proteins of muscle stage larva; their work dealt mainly
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with serine proteinases (Cwiklinski et al., 2009; Liu et al., 2007). The 45 kDa protein of T. spiralis is present in the - and ␥-stichocytes of the secretory organs of muscle larvae (Nagano et al., 2009). Bioinformatics analysis identified the 45 kDa protein is a family of trypsin like serine proteases (Robinson et al., 2009). In addition, Romaris et al. reported glycoprotein of T. spiralis, which was serine protease and localized around collagen capsule (Romaris et al., 2002). Todorova et al. showed that the proteinases secreted from adult worms of T. spiralis degraded fibrinogen and plasminogen, and degradation was susceptible to the action of serine, cysteine, and aspartyl proteinases inhibitors (Todorova and Stoyanov, 2000). Aspartic proteases are defined by the presence of catalytic aspartic acid residues in the clefts of their active sites, and include pepsins, renins, cathepsins D and E, and chymosins (Tcherepanova et al., 2000). In nematode parasites, aspartic proteases play a crucial role in degradation of host hemoglobin. Aspartic proteases in particular have been reported from hematophagous parasites: blood fluke Schistosoma mansoni and S. japonicum, hookworms Ancylostoma canium and Necator americanus, the trichostrongylid Haemonchus contortus, and filaria Onchocerca volvulus (Brindley et al., 2001; Brown et al., 1999; Hotez et al., 2002; Jolodar and Miller, 1997, 1998; Shaw et al., 2003; Smith et al., 2003; Verity et al., 1999; Williamson et al., 2002). Despite the large number of nematodes that are known to secrete aspartic proteases, the function of aspartic proteases during developmental stages remains unclear. In a previous study, using expressed sequence tag (EST) analysis, we identified an aspartic protease gene from the T. spiralis muscle stage larva cDNA library. Although many proteases from T. spiralis have been reported, most were associated with serine protease. In order to gain a better understanding of molecular pathogenesis of T. spiralis, we conducted recombinant production, molecular characterization, and expression analysis of a new aspartic protease of T. spiralis. 2. Materials and methods 2.1. T. spiralis experimental infection The strain of T. spiralis was maintained in our laboratory via serial passage in rats. For acquisition of muscle stage larva, eviscerated mouse carcasses were cut into pieces, followed by digestion in 0.5% pepsin 1% hydrochloride digestion fluid (artificial gastric juice) during 2 h at 37◦ C with stirring. Larvae were collected manually from muscle digested solution under microscopy and washed 6 times with sterile PBS. After collection, in order to prevent contamination with the host material, worms were thoroughly and carefully washed several times over a 3 h period in PBS. To obtain serial serum of parasite infected mice, mice were infected with muscle stage larva (250 ea/mouse) and sacrificed every week after infection. Parasite infected muscle tissue was obtained from the left calf muscle of infected mice. Methods described in previous reports were used to obtain ES protein and total somatic extract (TE) of T. spiralis muscle stage larva (Park et al., 2011a, 2011b). This study
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was approved by the Pusan National University Animal Care and Use Committee. 2.2. Isolation of adults and newborn larva Adult worms and new born larva were obtained by a modification of the method of Wranicz et al. and Ozkoc et al. (Ozkoc et al., 2009; Wranicz et al., 1998). To obtain the adult worms, 10 rats (Wistar strain) were infected with 5000 T. spiralis muscle larva. After a week, T. spiralis infected rats were sacrified and the entire small intestines were removed. After washing, the small intestine was cut into 2 cm sections and placed on a gauze (40 m) in a PBS contain beaker for 3 h at 37◦ C. After incubation, adult worms were collected from the bottom of the beaker. The worms were washed three times by PBS containing antibiotics and were cultivated in RPMI-1640 media containing 10% FBS and antibiotics at 37◦ C and 5% CO2 for 2 days. At the end of the incubation time, newborn larva was isolated from the adults by free sedimentation. 2.3. Identification and analysis of new aspartic protease from T. spiralis The aspartic protease gene (Ts-Asp, GenBank accession No. 339237490) was isolated by chance from the Trichinella spiralis (isolate code ISS623) muscle stage larva cDNA library during EST analysis of the parasite (Park et al., 2008; Sohn et al., 2003). The gene was analysis using proteomics databases (ExPASy Bioinformatics Resource Portal: http://www.espasy.ch.protomics). A phylogenetic tree was constructed using ClustalW2 Multiple Sequence Alignments (http://www.ebi.ac.uk/Tools/msa/clustalw2/) by default condition. 2.4. Preparation of recombinant aspartic protease After confirmation of the sequence of the PCR product, Ts-Asp was extracted for ligation into the pGEX4T-1 expression vector (GE healthcare, USA). The constructs had BamH I and Xho I restriction sites at their 5 - and 3 -ends. Following gene ligation, the constructed plasmids were transformed into the E. coli BL21 (DE3) strain. Protein expression can be induced by 1 mM isopropyl--D-thiogalactoside (IPTG) to the culture medium and cultured at 25 ◦ C for 16 h. The GST fusion Ts-Asp protein was purified by GST affinity column chromatography (Glutathione Sepharose 4B; GE healthcare, USA). Following removal of GST from the fusion protein by thrombin digestion, recombinant Ts-Asp (rTsAsp) was purified. Polyclonal anti-rTs-Asp antibody was produced in rats that were boosted four times with 250 ug of rTs-Asp and complete and incomplete Freund adjuvant (Sigma, USA). After two additional boosters, blood was collected after five weeks, and serum was stored at −20 ◦ C. 2.5. Protease activity test using zymogram Gelatin-gel was prepared with gelatin concentration of 0.1% (1 mg/ml). The GST fusion rTs-Asp or rTs-Asp, was mixed with 2 × sample buffer (1 M Tris pH 6.8, 1% bromphenol blue, SDS, glycerol, -mercaptoethanol) and
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allowed to sit for 10 min at room temperature. The gel was run with 1 × Tris-Glycine SDS running buffer (0.025 M Tris, 0.192 M Glycine, pH 8.5, 0.1% SDS) (125 V for 2 h). After running the gel, it was washed for removal of SDS, and proteinase activity was re-natured using zymogram renaturing buffer (2.5% Triton X-100). Zymogram developing buffer (0.05 M Tris–HCl ph 7.6, 0.2 M NaCl, 5 mM CaCl2, 0.2% Brij) was used for development of the gel for 30 min at room temperature. The buffer was then replaced with fresh 1 × zymogram developing buffer, followed by incubation of the gel at 37 ◦ C for 8 h. The gel was stained with Coomassie Blue R-250 for 30 min and de-stained using an appropriate de-staining solution.
