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Cysteine proteinase activity in various developmental stages of Clonorchis sinensis: A comparative analysis
Comp. Biochem. Physiol. Vol. 99B, No. 1, pp. 137-140, 1991 Printed in Great Britain
0305-0491/'91 $3.00 + 0.00 Pergamon Press plc
CYSTEINE PROTEINASE ACTIVITY IN VARIOUS DEVELOPMENTAL STAGES OF C L O N O R C H I S SINENSIS: A COMPARATIVE ANALYSIS CHUL YONG SONG* and AJAY A. REGEt~ *Department of Biology, Chung-Ang University, Seoul, Korea; and tVerna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA (Tel: 713-798-4531 (Received 9 October 1990) Abstract--1. Cysteine proteinase activity in acidic extracts of various developmental stages of Clonorchis sinensis (metacercariae, 1-, 2-, and 3-month old worms) was examined. All the activities were maximum at acidic pH and showed inhibitor susceptibilities similar to the vertebrate cysteine proteinases. 2. Specific activity of cysteine proteinase(s) was highest in metacercariae with either CBZ-phe-arg-AFC or Azocoll as the substrate. The immature and mature worms had similar (but less than metacercariae) levels of activity. 3. A soluble cysteine proteinase with a native molecular weight of approximately 20,000 + 1414 was partially purified from 1-, 2-, and 3-month worms. The molecular weight of similar activity in metacercariae was approximately 32,000. 4. Results suggest developmental regulation of cysteine proteinase activity in the life cycle of C. sinensis.
INTRODUCTION
Clonorchis sinensis is a trematode parasite that causes liver disease in its host. After oral infection with metacercariae, the parasites hatch out in the digestive tract and migrate up to the c o m m o n bile duct where they grow to maturity. During the development of Clonorchis sinensis, biliary epithelial hyperplasia and fibrosis develop around the worms, and dilation of the ducts into cyst-like cavities may occur. The biochemical mechanisms involved in both the nutrition of and the pathology caused by the parasite are not well understood. Proteolytic enzymes, including cysteine proteinases, are important to the host-parasite relationship and seem to play important roles in parasite physiology, pathogenesis, and evasion of host defense. Proteinases have been detected in various developmental stages of many trematode parasites. Thus, neutral proteases have been characterized in various developmental stages of Schistosoma mansoni (Auriault et al., 1982). Similarly, cysteine proteinases have been detected in the developmental stages of Fasciola hepatica (Dalton and Heffernan, 1989), and of Paragonimus westermani (Song and Dresden, 1990). However, very little is known about cysteine proteinase activity in C. sinensis. Earlier we reported the purification and characterization of a major cysteine proteinase from adult C. sinensis (Song et al., 1990). Here we present a comparative analysis of cysteine proteinase activity from various developmental stages of this parasite and demonstrate that some of these stages contain unique enzymes, suggesting their developmental regulation. ~:Author to whom correspondence should be addressed.
MATERIALS
AND METHODS
Parasites The metacercariae of Clonorchis sinensis were obtained from naturally infected Pseudorasbora parva collected in Kimhai-Gun, Korea. Rabbits were fed orally 300 metacercariae and were dissected sequentially on the first, second and third months after the experimental infection. The worms were harvested in the bile duct of the liver. The mean size of metacercariae was about 0.15 x 0.13 mm. The mean size of 1-month worms was 4.2 +0.16mm in length and 0.9 + 0.86 mm in width. The mean size of 2-month worms was 6.6+0.61 mm in length and 2.1 +0.17ram in width. The mean size of 3-month (adult) worms was 11.5 + 0.78 mm in length and 2.8 + 0.23 mm in width. The collected worms were washed several times with cold 0.85% saline, once again with distilled water, and lyophilized. Extraction of proteinases All procedures were carried out at 4°C unless otherwise stated. For the preparation of proteolytic enzymes, lyophilized C. sinensis metacercariae and worms (I0 mg/ml buffer) were homogenized in 0.1 M sodium citrate, pH 4.9, and processed as described previously (Song et al., 1990). The resulting supernatant fractions were subjected to further analysis. Assay of enzymatic activity Proteinase activities of the developmental stages (metacercariae, 1-month, 2-month, and 3-month worms) of C. sinensis were estimated using a low molecular weight, synthetic dipeptide substrate, carboxybenzoxyl-phenylalanine-arginine-7-amino-4-trifluoromethylcoumarin (CBZphe-arg-AFC; Enzyme Systems Products, Livermore, CA). The assay was done as previously described (Song et al., 1990). In some experiments, measurement of general proteolytic activity was done as previously described using Azocoll (Dresden et al., 1985). The assay mixtures contained 4 mg AzocoU, 5 mM DTT, and 50 #g of partially purified proteinase in a total volume of 1.0 ml buffer (0.1 M sodium citrate, 137
138
CHUL YONG SONGand AJAYA. REGE sulfonyl fluoride (PMSF), and 1,10-phenanthroline (all from Sigma), and ethylene diamine tetraacetate (EDTA, Yakuri Co., Japan).
