Molecular and immunological characterization of cathepsin L-like cysteine protease of Paragonimus pseudoheterotremus.

Cathepsin L is a cysteine protease belonging to the papain family. In parasitic trematodes, cathepsin L plays essential roles in parasite survival and host-parasite interactions. In this study, cathepsin L of the lung fluke Paragonimus pseudoheterotremus (PpsCatL) was identified and its molecular biological and immunological features characterized. A sequence analysis of PpsCatL showed that the gene encodes a 325-amino-acid protein that is most similar to P. westermani cathepsin L. The in silico three-dimensional structure suggests that PpsCatL is a pro-enzyme that becomes active when the propeptide is cleaved. A recombinant pro-PpsCatL lacking the signal peptide (rPpsCatL), with a molecular weight of 35 kDa, was expressed in E. coli and reacted with P. pseudoheterotremus-infected rat sera. The native protein was detected in crude worm antigens and excretory-secretory products and was localized in the cecum and in the lamellae along the intestinal tract of the adult parasite. Enzymatic activity of rPpsCatL showed that the protein could cleave the fluorogenic substrate Z-Phe-Arg-AMC after autocatalysis but was inhibited with E64. The immunodiagnostic potential of the recombinant protein was evaluated with an enzyme-linked immunosorbent assay (ELISA) and suggested that rPpsCatL can detect paragonimiasis with high sensitivity and specificity (100 and 95.6 %, respectively). This supports the further development of an rPpsCatL-ELISA as an immunodiagnostic tool.

Molecular and biochemical characterization of Paragonimus westermani tyrosinase.

Trematode tyrosinases (TYRs) play a major role in the tanning process during eggshell formation. We investigated the molecular and biochemical features of Paragonimus westermani TYR (PwTYR). The PwTYR cDNA was composed of 1568-bp encompassing a 1422-bp-long open reading frame (474-amino acid polypeptide). A strong phylogenetic relationship with Platyhelminthes and Deuterostomian orthologues was evident. The recombinant PwTYR expressed in prokaryotic cells promptly oxidized diphenol substrates, with a preferential affinity toward ortho-positioned hydroxyl groups. It demonstrated fairly weak activity for monophenol compounds. Diphenol oxidase activity was augmented with an increase of pH from 5.0 to 8.0, while monophenol oxidase activity was highest at an acidic pH and gradually decreased as pH increased. Transcription profile of PwTYR was temporally upregulated along with worm development. PwTYR was specifically localized in vitellocytes and eggs. The results suggested that conversion of tyrosine to L-dihydroxyphenylalanine by PwTYR monophenol oxidase activity might be rate-limiting step during the sclerotization process of P. westermani eggs. The pH-dependent pattern of monophenol and diphenol oxidase activity further proposes that the initial hydroxylation might slowly but steadily progress in acidic secreted vesicles of vitellocytes and the second oxidation process might be rapidly accelerated by neural or weak alkaline pH environments within the ootype.

Cloning and characterization of a new cysteine proteinase secreted by Paragonimus westermani adult worms.

The cysteine proteinases of Paragonimus westermani are known to play important roles in invasion and pathogenesis to hosts and in immune modulation and nutrient uptake. In this study, we have cloned a new cysteine proteinase of P. westermani, PwCP2, from adult worms and tested its diagnostic usefulness. The PwCP2 gene had an open reading frame of 816 base pairs and a conserved catalytic triad of cysteine, histidine, and asparagine residues. The mature form of recombinant PwCP2 (rPwCP2) lacking a proregion was overexpressed in Escherichia coli and used to produce antiserum. Western blot and immunohistochemical analyses using this antiserum showed that PwCP2 was expressed as a mature form, 24-kD product in a crude extract and in the excretory-secretory product of P. westermani, and was localized mainly in the intestinal epithelium of the adult worm. Western blot analysis using the rPwCP2 showed not only high sensitivity (90%) and specificity (100%) to sera from patients with paragonimiasis westermani, but also no cross-reactivity with sera from patients with clonorchiasis, sparganosis, or cysticercosis. Furthermore, an enzyme-linked immunosorbent assay using rPwCP2 exhibited a sensitivity of 93% and a specificity of 93% with sera of rats infected with P. westermani metacercariae. These results suggest that the excretory-secretory PwCP2 can be used for the diagnosis of paragonimiasis.