semidry blotting for 60 min at 400 mA. After blocking with skim milk, NC membranes were cut into strips and soaked in primary antibody solution (anti-rTs-Asp polyclonal antibodies or serum (1:1000 dilution) of T. spiralis infected mice 2, 3, and 4 weeks after infection) for 2 h at room temperature (RT). The membranes were then incubated in HRP-labeled secondary goat anti-rat IgG antibodies solution (1:2000 dilutions) for 1 h at RT. ECL western blotting detection reagent (GE healthcare, UK) was used for detection of positive reactions. The resultant complexes were processed for the detection system using an LAS-3000 (Fuzi, Japan). 2.9. Immunohistochemistry
2.6. Aspartic protease specific activity test For measurement of Ts-Asp activity, a SensoLyte® 520 Cathepsin D Assay Kit (Anaspec, Fremont, CA, USA) was used, according to the manufacturer’s instructions. Briefly, Cathepsin D (4 ng/10 l), pepstatin A, (400 M, Cathepsin D inhibitor), and rTs-Asp (10 g/20 l) mixtures were preincubated for 10 min at assay temperature, followed by addition of 50 l of Cathepsin D substrate solution to each well. After mixing well, fluorescence signal was measured at Ex/Em = 490 nm/520 nm for 10 min. Samples were monitored at 28 ◦ C using a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). 2.7. Real time RT-PCR After homogenization of T. spiralis muscle stage larva, adult worms, and newborn larva, each extracts were mixed with TRI-zol (Invitrogen, Germany), and RNA was extracted in accordance with the manufacturer’s protocols (Invitrogen, Germany). The first-strand cDNA was synthesized by using MMLV reverse transcriptase with oligo (dT)18 primers. Expression levels of the Ts-Asp gene were determined by real time RT-PCR using an iCyclerTM (Bio-Rad Laboratories, Inc., Hercules, California, USA). The Realtime PCR primers were made from Ts-Asp (GenBank accession No. 339237490) internal sequence (forward 5 - GCT GAT ACT GGC ACA TCG TTG ATA -3 , reverse 5 - GCA GCT GAC GTA ATA TTG ACC CAT -3 ). The GAPDH gene (GenBank accession No. AF452239, forward 5 - CAG GTG CTG TAT TAC GCT GTT -3 , reverse 5 - ACG CCA ATG CTT ACC AGA T -3 ) of T. spiralis was utilized as the reference gene. Amplification of all genes was performed under the following conditions: 1 min 30 s host start at 95 ◦ C, followed by denaturation at 95 ◦ C for 25 s, primer annealing at 55 ◦ C for 20 s, and elongation at 72 ◦ C and 30 s for 40 cycles. Fluorescent DNA-binding dye SYBR was monitored after each cycle at 55 ◦ C. An iCycler iQTM multi-color real-time PCR detection system (Bio-Rad Laboratories) was used for estimation of expression levels. Then, using the Gene-x program (Bio-Rad Laboratories), relative expression of the gene was calculated as the ratio to a housekeeping gene. 2.8. Western blot analysis SDS-PAGE (10%) was performed for separation of ES, TE, and rTs-Asp proteins. Following SDS-PAGE, proteins were transferred onto a nitrocellulose (NC) membrane by
Paraffin-embedded sections of left calf muscle were deparaffinized and hydrated. For antigen retrieval, slides were immersed in citrate buffer (0.01 M, pH 6.0) and heated twice in a microwave (700 W or high) for 5 min. Slides were then quenched with endogenous peroxidase by incubation with a 3% hydrogen peroxide solution for 5 min and washed three times in PBS for 5 min. Immunostaining of slides a with primary antibody (anti-rTs-Asp polyclonal antibody) (1:4000 dilution) was performed overnight at 4 ◦ C. After incubation with primary antibody, slides were washed three times in PBS for 5 min, followed by incubation with secondary antibody (Alexa 488 labeled anti-rat IgG for fluorescence image, or HRP labeled anti-rat IgG) for 1 h. The HRP labeled slides were subsequently washed four times in PBS for 5 min each, followed by development of the color reaction using Dako’s EnVisionTM System (DAKO, Carpinteria, CA, USA). Slides were stained with DAB and counterstained with Meyer’s hematoxylin (DAKO, Carpinteria, CA, USA) for 20 s, dehydrated, and mounted with Permount (Fisher Scientific, Pittsburgh, PA, USA). After three washes, Alexa 488 labeled slides were mounted with Dapi Fluoromount G (Southern Biotech). The tissue was imaged with a confocal microscope (Olympus, Fluoview 1000) and images were processed using software FV10-ASW 2.1. Background staining was checked by immunostaining of all slides without primary antibody and positive controls (rTs-Asp on slide) were used for confirmation of antibody specificity. 2.10. Statistical analysis All experiments were conducted in triplicate. Means ± SDs were calculated and the Student’s t test or ANOVA was used for determination of significant differences. PASW 18.0 was used for statistical analysis. 3. Results 3.1. Molecular characterization of an aspartic protease of T. spiralis The Ts-Asp gene sequence was 1281 bp long and was found to encode a protein of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95 (Genebank No. XP 003380300). Results of similarity analysis using the NCBI BLAST program showed that the deduced amino
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Fig. 1. Multiple amino acid sequence alignment of the aspartic protease (Ts-Asp) from T. spiralis with homologues from other helminthes. B. malayi BmAsp2 (Genebank accession no. BAC05689), N. americanus necepsin II (Genebank No. CAC00543), A. suum cathepsin D (Genebank No. ADY43078), A. simplex cathepsin D (Genebank No. ACY38599), A. ceylanicum cathepsin D (Genebank No. AAO22152), C. elegans Asp-4 (Genebank No. NP 510191), L. loa Asp-2 (Genebank No. XP 003148873), and S. ratti Asp-4 (Genebank No. ACR56788). Identical residues are shown in black boxes, conservative residues in dark gray boxes, similar residues in light gray boxes, and unrelated residues have a white background. Amino acid numbers are shown on the right. The putative signal sequences are shown in the box. The catalytic motif region is indicated with a dotted line (DTG) and the active site flap is represented by a solid line. Catalytic Asp residues are indicated with arrow heads (), and inhibitor binding residues are indicated by stars ().
acid sequence of Ts-Asp possessed homology with aspartic proteases from N. americanus (56%), Ascaris suum (55%), Anisakis simplex (54%), Ancylostoma ceylanicum (57%), C. elegans (55%), Brugia malayi (53%), Loa loa (53%), and Strongyloides ratti (54%) (Fig. 1). Ts-Asp nucleotide sequence predicted by NCBI gene Graphic viewer and Expasy proteomics tool. Ts-Asp contains signal pepetide sequence (from Met-1 to Gly-15) and two catalytic motifs (DTG), which include active residues (Asp-99, Asp-286) thought to be essential for aspartic protease activity. However, two active site flaps were not identical to those previously reported aspartic proteases of other parasites (Fig. 1), although most inhibitor binding sites were conserved (Asp99, Gly-101, Ser-103, Ile-142, Arg-143, and Ile-189). To examine the phylogenetic relationship of Ts-Asp with other
homologous aspartic proteases, a phylogenetic tree was constructed based on the deduced amino acid sequences from 26 aspartic proteases. Protein distance-based phylogenetic analysis of the eukaryotic aspartic protease amino acid sequences resulted in the creation of a dendrogram with three clusters (Fig. 2). Ts-Asp was located in Cluster I with cathepsin D of Schistosoma japonicum, A. suum, A. simplex, and A. ceylanicum; lysosomal aspartic protease of Culex quinquefasciatus; asp-2B of B. malayi and L. loa; asp-4 of S. ratti, and C. elegans; necepsin II of N. americanus. 3.2. rTs-Asp exhibited aspartic protease activity In order to understand about Ts-Asp, we performed purification of GSP fusion rTs-Asp (∼72 kDa) using a
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Fig. 2. Clustering of aspartic proteases using molecular phylogenetic analysis. An un-rooted phylogenetic tree was generated based on alignment of amino acid sequences from 26 aspartic proteases of nematode helminthes. The scale bar indicates an evolutionary distance of amino acid substitutions per position. T. spiralis lysosomal aspartic protease (Genebank No. XP 003380300) A. ceylanicum cathepsin D (Genebank No. AAO22152.1), A. simplex cathepsin D (Genebank No. ACY38599.1), A. suum cathepsin D (Genebank No. ADY43078.1), B. malayi Asp-1 (Genebank No. BAC05688) and Asp-2, C. briggsae Asp-2 (Genebank No. XP 002637505) and Asp-6 (Genebank No. XP 002635088), C. elegans 1a Asp (Genebank No. CAA08899), 2a Asp-2 (Genebank No. NP 872129), Asp-2 (Genebank No. NP 505384), Asp-4, C. remanei Asp-1 (Genebank No. XP 003093500), Asp-5 (Genebank No. XP 003110542), C. quinquefasciatus Asp (Genebank No. XP 001867326), H. contortus Asp (Genebank No. CAE12199), L. loa Asp-2 and Asp-6 (Genebank No. EFO15484), N. americanus necepsin I (Genebank No. CAC00542) and necepsin II, S. japonicum cathepsin D (Genebank No. CAX72322), Steinernema carpocapsae Asp (Genebank No. ADJ94115), St. feltiae Asp (Genebank No. ACS32298), S. ratti Asp-4 and Asp-2B (Genebank No. ACR56786), and S. stercoralis Asp (Genebank No. AAD09345).
prokaryotic expression system (Fig. 3A). After thrombin digestion, rTs-Asp was appeared at approximately 45 kDa. Gelatin zymogram analysis was performed for evaluation of proteolytic activity. As a result, protease activity of approximately 43 kDa (rTs-Asp) and 70 kDa (GST – rTsAsp fusion protein) was detected (Fig. 3B). This size shift effect may be due to differences in sample preparation (native form or denatured form) between zymogram and SDS-PAGE. For determination of rTs-Asp aspartic protease activity, aspartic protease activity of rTs-Asp was evaluated using assay kits. After a 10 min incubation period, rTs-Asp had digested approximately 60–70% of aspartic specific substrate; however, complete inhibition of this activity was observed by treatment with pepstatin (aspartic protease specific inhibitor) (Fig. 3C).
3.3. Abundant expression of Ts-Asp was observed in muscle stage larva In order to determine when Ts-Asp mRNA showed the greatest expression, real time RT-PCR was performed on first-strand cDNA from newborn larva, muscle stage larva, and adult worms of T. spiralis. As shown in Fig. 4, the highest level of expression of the Ts-Asp gene was observed in muscle stage larva of T. spiralis.
3.4. Ts-Asp was secreted and elicited specific antibody production To determine whether or not T. spiralis muscle stage larvae secrete Ts-Asp, SDS-PAGE was performed under reducing conditions for resolution of TE and ES products from muscle larva, followed by blotting to NC membranes. Immunoblotting was performed using rat antiserum against rTs-Asp. Approximately 45 kDa protein, which was reacted with rTs-Asp polyclonal antibody, was observed in both TE and ES proteins (Fig. 5A). To determine whether or not Ts-Asp has antigenecity, western blotting was performed using serum of T. spiralis infected mice 2, 3, and 4 weeks after infection. Anti Ts-Asp specific antibody was detected in serum at 3 and 4 weeks after T. spiralis infection of mice (Fig. 5B). These results suggested that Ts-Asp might have antigenicity and be secreted by muscle stage larva. 3.5. Ts-Asp was localized in genital primordium and stichocytes. To determine the location of Ts-Asp secretion, we performed immunohistochemistry to evaluate localization of Ts-Asp specific antiserum in muscle tissue of T. spiralis muscle stage larva infected mice. Results showed that anti
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Fig. 3. Analysis of purification and proteolytic activity of rTs-Asp. SDS-PAGE was performed for separation of the purified GST fusion rTs-Asp (A). M; protein molecular marker, lane 1; inclusion body of Ts-Asp transformed BL21 cell lysate, lane 2; supernatant of Ts-Asp transformed BL21 cell lysate, lane 3; purified GST fusion rTs-asp. Gelatin Zymogram was used for analysis of proteolytic activity (B). M; protein molecular marker, lane 1; supernatant of Ts-Asp transformed BL21 cell lysate, lane 2; 1 g of GST–rTs-Asp fusion protein after thrombin digestion. lane 3; 10 g GST–rTs-Asp fusion protein after thrombin digestion. Arrowhead; GST–rTs-Asp fusion protein, Arrow; rTs-Asp. An aspartic protease assay kit was used for measurement of spartic protease specific activity of rTs-Asp (C). C; (substrate + Cathepsin D), T; (substrate + rTs-Asp), C + I (substrate + Cathepsin D + pepstatin), T + I (substrate + rTs-Asp + pepstatin).
rTs-Asp serum was strongly reacted with the genital primordium and also they was slightly reacted with some stichocytes of muscle stage larva (Fig. 6). 4. Discussion
Fig. 4. Real-time RT-PCR analysis of expression of Ts-Asp during developmental stages. N; new born larva, M; muscle stage larva, A; adult worm (*; p < 0.05, experiments was performed in triplicate).
In this study, we characterized an aspartic protease released by nematode parasite T. spiralis. This is the first report on characterization study about aspartic protease of T. spiralis. Based on the alignment and comparison of the Ts-Asp protein sequence deduced from full length cDNA sequences in the protein databases using the BLAST program, we found that Ts-Asp shared significant identity (above 50%) with aspartic proteases of other nematodes, including N. americanus, A. suum, A. simplex, A. ceylanicum, C. elegans, B. malayi, L. loa, and S. ratti (Fig. 1). In addition, Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, which is a common
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Fig. 5. Detection of Ts-Asp in ES proteins and specific antibody against Ts-Asp using western blot analysis. One hundred g of TE and ES protein of T. spiralis were separated by SDS-PAGE. The polyclonal antibody anti-rTs-Asp (1:1000 dilution) was used as primary antibody (A). Ten g of GST fusion rTs-Asp was loaded on SDS-PAGE, and the proteins was reacted with mice serum obtained from every weeks after T. spiralis infection (B) (N; serum of non-infected mice, 2–4 weeks; serum of mice 2–4 weeks after T. spiralis infection).
Fig. 6. Localization of Ts-ASP in muscle stage larva using immunohistochemistry. Muscle tissue was obtained from T. spiralis infected mice at 6 weeks; the tissue was reacted with anti-rTs-Asp antibody (A and B) and with PBS (C). Arrow indicated reactive regions of muscle stage larva. Arrow head indicated midgut of muscle stage larva (Bar is 50 m).
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characteristic of aspartic proteases. Also, rTs-Asp could digest aspartic specific substrate; this activity was completely inhibited by aspartic protease inhibitor (Fig. 3C). These results demonstrated that Ts-Asp could be an aspartic protease isolated from T. spiralis. Although a large number of aspartic protease genes have been identified from several nematodes, only a few detailed phylogenetic studies of these genes have been reported. Mello et al. demonstrated that most nematode aspartic proteases were grouped into three clusters (Mello et al., 2009). They also suggested that at least three independent duplications have occurred in this part of the protease family; one in the lineage containing C. elegans Asp-1a, another in the predecessor of H. contortus, and a distinct set of events in the branch leading to S. ratti asp-4 (Mello et al., 2009). They found that the cluster is closely related to the location of aspartic protease gene within the genome; however, functional differences between the clusters have not been revealed. In our study, Ts-Asp was clustered with several cathepsin D type aspartic proteases (Fig. 2); this cluster was also reported by Mello et al. (2009). Most cathepsin D aspartic proteases have been identified from blood feeding nematodes or tissue invading nematodes; their function was related to the digestion of hemoglobin and the invasion of host tissue (Balasubramanian et al., 2012; Brinkworth et al., 2000; Gallego et al., 1998; Harrop et al., 1996; Williamson et al., 2003). Although T. spiralis can also invade tissue and feed on blood materials during their life cycle, it is not clear whether or not TsAsp is closely related to the blood feeding and the tissue invasion. Many nematodes are known to secrete aspartic proteases; however, the function of these molecules is not yet clear. The presence of several aspartic protease homologues in the free-living nematode C. elegans (Geier et al., 1999) suggests that these proteases could be related to the development of embryo and larvae (Tcherepanova et al., 2000). In parasites, aspartic proteases may be related not only to development of worms, but also to parasitism such as degradation of the extracellular matrix, digestion of hemoglobin, and so on (Dzik, 2006). Yang et al. suggested that aspartic protease has a definite role in embryo development and the pre-parasitic muscle larva stage of A. caninum (Yang et al., 2009). In this study, Ts-Asp was more likely to be expressed in genital primodium (Fig. 6). These results perhaps indicated that Ts-Asp was able to have a role in genital development of muscle larva. The expression levels of Ts-Asp across the life cycle of T. spiralis were measured by Real time RT-PCR. As results, the highest levels of expression of Ts-Asp were observed in muscle stage larva of T. spiralis (Fig. 4). Also, detection of anti-Ts-Asp specific antibodies in serum of parasite infected mice started to appear 3 weeks after infection (Fig. 5). Therefore, primary secretion of Ts-Asp might have begun once newborn larvae had reached muscle tissue. In conclusion, we firstly characterized an aspartic protease Ts-Asp of T. spiralis, which was abundant expressed by muscle stage larva. In order to better understand the exact function and mechanisms of Ts-Asp, more detailed functional and mechanism studies will be needed.