[Cloning and identification of the gene fragments of Paragonimus westermani].

OBJECTIVE: To screen and identify the recombinants from the cDNA library of the adult Paragonimus westermani(PwA) for immunodiagnosis and immunoprophylaxis. METHODS: PwA cDNA library was screened with the PwA antigen immunized rabbit sera(IRS) pre-absorbed by the extract of E. coli XL1-Blue. The recombinants from positive clones were amplified by PCR, sequenced and cut off by KpmI/BamHI and, then sub-cloned into pRESETB vector. The fusion protein was expressed, analysed by SDS-PAGE and identified by Western blotting with immune rabbit serum against worm antigen of Paragonimus westermani. RESULTS: The inserted cDNA fragment from the positive clone Pw-2 was about 800 bp, which contained an open reading frame(ORF) encoding Pw pre-procathepsin L belonging to cysteinase family. Expression product of Pw-2 was a fusion protein of 32 kDa, which can be recognized by immune rabbit serum against worm antigen of Paragominus westermani. CONCLUSION: A recombinant plasmid Pw-2 encodes Pw pre-procathepsin L is constructed.

[cDNA cloning and location of Pagumogonimus skrj abini cysteine protease].

OBJECTIVE: To clone the cysteine protease cDNA fragment from Pagumogonimus skrjabini adults and locate the tissue of the adult worm where cysteine protease is expressed. METHODS: The cysteine protease cDNA fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with degenerated primers. The production was TA-cloned into the pUCm-T vector and sequenced. DNASIS program was used to analyse the nucleotide sequence and deduce the amino acid sequence, which was aligned with the correlated parasite cysteine protease afterwards. The digoxin labeled cRNA probe was synthesised by in vitro transcription with the cloned cDNA as template. The frozen sections of the adult worms were analysed by hybridization in situ to locate the gene expression. RESULTS: A 495 bp cDNA fragment was amplified by RT-PCR and sequenced. An amino acid sequence was deduced by DNASIS. Sequence analysis and alignment showed significant homologies with the correlated parasite cysteine proteinases and conservation of Cys, His and Asn residues that from a catalytic triad. In the hybridization in situ analysis, intestinal epithelium was stained positively on transverse section of adult worms. CONCLUSION: The cysteine proteinase cDNA fragment from Pagumogonimus skrjabini adults was cloned. There are some key sites which are correlated to the function of cysteine protease in the cDNA fragment. Cysteine protease is mainly expressed in intestinal epithelium of P. skrjabini.

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae.

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.

Characterization and classification of five cysteine proteinases expressed by Paragonimus westermani adult worm.

Three new members of the cysteine proteinase gene family of Paragonimus westermani have been isolated and classified. Comparisons of the predicted amino acid sequences of PwCP2 (U69121), PwCP4 (U56958), and PwCP5 (U33215) were performed with those of the previously reported PwCP1 (U69120) and PwCP3 (U56865) sequence. The amino acid alignment showed conservation of the cysteine, histidine, and asparagine residue that form the catalytic triad. With 57 cysteine proteinases including PwCP1-5, we conducted phylogenetic analysis using neighbor joining method (NJ). A resultant unrooted tree revealed that PwCP1-5 were clustered with cruzipain-like or cathepsin L-like cysteine proteinases. More detailed phylogenetic analyses with a reduced alignment set (22 cysteine proteinases) were performed by NJ and maximum parsimony (MP) methods. The results showed coincidently that PwCP1, 2, 3, and 4 belonged to the group of previously reported cruzipain-like cysteine proteinases (bootstrapping values of 97 and 100% in the MP and NJ trees) but PwCP5 to cathepsin L-like cysteine proteinases (the value of 76 and 100% in MP and NJ trees). Within the cruzipain-like clade, PwCP2 and 4 were found to be the most closely related. PwCP 2, 3, and 4 have five of six cruzipain signature sequences known previously, whereas PwCP5 do not have any cruzipain sequences in the corresponding sites. We found that two signature candidate sites (Gly 174, Asn 175--human cathepsin L numbering) for cathepsin L-like group are conserved in PwCP5, which are conserved within cathepsin L-like group and also different from those of cruzipain and other cysteine proteinase groups. PwCP5 has three-residue insertion (hydrophilic residues, Ser-Tyr-Gly) within the position corresponding to S3 subsite of SmCL2. Compared to the two-residue insertion (Tyr-Gly) in SmCL2, the three-residue insertion appeared in PwCP5 may bring bigger difference in substrate specificity between PwCP1-4 (cruzipain) and PwCP5 (cathepsin L-like). Such presumption is quite plausible considering extremely lower amino acid sequence similarity (18.2%) between PwCP1-4 and PwCP5. The present study is worthy of reporting one another case, the third organism after Schistosoma mansoni and Schistosoma japonicum, which has the two kinds of genes encoding both the cruzipain and cathepsin L-like cysteine proteinases. In addition, the fact that most of cysteine proteinases from P. westermani are cruzipain-like type implies strongly that a new powerful drug for paragonimiasis could be designed and developed if we focus on the exploration of anti-agents against P. westermani cruzipain-like cysteine proteinases.