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Acknowledgement This work was supported by the Bio-Scientific Research Grant funded by the Pusan National University (PNU, BioScientific Research Grant) (PNU-2010-101-251) References Balasubramanian, N., Toubarro, D., Nascimento, G., Ferreira, R., Simoes, N., 2012. Purification, molecular characterization and gene expression analysis of an aspartic protease (Sc-ASP113) from the nematode Steinernema carpocapsae during the parasitic stage. Mol. Biochem. Parasitol. 182, 37–44. Brindley, P.J., Kalinna, B.H., Wong, J.Y., Bogitsh, B.J., King, L.T., Smyth, D.J., Verity, C.K., Abbenante, G., Brinkworth, R.I., Fairlie, D.P., Smythe, M.L., Milburn, P.J., Bielefeldt-Ohmann, H., Zheng, Y., McManus, D.P., 2001. Proteolysis of human hemoglobin by schistosome cathepsin D. Mol. Biochem. Parasitol. 112, 103–112. Brinkworth, R.I., Harrop, S.A., Prociv, P., Brindley, P.J., 2000. Host specificity in blood feeding parasites: a defining contribution by haemoglobindegrading enzymes? Int. J. Parasitol. 30, 785–790. Brown, A., Girod, N., Billett, E.E., Pritchard, D.I., 1999. Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration. Am. J. Trop. Med. Hyg. 60, 840–847. Capo, V., Despommier, D.D., 1996. Clinical aspects of infection with Trichinella spp. Clin. Microbiol. Rev. 9, 47–54. Cwiklinski, K., Meskill, D., Robinson, M.W., Pozio, E., Appleton, J.A., Connolly, B., 2009. Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene. Vet. Parasitol. 159, 268–271. Despommier, D., 1975. Adaptive changes in muscle fibers infected with Trichinella spiralis. Am. J. Pathol. 78, 477–496. Despommier, D.D., 1993. Trichinella spiralis and the concept of niche. J. Parasitol. 79, 472–482. Dzik, J.M., 2006. Molecules released by helminth parasites involved in host colonization. Acta Biochim. Pol. 53, 33–64. Gallego, S.G., Slade, R.W., Brindley, P.J., 1998. A cDNA encoding a pepsinogen-like, aspartic protease from the human roundworm parasite Strongyloides stercoralis. Acta Trop. 71, 17–26. Geier, G., Banaj, H.J., Heid, H., Bini, L., Pallini, V., Zwilling, R., 1999. Aspartyl proteases in Caenorhabditis elegans Isolation, identification and characterization by a combined use of affinity chromatography, two-dimensional gel electrophoresis, microsequencing and databank analysis. Eur. J. Biochem. 264, 872–879. Harrop, S.A., Prociv, P., Brindley, P.J., 1996. Acasp, a gene encoding a cathepsin D-like aspartic protease from the hookworm Ancylostoma caninum. Biochem. Biophys. Res. Commun. 227, 294–302. Hotez, P.J., Ashcom, J., Bin, Z., Bethony, J., Williamson, A., Hawdon, J.M., Jianjun, F., Dobardzic, A., Rizo, I., Bolden, J., Jin, Q., Yan, W., Dobardzic, R., Chung-Debose, S., Crowell, M., Datu, B., Delaney, A., Dragonovski, D., Jiang, Y., Yueyuan, L., Ghosh, K., Loukas, A., Brandt, W., Russell, P.K., Zook, B.C., 2002. Effect of vaccinations with recombinant fusion proteins on Ancylostoma caninum habitat selection in the canine intestine. J. Parasitol. 88, 684–690. Jolodar, A., Miller, D.J., 1997. Preliminary characterisation of an Onchocerca volvulus aspartic protease. Int. J. Parasitol. 27, 1087–1090. Jolodar, A., Miller, D.J., 1998. Identification of a novel family of nonlysosomal aspartic proteases in nematodes. Biochim. Biophys. Acta 1382, 13–16. Liu, M.Y., Wang, X.L., Fu, B.Q., Li, C.Y., Wu, X.P., Le Rhun, D., Chen, Q.J., Boireau, P., 2007. Identification of stage-specifically expressed genes of Trichinella spiralis by suppression subtractive hybridization. Parasitology 134, 1443–1455. Mello, L.V., O’Meara, H., Rigden, D.J., Paterson, S., 2009. Identification of novel aspartic proteases from Strongyloides ratti and characterisation of their evolutionary relationships, stage-specific expression and molecular structure. BMC Genomics 10, 611. Nagano, I., Wu, Z., Takahashi, Y., 2009. Functional genes and proteins of Trichinella spp. Parasitol. Res. 104, 197–207. Ozkoc, S., Tuncay, S., Delibas, S.B., Akisu, C., 2009. In vitro effects of resveratrol on Trichinella spiralis. Parasitol. Res. 105, 1139–1143. Park, H.K., Chang, S.W., Kang, S.W., Cho, M.K., Choi, S.H., Hong, Y.C., Lee, Y.S., Jeong, H.J., Yu, H.S., 2008. Expressed sequence tags of Trichinella spiralis muscle stage larvae. Korean J. Parasitol. 46, 59–63. Park, H.K., Cho, M.K., Choi, S.H., Kim, Y.S., Yu, H.S., 2011a. Trichinella spiralis: infection reduces airway allergic inflammation in mice. Exp. Parasitol. 127, 539–544.
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Park, M.K., Cho, M.K., Kang, S.A., Park, H.K., Kim, Y.S., Kim, K.U., Ahn, S.C., Kim, D.H., Yu, H.S., 2011b. Protease-activated receptor 2 is involved in Th2 responses against Trichinella spiralis infection. Korean J. Parasitol. 49, 235–243. Robinson, M.W., Menon, R., Donnelly, S.M., Dalton, J.P., Ranganathan, S., 2009. An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host. Mol. Cell Proteomics 8, 1891–1907. Romaris, F., North, S.J., Gagliardo, L.F., Butcher, B.A., Ghosh, K., Beiting, D.P., Panico, M., Arasu, P., Dell, A., Morris, H.R., Appleton, J.A., 2002. A putative serine protease among the excretory–secretory glycoproteins of L1 Trichinella spiralis. Mol. Biochem. Parasitol. 122, 149–160. Shaw, R.J., McNeill, M.M., Maass, D.R., Hein, W.R., Barber, T.K., Wheeler, M., Morris, C.A., Shoemaker, C.B., 2003. Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis. Int. J. Parasitol. 33, 1233–1243. Smith, W.D., Skuce, P.J., Newlands, G.F., Smith, S.K., Pettit, D., 2003. Aspartyl proteases from the intestinal brush border of Haemonchus contortus as protective antigens for sheep. Parasite Immunol. 25, 521–530. Sohn, W.M., Huh, S., Chung, D.I., Pozio, E., 2003. Molecular identification of Korean Trichinella isolates. Korean J. Parasitol. 41, 125–127. Tcherepanova, I., Bhattacharyya, L., Rubin, C.S., Freedman, J.H., 2000. Aspartic proteases from the nematode Caenorhabditis elegans
Structural organization and developmental and cell-specific expression of asp-1. J. Biol. Chem. 275, 26359–26369. Todorova, V.K., Stoyanov, D.I., 2000. Partial characterization of serine proteinases secreted by adult Trichinella spiralis. Parasitol. Res. 86, 684–687. Verity, C.K., McManus, D.P., Brindley, P.J., 1999. Developmental expression of cathepsin D aspartic protease in Schistosoma japonicum. Int. J. Parasitol. 29, 1819–1824. Williamson, A.L., Brindley, P.J., Abbenante, G., Datu, B.J., Prociv, P., Berry, C., Girdwood, K., Pritchard, D.I., Fairlie, D.P., Hotez, P.J., Zhan, B., Loukas, A., 2003. Hookworm aspartic protease Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion. J. Infect. Dis. 187, 484–494. Williamson, A.L., Brindley, P.J., Abbenante, G., Prociv, P., Berry, C., Girdwood, K., Pritchard, D.I., Fairlie, D.P., Hotez, P.J., Dalton, J.P., Loukas, A., 2002. Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity. FASEB. J. 16, 1458–1460. Wranicz, M.J., Gustowska, L., Gabryel, P., Kucharska, E., Cabaj, W., 1998. Trichinella spiralis: induction of the basophilic transformation of muscle cells by synchronous newborn larvae. Parasitol. Res. 84, 403–407. Yang, Y., Wei, H., Qin, W., Zheng, J., 2009. Expression and characterization of aspartic protease gene in eggs and larvae stage of Ancylostoma caninum. Parasitol. Res. 104, 1327–1333.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis Jong Nam Park a,b,1 , Sang Kyun Park a,1 , Min Kyoung Cho a , Mi-Kyung Park a , Shin Ae Kang a , Dong-Hee Kim c , Hak Sun Yu a,∗ a b c
Department of Parasitology, School of Medicine, Pusan National University, Yangsan 626-870, South Korea Park Jong Nam Internal Medicine Clinic, Busan 604-030, South Korea Department of Nursing, College of Nursing, Pusan National University, 626-870, South Korea
a r t i c l e
i n f o
Article history: Received 23 March 2012 Received in revised form 18 June 2012 Accepted 25 June 2012
Keywords: Trichinella spiralis Aspartic protease Ts-Asp Muscle stage larva Excretory–secretory protein
a b s t r a c t In a previous study, we identified an aspartic protease gene (Ts-Asp) from the Trichinella spiralis muscle stage larva cDNA library. The gene sequence of Ts-Asp was 1281 bp long and was found to encode a protein consisting of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95. The deduced Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, a common characteristic of aspartic proteases. In addition, the deduced amino acid sequence of Ts-Asp was found to possess significant homology (above 50%) with aspartic proteases from nematode parasites. Results of phylogenetic analysis indicated a close relationship of Ts-Asp with cathepsin D aspartic proteases. For production of recombinant Ts-Asp (rTs-Asp), the pGEX4T expression system was used. Like other proteases, the purified rTs-Asp was able to digest collagen matrix in vitro. Abundant expression of Ts-Asp was observed in muscle stage larva. Ts-Asp was detected in ES proteins, and was able to elicit the production of specific antibodies. It is the first report of molecular characterization of aspartic protease isolated from T. spiralis. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Infection by the parasitic nematode T. spiralis occurs upon consumption of meat contaminated with infective, first-stage larvae. The parasite is released from muscle by digestive enzymes in the stomach of the host (Capo and Despommier, 1996). Larvae of T. spiralis invade the epithelium of the small intestine, where they undergo four molts, maturation, mating, and reproduction (Despommier, 1993). Newborn larvae travel via the bloodstream, eventually entering skeletal muscle, where
∗ Corresponding author at: Department of Parasitology, School of Medicine, Pusan National University, Beomeo-ri, Mulgeum-eup, Yangsansi, Gyeongsangnam-do 626-870, South Korea. Tel.: +82 51 510 8022; fax: +82 51 980 0872. E-mail address: [email protected] (H.S. Yu). 1 Jong Nam Park and Sang Kyun Park equally contributed to this study. 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.06.029
each larva invades a single, terminally differentiated muscle cell (Despommier, 1975). Over a period of 20 days, the parasite modifies the infected myotube by reentry into the cell cycle, remodeling of the cytoplasmic matrix, synthesis of a collagen capsule, and formation of a capillary net around the altered cell (Despommier, 1993). It is remarkable how T. spiralis make a new architecture in the host tissue, by morphological remodeling. The formation of nurse cell is thought to be orchestrated by molecules (excretory–secretory proteins; ES proteins) elaborated by the parasite (Despommier, 1993). Proteinases secreted by parasitic organisms may be involved in a wide variety of adaptive functions such as tissue penetration, larval migration, immune evasion, retardation of blood coagulation, digestion, molting, and degradation of cellular matrix (Nagano et al., 2009). Recently, several authors have reported on proteinases in ES proteins of muscle stage larva; their work dealt mainly
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with serine proteinases (Cwiklinski et al., 2009; Liu et al., 2007). The 45 kDa protein of T. spiralis is present in the - and ␥-stichocytes of the secretory organs of muscle larvae (Nagano et al., 2009). Bioinformatics analysis identified the 45 kDa protein is a family of trypsin like serine proteases (Robinson et al., 2009). In addition, Romaris et al. reported glycoprotein of T. spiralis, which was serine protease and localized around collagen capsule (Romaris et al., 2002). Todorova et al. showed that the proteinases secreted from adult worms of T. spiralis degraded fibrinogen and plasminogen, and degradation was susceptible to the action of serine, cysteine, and aspartyl proteinases inhibitors (Todorova and Stoyanov, 2000). Aspartic proteases are defined by the presence of catalytic aspartic acid residues in the clefts of their active sites, and include pepsins, renins, cathepsins D and E, and chymosins (Tcherepanova et al., 2000). In nematode parasites, aspartic proteases play a crucial role in degradation of host hemoglobin. Aspartic proteases in particular have been reported from hematophagous parasites: blood fluke Schistosoma mansoni and S. japonicum, hookworms Ancylostoma canium and Necator americanus, the trichostrongylid Haemonchus contortus, and filaria Onchocerca volvulus (Brindley et al., 2001; Brown et al., 1999; Hotez et al., 2002; Jolodar and Miller, 1997, 1998; Shaw et al., 2003; Smith et al., 2003; Verity et al., 1999; Williamson et al., 2002). Despite the large number of nematodes that are known to secrete aspartic proteases, the function of aspartic proteases during developmental stages remains unclear. In a previous study, using expressed sequence tag (EST) analysis, we identified an aspartic protease gene from the T. spiralis muscle stage larva cDNA library. Although many proteases from T. spiralis have been reported, most were associated with serine protease. In order to gain a better understanding of molecular pathogenesis of T. spiralis, we conducted recombinant production, molecular characterization, and expression analysis of a new aspartic protease of T. spiralis. 2. Materials and methods 2.1. T. spiralis experimental infection The strain of T. spiralis was maintained in our laboratory via serial passage in rats. For acquisition of muscle stage larva, eviscerated mouse carcasses were cut into pieces, followed by digestion in 0.5% pepsin 1% hydrochloride digestion fluid (artificial gastric juice) during 2 h at 37◦ C with stirring. Larvae were collected manually from muscle digested solution under microscopy and washed 6 times with sterile PBS. After collection, in order to prevent contamination with the host material, worms were thoroughly and carefully washed several times over a 3 h period in PBS. To obtain serial serum of parasite infected mice, mice were infected with muscle stage larva (250 ea/mouse) and sacrificed every week after infection. Parasite infected muscle tissue was obtained from the left calf muscle of infected mice. Methods described in previous reports were used to obtain ES protein and total somatic extract (TE) of T. spiralis muscle stage larva (Park et al., 2011a, 2011b). This study
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was approved by the Pusan National University Animal Care and Use Committee. 2.2. Isolation of adults and newborn larva Adult worms and new born larva were obtained by a modification of the method of Wranicz et al. and Ozkoc et al. (Ozkoc et al., 2009; Wranicz et al., 1998). To obtain the adult worms, 10 rats (Wistar strain) were infected with 5000 T. spiralis muscle larva. After a week, T. spiralis infected rats were sacrified and the entire small intestines were removed. After washing, the small intestine was cut into 2 cm sections and placed on a gauze (40 m) in a PBS contain beaker for 3 h at 37◦ C. After incubation, adult worms were collected from the bottom of the beaker. The worms were washed three times by PBS containing antibiotics and were cultivated in RPMI-1640 media containing 10% FBS and antibiotics at 37◦ C and 5% CO2 for 2 days. At the end of the incubation time, newborn larva was isolated from the adults by free sedimentation. 2.3. Identification and analysis of new aspartic protease from T. spiralis The aspartic protease gene (Ts-Asp, GenBank accession No. 339237490) was isolated by chance from the Trichinella spiralis (isolate code ISS623) muscle stage larva cDNA library during EST analysis of the parasite (Park et al., 2008; Sohn et al., 2003). The gene was analysis using proteomics databases (ExPASy Bioinformatics Resource Portal: http://www.espasy.ch.protomics). A phylogenetic tree was constructed using ClustalW2 Multiple Sequence Alignments (http://www.ebi.ac.uk/Tools/msa/clustalw2/) by default condition. 2.4. Preparation of recombinant aspartic protease After confirmation of the sequence of the PCR product, Ts-Asp was extracted for ligation into the pGEX4T-1 expression vector (GE healthcare, USA). The constructs had BamH I and Xho I restriction sites at their 5 - and 3 -ends. Following gene ligation, the constructed plasmids were transformed into the E. coli BL21 (DE3) strain. Protein expression can be induced by 1 mM isopropyl--D-thiogalactoside (IPTG) to the culture medium and cultured at 25 ◦ C for 16 h. The GST fusion Ts-Asp protein was purified by GST affinity column chromatography (Glutathione Sepharose 4B; GE healthcare, USA). Following removal of GST from the fusion protein by thrombin digestion, recombinant Ts-Asp (rTsAsp) was purified. Polyclonal anti-rTs-Asp antibody was produced in rats that were boosted four times with 250 ug of rTs-Asp and complete and incomplete Freund adjuvant (Sigma, USA). After two additional boosters, blood was collected after five weeks, and serum was stored at −20 ◦ C. 2.5. Protease activity test using zymogram Gelatin-gel was prepared with gelatin concentration of 0.1% (1 mg/ml). The GST fusion rTs-Asp or rTs-Asp, was mixed with 2 × sample buffer (1 M Tris pH 6.8, 1% bromphenol blue, SDS, glycerol, -mercaptoethanol) and
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allowed to sit for 10 min at room temperature. The gel was run with 1 × Tris-Glycine SDS running buffer (0.025 M Tris, 0.192 M Glycine, pH 8.5, 0.1% SDS) (125 V for 2 h). After running the gel, it was washed for removal of SDS, and proteinase activity was re-natured using zymogram renaturing buffer (2.5% Triton X-100). Zymogram developing buffer (0.05 M Tris–HCl ph 7.6, 0.2 M NaCl, 5 mM CaCl2, 0.2% Brij) was used for development of the gel for 30 min at room temperature. The buffer was then replaced with fresh 1 × zymogram developing buffer, followed by incubation of the gel at 37 ◦ C for 8 h. The gel was stained with Coomassie Blue R-250 for 30 min and de-stained using an appropriate de-staining solution.