Cysteine protease secreted by Paragonimus westermani attenuates effector functions of human eosinophils stimulated with immunoglobulin G.

An immunoglobulin G (IgG)-coated surface, such as that found on helminth parasites, is one of the most effective physiologic stimuli for eosinophil activation. The cysteine proteases secreted by tissue-invasive helminth larvae play an important role in evasion of the immune response by degrading the host immunoglobulins. In this study, we investigated whether cysteine proteases in the excretory-secretory product (ESP) produced by Paragonimus westermani newly excysted metacercariae (PwNEM), which cause pulmonary or extrapulmonary paragonimiasis in human beings, could modify effector functions of human eosinophils stimulated with IgG. We coated 96-well plates with human IgG in the absence or presence of the ESP produced by PwNEM. When eosinophils were incubated in the wells coated with IgG in the presence of the ESP, eosinophil degranulation and superoxide production were significantly reduced compared with results for cells incubated in wells coated with IgG alone. This inhibitory effect of the ESP on IgG-induced superoxide production was dose dependent and was significantly abolished by pretreatment of the ESP with heat. These findings suggest that the cysteine proteases secreted by PwNEM attenuate both activation and degranulation of eosinophils stimulated with IgG. Thus, the cysteine proteases produced by tissue-invasive helminth larvae play crucial roles in evasion of IgG-dependent eosinophil helminthotoxicity and in reduction of eosinophil-associated tissue inflammation during the migratory period.

Structural and immunological characteristics of a 28-kilodalton cruzipain-like cysteine protease of Paragonimus westermani expressed in the definitive host stage.

A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis.

Proteolytic activity of cysteine protease in excretory-secretory product of Paragonimus westermani newly excysted metacercariae pivotally regulates IL-8 production of human eosinophils.

This study investigated the effect of the excretory-secretory product (ESP) of Paragonimus westermani newly excysted metacercariae (PwNEM) on IL-8 production of human mature eosinophils. Treatment of eosinophils with lower concentrations (0.3 and 1 microg/ml) of the ESP significantly (P < 0.01) induced IL-8 production, whereas treatment of cells with higher concentrations (3 and 10 microg/ml) did not. This effect of the ESP was concentration-dependent. Interestingly, the amount of IL-8 production released into the culture supernatants was inversely correlated with the rate of eosinophil survival. When eosinophils were cultured with the same concentrations of the ESP for 24 h, the ESP resulted in eosinophil death in a dose-dependent manner. To investigate whether high proteolytic activity of proteases in the ESP could cause little production of IL-8, 10 microg/ml of ESP was pretreated with heat at 100 degrees C for 10 min or 56 degrees C for 30 min to reduce its proteolytic activity. IL-8 production of eosinophils incubated with heat-treated ESP was markedly increased comparable to that of cells treated with the lowest concentration used in this study. These findings suggest that the protease in the ESP of PwNEM pivotally regulates IL-8 production by controlling of eosinophil survival, depending on the amount of ESP released in vivo. Thus, the cysteine protease in the ESP of PwNEM could provide a novel role to control recruitment of inflammatory cells in larval-infected lesions in human paragonimiasis.

Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage.

We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST.

Cloning and expression of a cysteine proteinase gene from Paragonimus westermani adult worms.

A gene encoding a cysteine proteinase from Paragonimus westermani has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from the conserved active site of the cysteine proteinase. The 5' and 3' regions of the gene were amplified using a PCR technique for the rapid amplification of cDNA ends. The cloned gene has an open reading frame of 687 bp and deduced amino acid sequence of 229. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form a catalytic triad. Analysis of the expressed protein on sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa. The expressed protein reacted with the sera of patients with paragonimiasis but not with the sera of fascioliasis and clonorchiasis. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of paragonimiasis.

Comparative studies on protein, isoenzyme and DNA restriction endonuclease profiles of Thai Paragonimus metacercariae.

General proteins and 14 enzymes from metacercariae of Paragonimus heterotremus, P. siamensis and P. westermani were determined by vertical polyacrylamide gel electrophoresis. The isoenzyme profiles showed considerable interspecific polymorphism for general protein (PT), malate dehydrogenase (MDH), malic enzyme (ME) and tetrazolium oxidase (TO) while those of glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) showed similarity. The Pt-6 and To-I loci can be used as identification markers for these three species. The preliminary study of the molecular biology of Paragonimus heterotremus P. siamensis and P. westermani was based on analysis of metacercarial genomic DNA with restriction endonuclease Pst I. Agarose gel electrophoresis revealed restriction fragment length differences among the three species studied. The DNA restriction fragments were approximately 4-6 fragments, ranging from 5.35 to 14.67 kb. Among these. P westermani shared two homologous fragments with P. siamensis, ie, 5.35 and 7.22 kh, none with P. heterotremus, while P. heterotremus shared only one with P. siamensis, ie, 8.16 kb. Thus, the DNA restriction fragment length differences can be used to differentiate among these three species.

Cysteine protease activities during maturation stages of Paragonimus westermani.

In mature Paragonimus westermani, specific activity of parasitic cysteine protease declines. To clarify which of the known 17-, 27-, and 28-kDa enzyme activities is decreased, the cysteine proteases were purified from the crude extracts of metacercariae, 4- and 7-wk juveniles, and 16-wk adults by gel filtration, ion-exchange, and affinity matrix chromatographies; the enzyme activity was monitored with the fluorogenic substrate, Cbz-phe-arg-AMC. In addition to 3 known enzymes, 2 other cysteine proteases at 15 and 53 kDa were identified in juveniles and adults and were purified. The 2 novel enzymes were most active in 0.1 M ionic strength and pH 5-6 and were inhibited by N-(N-[L-3-transcarboxyrane-2-carbonyl]-L-leucyl)agamatine, iodoacetamide, and leupeptin. Of the 5 enzymes, specific activities of metacercarial 27- and 28-kDa enzymes were lowered from metacercaria to 16 wk. Between 4 and 16 wk, activities of 3 cysteine proteases of juveniles and adults were additionally exhibited. The activity changes of 5 different cysteine proteases may be associated with migration and immune evasion during the maturation stage of P. westermani when the parasite environment is changing.

Activities of different cysteine proteases of Paragonimus westermani in cleaving human IgG.

Cleaving host immunoglobulins is a well known mechanism of evading host immune reactions exploited by helminth parasites. Secreted cysteine proteases of helminth are a part of enzymes cleaving host IgG. Paragonimus westermani produces at least six different species of the cysteine protease in its developmental stages. This study was undertaken to evaluate comparatively the activities against human IgG by the different enzymes. When the metacercariae, which secrete 27 and 28 kDa cysteine proteases, were incubated in human IgG solution, IgG was degraded at its hinge region. Further incubation resulted complete hydrolysis. From 4-week and 7-week old juveniles and 16-week old adults of P. westermani, five different enzymes at 15, 17, 27, 28 and 53 kDa have been purified, if the enzyme with the same molecular mass is regarded to be identical. In cleaving human IgG, each cysteine protease exhibited decreasing activities with age.

Cloning of a cysteine proteinase cDNA of adult Paragonimus westermani by polymerase chain reaction.