semidry blotting for 60 min at 400 mA. After blocking with skim milk, NC membranes were cut into strips and soaked in primary antibody solution (anti-rTs-Asp polyclonal antibodies or serum (1:1000 dilution) of T. spiralis infected mice 2, 3, and 4 weeks after infection) for 2 h at room temperature (RT). The membranes were then incubated in HRP-labeled secondary goat anti-rat IgG antibodies solution (1:2000 dilutions) for 1 h at RT. ECL western blotting detection reagent (GE healthcare, UK) was used for detection of positive reactions. The resultant complexes were processed for the detection system using an LAS-3000 (Fuzi, Japan). 2.9. Immunohistochemistry
2.6. Aspartic protease specific activity test For measurement of Ts-Asp activity, a SensoLyte® 520 Cathepsin D Assay Kit (Anaspec, Fremont, CA, USA) was used, according to the manufacturer’s instructions. Briefly, Cathepsin D (4 ng/10 l), pepstatin A, (400 M, Cathepsin D inhibitor), and rTs-Asp (10 g/20 l) mixtures were preincubated for 10 min at assay temperature, followed by addition of 50 l of Cathepsin D substrate solution to each well. After mixing well, fluorescence signal was measured at Ex/Em = 490 nm/520 nm for 10 min. Samples were monitored at 28 ◦ C using a SpectraMax M2 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). 2.7. Real time RT-PCR After homogenization of T. spiralis muscle stage larva, adult worms, and newborn larva, each extracts were mixed with TRI-zol (Invitrogen, Germany), and RNA was extracted in accordance with the manufacturer’s protocols (Invitrogen, Germany). The first-strand cDNA was synthesized by using MMLV reverse transcriptase with oligo (dT)18 primers. Expression levels of the Ts-Asp gene were determined by real time RT-PCR using an iCyclerTM (Bio-Rad Laboratories, Inc., Hercules, California, USA). The Realtime PCR primers were made from Ts-Asp (GenBank accession No. 339237490) internal sequence (forward 5 - GCT GAT ACT GGC ACA TCG TTG ATA -3 , reverse 5 - GCA GCT GAC GTA ATA TTG ACC CAT -3 ). The GAPDH gene (GenBank accession No. AF452239, forward 5 - CAG GTG CTG TAT TAC GCT GTT -3 , reverse 5 - ACG CCA ATG CTT ACC AGA T -3 ) of T. spiralis was utilized as the reference gene. Amplification of all genes was performed under the following conditions: 1 min 30 s host start at 95 ◦ C, followed by denaturation at 95 ◦ C for 25 s, primer annealing at 55 ◦ C for 20 s, and elongation at 72 ◦ C and 30 s for 40 cycles. Fluorescent DNA-binding dye SYBR was monitored after each cycle at 55 ◦ C. An iCycler iQTM multi-color real-time PCR detection system (Bio-Rad Laboratories) was used for estimation of expression levels. Then, using the Gene-x program (Bio-Rad Laboratories), relative expression of the gene was calculated as the ratio to a housekeeping gene. 2.8. Western blot analysis SDS-PAGE (10%) was performed for separation of ES, TE, and rTs-Asp proteins. Following SDS-PAGE, proteins were transferred onto a nitrocellulose (NC) membrane by
Paraffin-embedded sections of left calf muscle were deparaffinized and hydrated. For antigen retrieval, slides were immersed in citrate buffer (0.01 M, pH 6.0) and heated twice in a microwave (700 W or high) for 5 min. Slides were then quenched with endogenous peroxidase by incubation with a 3% hydrogen peroxide solution for 5 min and washed three times in PBS for 5 min. Immunostaining of slides a with primary antibody (anti-rTs-Asp polyclonal antibody) (1:4000 dilution) was performed overnight at 4 ◦ C. After incubation with primary antibody, slides were washed three times in PBS for 5 min, followed by incubation with secondary antibody (Alexa 488 labeled anti-rat IgG for fluorescence image, or HRP labeled anti-rat IgG) for 1 h. The HRP labeled slides were subsequently washed four times in PBS for 5 min each, followed by development of the color reaction using Dako’s EnVisionTM System (DAKO, Carpinteria, CA, USA). Slides were stained with DAB and counterstained with Meyer’s hematoxylin (DAKO, Carpinteria, CA, USA) for 20 s, dehydrated, and mounted with Permount (Fisher Scientific, Pittsburgh, PA, USA). After three washes, Alexa 488 labeled slides were mounted with Dapi Fluoromount G (Southern Biotech). The tissue was imaged with a confocal microscope (Olympus, Fluoview 1000) and images were processed using software FV10-ASW 2.1. Background staining was checked by immunostaining of all slides without primary antibody and positive controls (rTs-Asp on slide) were used for confirmation of antibody specificity. 2.10. Statistical analysis All experiments were conducted in triplicate. Means ± SDs were calculated and the Student’s t test or ANOVA was used for determination of significant differences. PASW 18.0 was used for statistical analysis. 3. Results 3.1. Molecular characterization of an aspartic protease of T. spiralis The Ts-Asp gene sequence was 1281 bp long and was found to encode a protein of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95 (Genebank No. XP 003380300). Results of similarity analysis using the NCBI BLAST program showed that the deduced amino
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Fig. 1. Multiple amino acid sequence alignment of the aspartic protease (Ts-Asp) from T. spiralis with homologues from other helminthes. B. malayi BmAsp2 (Genebank accession no. BAC05689), N. americanus necepsin II (Genebank No. CAC00543), A. suum cathepsin D (Genebank No. ADY43078), A. simplex cathepsin D (Genebank No. ACY38599), A. ceylanicum cathepsin D (Genebank No. AAO22152), C. elegans Asp-4 (Genebank No. NP 510191), L. loa Asp-2 (Genebank No. XP 003148873), and S. ratti Asp-4 (Genebank No. ACR56788). Identical residues are shown in black boxes, conservative residues in dark gray boxes, similar residues in light gray boxes, and unrelated residues have a white background. Amino acid numbers are shown on the right. The putative signal sequences are shown in the box. The catalytic motif region is indicated with a dotted line (DTG) and the active site flap is represented by a solid line. Catalytic Asp residues are indicated with arrow heads (), and inhibitor binding residues are indicated by stars ().
acid sequence of Ts-Asp possessed homology with aspartic proteases from N. americanus (56%), Ascaris suum (55%), Anisakis simplex (54%), Ancylostoma ceylanicum (57%), C. elegans (55%), Brugia malayi (53%), Loa loa (53%), and Strongyloides ratti (54%) (Fig. 1). Ts-Asp nucleotide sequence predicted by NCBI gene Graphic viewer and Expasy proteomics tool. Ts-Asp contains signal pepetide sequence (from Met-1 to Gly-15) and two catalytic motifs (DTG), which include active residues (Asp-99, Asp-286) thought to be essential for aspartic protease activity. However, two active site flaps were not identical to those previously reported aspartic proteases of other parasites (Fig. 1), although most inhibitor binding sites were conserved (Asp99, Gly-101, Ser-103, Ile-142, Arg-143, and Ile-189). To examine the phylogenetic relationship of Ts-Asp with other
homologous aspartic proteases, a phylogenetic tree was constructed based on the deduced amino acid sequences from 26 aspartic proteases. Protein distance-based phylogenetic analysis of the eukaryotic aspartic protease amino acid sequences resulted in the creation of a dendrogram with three clusters (Fig. 2). Ts-Asp was located in Cluster I with cathepsin D of Schistosoma japonicum, A. suum, A. simplex, and A. ceylanicum; lysosomal aspartic protease of Culex quinquefasciatus; asp-2B of B. malayi and L. loa; asp-4 of S. ratti, and C. elegans; necepsin II of N. americanus. 3.2. rTs-Asp exhibited aspartic protease activity In order to understand about Ts-Asp, we performed purification of GSP fusion rTs-Asp (∼72 kDa) using a
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Fig. 2. Clustering of aspartic proteases using molecular phylogenetic analysis. An un-rooted phylogenetic tree was generated based on alignment of amino acid sequences from 26 aspartic proteases of nematode helminthes. The scale bar indicates an evolutionary distance of amino acid substitutions per position. T. spiralis lysosomal aspartic protease (Genebank No. XP 003380300) A. ceylanicum cathepsin D (Genebank No. AAO22152.1), A. simplex cathepsin D (Genebank No. ACY38599.1), A. suum cathepsin D (Genebank No. ADY43078.1), B. malayi Asp-1 (Genebank No. BAC05688) and Asp-2, C. briggsae Asp-2 (Genebank No. XP 002637505) and Asp-6 (Genebank No. XP 002635088), C. elegans 1a Asp (Genebank No. CAA08899), 2a Asp-2 (Genebank No. NP 872129), Asp-2 (Genebank No. NP 505384), Asp-4, C. remanei Asp-1 (Genebank No. XP 003093500), Asp-5 (Genebank No. XP 003110542), C. quinquefasciatus Asp (Genebank No. XP 001867326), H. contortus Asp (Genebank No. CAE12199), L. loa Asp-2 and Asp-6 (Genebank No. EFO15484), N. americanus necepsin I (Genebank No. CAC00542) and necepsin II, S. japonicum cathepsin D (Genebank No. CAX72322), Steinernema carpocapsae Asp (Genebank No. ADJ94115), St. feltiae Asp (Genebank No. ACS32298), S. ratti Asp-4 and Asp-2B (Genebank No. ACR56786), and S. stercoralis Asp (Genebank No. AAD09345).
prokaryotic expression system (Fig. 3A). After thrombin digestion, rTs-Asp was appeared at approximately 45 kDa. Gelatin zymogram analysis was performed for evaluation of proteolytic activity. As a result, protease activity of approximately 43 kDa (rTs-Asp) and 70 kDa (GST – rTsAsp fusion protein) was detected (Fig. 3B). This size shift effect may be due to differences in sample preparation (native form or denatured form) between zymogram and SDS-PAGE. For determination of rTs-Asp aspartic protease activity, aspartic protease activity of rTs-Asp was evaluated using assay kits. After a 10 min incubation period, rTs-Asp had digested approximately 60–70% of aspartic specific substrate; however, complete inhibition of this activity was observed by treatment with pepstatin (aspartic protease specific inhibitor) (Fig. 3C).
3.3. Abundant expression of Ts-Asp was observed in muscle stage larva In order to determine when Ts-Asp mRNA showed the greatest expression, real time RT-PCR was performed on first-strand cDNA from newborn larva, muscle stage larva, and adult worms of T. spiralis. As shown in Fig. 4, the highest level of expression of the Ts-Asp gene was observed in muscle stage larva of T. spiralis.
3.4. Ts-Asp was secreted and elicited specific antibody production To determine whether or not T. spiralis muscle stage larvae secrete Ts-Asp, SDS-PAGE was performed under reducing conditions for resolution of TE and ES products from muscle larva, followed by blotting to NC membranes. Immunoblotting was performed using rat antiserum against rTs-Asp. Approximately 45 kDa protein, which was reacted with rTs-Asp polyclonal antibody, was observed in both TE and ES proteins (Fig. 5A). To determine whether or not Ts-Asp has antigenecity, western blotting was performed using serum of T. spiralis infected mice 2, 3, and 4 weeks after infection. Anti Ts-Asp specific antibody was detected in serum at 3 and 4 weeks after T. spiralis infection of mice (Fig. 5B). These results suggested that Ts-Asp might have antigenicity and be secreted by muscle stage larva. 3.5. Ts-Asp was localized in genital primordium and stichocytes. To determine the location of Ts-Asp secretion, we performed immunohistochemistry to evaluate localization of Ts-Asp specific antiserum in muscle tissue of T. spiralis muscle stage larva infected mice. Results showed that anti
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Fig. 3. Analysis of purification and proteolytic activity of rTs-Asp. SDS-PAGE was performed for separation of the purified GST fusion rTs-Asp (A). M; protein molecular marker, lane 1; inclusion body of Ts-Asp transformed BL21 cell lysate, lane 2; supernatant of Ts-Asp transformed BL21 cell lysate, lane 3; purified GST fusion rTs-asp. Gelatin Zymogram was used for analysis of proteolytic activity (B). M; protein molecular marker, lane 1; supernatant of Ts-Asp transformed BL21 cell lysate, lane 2; 1 g of GST–rTs-Asp fusion protein after thrombin digestion. lane 3; 10 g GST–rTs-Asp fusion protein after thrombin digestion. Arrowhead; GST–rTs-Asp fusion protein, Arrow; rTs-Asp. An aspartic protease assay kit was used for measurement of spartic protease specific activity of rTs-Asp (C). C; (substrate + Cathepsin D), T; (substrate + rTs-Asp), C + I (substrate + Cathepsin D + pepstatin), T + I (substrate + rTs-Asp + pepstatin).
rTs-Asp serum was strongly reacted with the genital primordium and also they was slightly reacted with some stichocytes of muscle stage larva (Fig. 6). 4. Discussion
Fig. 4. Real-time RT-PCR analysis of expression of Ts-Asp during developmental stages. N; new born larva, M; muscle stage larva, A; adult worm (*; p < 0.05, experiments was performed in triplicate).
In this study, we characterized an aspartic protease released by nematode parasite T. spiralis. This is the first report on characterization study about aspartic protease of T. spiralis. Based on the alignment and comparison of the Ts-Asp protein sequence deduced from full length cDNA sequences in the protein databases using the BLAST program, we found that Ts-Asp shared significant identity (above 50%) with aspartic proteases of other nematodes, including N. americanus, A. suum, A. simplex, A. ceylanicum, C. elegans, B. malayi, L. loa, and S. ratti (Fig. 1). In addition, Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, which is a common
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Fig. 5. Detection of Ts-Asp in ES proteins and specific antibody against Ts-Asp using western blot analysis. One hundred g of TE and ES protein of T. spiralis were separated by SDS-PAGE. The polyclonal antibody anti-rTs-Asp (1:1000 dilution) was used as primary antibody (A). Ten g of GST fusion rTs-Asp was loaded on SDS-PAGE, and the proteins was reacted with mice serum obtained from every weeks after T. spiralis infection (B) (N; serum of non-infected mice, 2–4 weeks; serum of mice 2–4 weeks after T. spiralis infection).
Fig. 6. Localization of Ts-ASP in muscle stage larva using immunohistochemistry. Muscle tissue was obtained from T. spiralis infected mice at 6 weeks; the tissue was reacted with anti-rTs-Asp antibody (A and B) and with PBS (C). Arrow indicated reactive regions of muscle stage larva. Arrow head indicated midgut of muscle stage larva (Bar is 50 m).
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characteristic of aspartic proteases. Also, rTs-Asp could digest aspartic specific substrate; this activity was completely inhibited by aspartic protease inhibitor (Fig. 3C). These results demonstrated that Ts-Asp could be an aspartic protease isolated from T. spiralis. Although a large number of aspartic protease genes have been identified from several nematodes, only a few detailed phylogenetic studies of these genes have been reported. Mello et al. demonstrated that most nematode aspartic proteases were grouped into three clusters (Mello et al., 2009). They also suggested that at least three independent duplications have occurred in this part of the protease family; one in the lineage containing C. elegans Asp-1a, another in the predecessor of H. contortus, and a distinct set of events in the branch leading to S. ratti asp-4 (Mello et al., 2009). They found that the cluster is closely related to the location of aspartic protease gene within the genome; however, functional differences between the clusters have not been revealed. In our study, Ts-Asp was clustered with several cathepsin D type aspartic proteases (Fig. 2); this cluster was also reported by Mello et al. (2009). Most cathepsin D aspartic proteases have been identified from blood feeding nematodes or tissue invading nematodes; their function was related to the digestion of hemoglobin and the invasion of host tissue (Balasubramanian et al., 2012; Brinkworth et al., 2000; Gallego et al., 1998; Harrop et al., 1996; Williamson et al., 2003). Although T. spiralis can also invade tissue and feed on blood materials during their life cycle, it is not clear whether or not TsAsp is closely related to the blood feeding and the tissue invasion. Many nematodes are known to secrete aspartic proteases; however, the function of these molecules is not yet clear. The presence of several aspartic protease homologues in the free-living nematode C. elegans (Geier et al., 1999) suggests that these proteases could be related to the development of embryo and larvae (Tcherepanova et al., 2000). In parasites, aspartic proteases may be related not only to development of worms, but also to parasitism such as degradation of the extracellular matrix, digestion of hemoglobin, and so on (Dzik, 2006). Yang et al. suggested that aspartic protease has a definite role in embryo development and the pre-parasitic muscle larva stage of A. caninum (Yang et al., 2009). In this study, Ts-Asp was more likely to be expressed in genital primodium (Fig. 6). These results perhaps indicated that Ts-Asp was able to have a role in genital development of muscle larva. The expression levels of Ts-Asp across the life cycle of T. spiralis were measured by Real time RT-PCR. As results, the highest levels of expression of Ts-Asp were observed in muscle stage larva of T. spiralis (Fig. 4). Also, detection of anti-Ts-Asp specific antibodies in serum of parasite infected mice started to appear 3 weeks after infection (Fig. 5). Therefore, primary secretion of Ts-Asp might have begun once newborn larvae had reached muscle tissue. In conclusion, we firstly characterized an aspartic protease Ts-Asp of T. spiralis, which was abundant expressed by muscle stage larva. In order to better understand the exact function and mechanisms of Ts-Asp, more detailed functional and mechanism studies will be needed.