Cysteine proteinase cDNA fragment from adult mammalian trematode, Paragonimus westermani was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 804 bp. The deduced amino acid sequence consists of 268 amino acids. Sequence analysis and alignment showed significant sequence similarity to other eukaryotic cysteine proteinases and conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. The cysteine proteinase cDNA fragment was also subcloned in the expression vector pET and expressed as a C-terminal His-tag fusion protein in Escherichia coli.

Enzyme-linked immunosorbent assay using cysteine proteinase antigens for immunodiagnosis of human paragonimiasis.

An enzyme-linked immunsorbent assay (ELISA) using worm extract antigens from lung flukes of Paragonimus westermani provided good sensitivity to sera from patients with paragonimiasis westermani but high cross-reactivity with sera from most fascioliasis patients and some patients with onchocerciasis or clonorchiasis. To improve the specificity, we tested an ELISA using fluke cysteine proteinases as antigens. Cysteine proteinases were partially purified from the excretory/secretory products of P. westermani by 40-75% ammonium sulfate fractionation, hydrophobic chromatography, and arginine affinity chromatography. An ELISA using the enzyme preparation not only had increased sensitivity to paragonimiasis westermani sera but also reduced cross-reactivity with the fascioliasis, onchocerciasis, and clonorchiasis sera to negligible levels. The reactivity of the ELISA to paragonimiasis miyazakii sera was similar to that of paragonimiasis westermani sera. A proteinase preparation from P. ohirai, which can be obtained easily from infected rats, provided similar results. Therefore, the ELISA using cysteine proteinases of Paragonimus could not distinguish the parasite species with which patients were infected, but it is a valuable assay with which to immunodiagnose paragonimiasis even when the proteinases are prepared from nonhuman species.

A cysteine protease of Paragonimus westermani eggs.

Protease activity was identified in crude extracts of Paragonimus westermani eggs which were purified from infected dog lungs, isolated on 14 weeks after metacercarial challenge. The eggs were used after removing possibly contaminated host or worm tissues on their shell surfaces. In the crude egg extracts, high proteolytic activities against carboxybenzoyl-phenylalanyl-arginyl-4-methoxy-beta-naphthylamide (Cbz-phe-arg-MNA) and Azocoll were detected whereas those against succinyl-alanyl-prolyl- phenylalanyl-p-nitroanilide (Suc-ala-pro-phe-pNA) were not revealed. The enzyme exhibited the maximal activity at pH 6. Its activity was inhibited by specific cysteine protease inhibitors, 10(-5) M 1-trans-epoxysuccinylleucylamido (4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA) while potentiated by 6.5-fold in the presence of 2.5 mM dithiothreitol (DTT). When the enzyme was purified partially by Sephacryl S-300 High Resolution gel filtration, it migrated as a single homogeneous band at 35 kDa. The 35 kDa cysteine protease has been recognized neither in the metacercariae nor in the adult. These findings indicated the presence of at least one protease of cathepsin family in immature eggs of P. westermani.

Purification and characterization of acid cysteine protease from metacercariae of the mammalian trematode parasite Paragonimus westermani.

Acid cysteine protease was purified from metacercariae of the mammalian trematode parasite Paragonimus westermani. The purified enzyme had a molecular mass of 27 kDa and was a monomeric polypeptide. The protease had an absolute requirement for a reducing agent for full activity towards fluorescein-isothiocyanate-labeled hemoglobin, and it was active in the acidic pH range, with an optimum pH of 4.0. While acidic proteolysis was insensitive to the aspartic protease inhibitor pepstatin A, activity was significantly inhibited by the cysteine protease inhibitors, leupeptin, chymostatin and L-trans-epoxy-succinyl-L-leucylamido(4-guanidino)-butane. The sensitivity of the enzyme to the inhibitors was similar to that of cathepsins B and L, but the specificity of the protease towards chromogenic substrates was slightly different from that of the cathepsins. The purified enzyme was highly specific for N-substituted peptidyl substrates containing arginine in the P1 position and phenylalanine in the P2 position, and the protease extensively degraded human native proteins, such as human serum albumin, immunoglobulins, complement components and also endogenous protease inhibitors. Since the protease hydrolyzes both soluble proteins and components of human defense systems, it may facilitate parasite nutrition and evasion of host defense mechanisms.