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Acknowledgement This work was supported by the Bio-Scientific Research Grant funded by the Pusan National University (PNU, BioScientific Research Grant) (PNU-2010-101-251) References Balasubramanian, N., Toubarro, D., Nascimento, G., Ferreira, R., Simoes, N., 2012. Purification, molecular characterization and gene expression analysis of an aspartic protease (Sc-ASP113) from the nematode Steinernema carpocapsae during the parasitic stage. Mol. Biochem. Parasitol. 182, 37–44. Brindley, P.J., Kalinna, B.H., Wong, J.Y., Bogitsh, B.J., King, L.T., Smyth, D.J., Verity, C.K., Abbenante, G., Brinkworth, R.I., Fairlie, D.P., Smythe, M.L., Milburn, P.J., Bielefeldt-Ohmann, H., Zheng, Y., McManus, D.P., 2001. Proteolysis of human hemoglobin by schistosome cathepsin D. Mol. Biochem. Parasitol. 112, 103–112. Brinkworth, R.I., Harrop, S.A., Prociv, P., Brindley, P.J., 2000. Host specificity in blood feeding parasites: a defining contribution by haemoglobindegrading enzymes? Int. J. Parasitol. 30, 785–790. Brown, A., Girod, N., Billett, E.E., Pritchard, D.I., 1999. Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration. Am. J. Trop. Med. Hyg. 60, 840–847. Capo, V., Despommier, D.D., 1996. Clinical aspects of infection with Trichinella spp. Clin. Microbiol. Rev. 9, 47–54. Cwiklinski, K., Meskill, D., Robinson, M.W., Pozio, E., Appleton, J.A., Connolly, B., 2009. Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene. Vet. Parasitol. 159, 268–271. Despommier, D., 1975. Adaptive changes in muscle fibers infected with Trichinella spiralis. Am. J. Pathol. 78, 477–496. Despommier, D.D., 1993. Trichinella spiralis and the concept of niche. J. Parasitol. 79, 472–482. Dzik, J.M., 2006. Molecules released by helminth parasites involved in host colonization. Acta Biochim. Pol. 53, 33–64. Gallego, S.G., Slade, R.W., Brindley, P.J., 1998. A cDNA encoding a pepsinogen-like, aspartic protease from the human roundworm parasite Strongyloides stercoralis. Acta Trop. 71, 17–26. Geier, G., Banaj, H.J., Heid, H., Bini, L., Pallini, V., Zwilling, R., 1999. Aspartyl proteases in Caenorhabditis elegans Isolation, identification and characterization by a combined use of affinity chromatography, two-dimensional gel electrophoresis, microsequencing and databank analysis. Eur. J. Biochem. 264, 872–879. Harrop, S.A., Prociv, P., Brindley, P.J., 1996. Acasp, a gene encoding a cathepsin D-like aspartic protease from the hookworm Ancylostoma caninum. Biochem. Biophys. Res. Commun. 227, 294–302. Hotez, P.J., Ashcom, J., Bin, Z., Bethony, J., Williamson, A., Hawdon, J.M., Jianjun, F., Dobardzic, A., Rizo, I., Bolden, J., Jin, Q., Yan, W., Dobardzic, R., Chung-Debose, S., Crowell, M., Datu, B., Delaney, A., Dragonovski, D., Jiang, Y., Yueyuan, L., Ghosh, K., Loukas, A., Brandt, W., Russell, P.K., Zook, B.C., 2002. Effect of vaccinations with recombinant fusion proteins on Ancylostoma caninum habitat selection in the canine intestine. J. Parasitol. 88, 684–690. Jolodar, A., Miller, D.J., 1997. Preliminary characterisation of an Onchocerca volvulus aspartic protease. Int. J. Parasitol. 27, 1087–1090. Jolodar, A., Miller, D.J., 1998. Identification of a novel family of nonlysosomal aspartic proteases in nematodes. Biochim. Biophys. Acta 1382, 13–16. Liu, M.Y., Wang, X.L., Fu, B.Q., Li, C.Y., Wu, X.P., Le Rhun, D., Chen, Q.J., Boireau, P., 2007. Identification of stage-specifically expressed genes of Trichinella spiralis by suppression subtractive hybridization. Parasitology 134, 1443–1455. Mello, L.V., O’Meara, H., Rigden, D.J., Paterson, S., 2009. Identification of novel aspartic proteases from Strongyloides ratti and characterisation of their evolutionary relationships, stage-specific expression and molecular structure. BMC Genomics 10, 611. Nagano, I., Wu, Z., Takahashi, Y., 2009. Functional genes and proteins of Trichinella spp. Parasitol. Res. 104, 197–207. Ozkoc, S., Tuncay, S., Delibas, S.B., Akisu, C., 2009. In vitro effects of resveratrol on Trichinella spiralis. Parasitol. Res. 105, 1139–1143. Park, H.K., Chang, S.W., Kang, S.W., Cho, M.K., Choi, S.H., Hong, Y.C., Lee, Y.S., Jeong, H.J., Yu, H.S., 2008. Expressed sequence tags of Trichinella spiralis muscle stage larvae. Korean J. Parasitol. 46, 59–63. Park, H.K., Cho, M.K., Choi, S.H., Kim, Y.S., Yu, H.S., 2011a. Trichinella spiralis: infection reduces airway allergic inflammation in mice. Exp. Parasitol. 127, 539–544.
518
J.N. Park et al. / Veterinary Parasitology 190 (2012) 510–518
Park, M.K., Cho, M.K., Kang, S.A., Park, H.K., Kim, Y.S., Kim, K.U., Ahn, S.C., Kim, D.H., Yu, H.S., 2011b. Protease-activated receptor 2 is involved in Th2 responses against Trichinella spiralis infection. Korean J. Parasitol. 49, 235–243. Robinson, M.W., Menon, R., Donnelly, S.M., Dalton, J.P., Ranganathan, S., 2009. An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host. Mol. Cell Proteomics 8, 1891–1907. Romaris, F., North, S.J., Gagliardo, L.F., Butcher, B.A., Ghosh, K., Beiting, D.P., Panico, M., Arasu, P., Dell, A., Morris, H.R., Appleton, J.A., 2002. A putative serine protease among the excretory–secretory glycoproteins of L1 Trichinella spiralis. Mol. Biochem. Parasitol. 122, 149–160. Shaw, R.J., McNeill, M.M., Maass, D.R., Hein, W.R., Barber, T.K., Wheeler, M., Morris, C.A., Shoemaker, C.B., 2003. Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis. Int. J. Parasitol. 33, 1233–1243. Smith, W.D., Skuce, P.J., Newlands, G.F., Smith, S.K., Pettit, D., 2003. Aspartyl proteases from the intestinal brush border of Haemonchus contortus as protective antigens for sheep. Parasite Immunol. 25, 521–530. Sohn, W.M., Huh, S., Chung, D.I., Pozio, E., 2003. Molecular identification of Korean Trichinella isolates. Korean J. Parasitol. 41, 125–127. Tcherepanova, I., Bhattacharyya, L., Rubin, C.S., Freedman, J.H., 2000. Aspartic proteases from the nematode Caenorhabditis elegans
Structural organization and developmental and cell-specific expression of asp-1. J. Biol. Chem. 275, 26359–26369. Todorova, V.K., Stoyanov, D.I., 2000. Partial characterization of serine proteinases secreted by adult Trichinella spiralis. Parasitol. Res. 86, 684–687. Verity, C.K., McManus, D.P., Brindley, P.J., 1999. Developmental expression of cathepsin D aspartic protease in Schistosoma japonicum. Int. J. Parasitol. 29, 1819–1824. Williamson, A.L., Brindley, P.J., Abbenante, G., Datu, B.J., Prociv, P., Berry, C., Girdwood, K., Pritchard, D.I., Fairlie, D.P., Hotez, P.J., Zhan, B., Loukas, A., 2003. Hookworm aspartic protease Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion. J. Infect. Dis. 187, 484–494. Williamson, A.L., Brindley, P.J., Abbenante, G., Prociv, P., Berry, C., Girdwood, K., Pritchard, D.I., Fairlie, D.P., Hotez, P.J., Dalton, J.P., Loukas, A., 2002. Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity. FASEB. J. 16, 1458–1460. Wranicz, M.J., Gustowska, L., Gabryel, P., Kucharska, E., Cabaj, W., 1998. Trichinella spiralis: induction of the basophilic transformation of muscle cells by synchronous newborn larvae. Parasitol. Res. 84, 403–407. Yang, Y., Wei, H., Qin, W., Zheng, J., 2009. Expression and characterization of aspartic protease gene in eggs and larvae stage of Ancylostoma caninum. Parasitol. Res. 104, 1327–1333.