100
RESULTS 5O
0-03 0.135
0.1 0.2 0.3 Molctriiy
0.4
0.5
Fig. 1. Effect of buffer molarity on cysteine proteinase activity in C. sinensis metacerceriae. C. sinensis metacercariae were used to prepare supernatant fractions by homogenization in 0.1 M sodium citrate, pH 4.9, and assayed for proteinase activity using CBZ-phe-arg-AFC in the presence of 5 mM DTT. Assay buffers were varied over a range of 0.014).5 M sodium citrate, pH 4.9, and relative activity was measured. Maximum activity is shown as 100%. pH 4.9). After incubating at 37°C for 5-14 hr, the unhydrolyzed substrate was removed by centrifugation, and the absorbance of the supernatant was measured at 540 nm. Purification of cysteine proteinase using AcA54 gel filtration chromatograph 3, The crude supernatant fractions were subjected to molecular sieve chromatography using AcA54 Ultrogel (1.6 x 40cm; LKB, Sweden), previously calibrated with proteins of known molecular weight. Fractionation was done with 0.1 M sodium acetate, pH 4.9, as previously described (Song et aL, 1990). Fractions (1.4 ml), collected at a flow rate of 10.0 ml/hr, were assayed for activity and protein content. Fractions with high activity were pooled and concentrated using polyethylene glycol (M r = 20,000) (WAKO, Japan). Quantification of protein content Protein content of each developmental stage was determined by the protein dye binding method of Bradford (1976). Bovine serum albumin (1-20 #g) was used as standard. Effects of proteinase inhibitors Partially purified proteinase samples (Peak I) were preincubated with inhibitors in 0.1 M sodium citrate, pH 4.9, containing 5 mM DTT, at 37°C for 30min. After incubation, the enzyme activity was measured using CBZ-phearg-AFC. The inhibitors tested were iodoacetic acid (1 mM; Wha Kwang Lab., Japan), t.-trans-epoxysuccinyl-leucylamido (4guanidino) butane (E-64; Enzyme Systems Products, Livermore, CA), leupeptin and pepstatin (both from Peninsula Lab., CA), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-c~-p-tosyl-L-lysine-chloromethyl ketone (TLCK), diisopropyl fluorophosphate (DFP), phenylmethyl
During preliminary studies of cysteine proteinases in the developmental stages of C. sinensis, the dependence of activity on ionic strength was tested with homogenates prepared in sodium citrate, pH 4.9. The ionic strength of assay buffers was varied over a range of 0.01~).5 M. As shown in Fig. 1, for metacercariae optimal activity against Z-phe-arg-AFC was obtained in 0.1 M sodium citrate, p H 4.9. Similar results were also obtained for the other three developmental stages studied. All of the activities were completely thiol dependent (data not shown). When specific activity of cysteine proteinases from crude extracts of the various developmental stages was assessed using CBZ-phe-arg-AFC, it was found that metacercariae had the highest specific activity (Table 1). When Azocoll was used as the substrate, the results were similar in that metacercariae had the highest specific activity, and the l-month, 2-month and 3-month old worms all had approximately similar specific activity. Further information about the nature of these proteolytic enzymes was obtained by testing activity on CBZ-phe-arg-AFC in the presence of various proteinase inhibitors. The results of these experiments are shown in Table 2. Out of the 10 inhibitors tested, iodoacetic acid, E-64, leupeptin, T P C K and T L C K showed significant inhibition of proteinase activity from all four developmental stages. N o significant inhibition was seen with D F P and P M S F (inhibitors of serine proteinase), pepstatin (aspartic proteinase) or 1,10-phenathroline and E D T A (inhibitors of metailo proteinases). For further characterization of cysteine proteinase activity, the supernatant fractions from the developmental stages (metacercariae, 1-month, 2-month, and 3-month worms) were subjected to molecular sieve chromatography on Ultrogel AcA54. The results for metacercariae are shown in Fig. 2. One major peak of activity was obtained with CBZ-phe-arg-AFC as the substrate. This peak had a molecular weight of approximately 32,000. Extracts from worms at 1, 2, and 3 months of maturation each yielded a single peak of activity. Figure 3 shows the chromatographic profiles for 1-month and 3-month-old worms. Each peak had an approximate molecular weight of 2 0 , 0 0 0 _ 1414. An elution profile of extracts from 2-month old worms was identical to those of 1-month and 3-month old worms (not shown).
Table 1. Specificactivity of cysteine proteinases in the developmentalstages of C. sinensis Specific activity (unit/mg) Metacercariae l-month 2-month 3-month Substrate CBZ-phe-arg-AFC 67.9 51.9 53.8 54.2 Azoeoll 0.896 0.609 0.454 0.787 Equivalent amounts of frozen tissue were extracted in 0.1 M sodium citrate, pH 4.9, and centrifuged. Supernatant fractions were assayed in the presence of 5 mM DTT. A unit is definedas nmoles AFC/hr for CBZ-phe-arg-AFC,and A540~m/hrfor Azocoll.
139
Cysteine proteinases in Clonorchis Table 2. Effects of inhibitors on proteinases from developmental stages of C. sinensis % Relative activity* Metacercariae 1-month 2-month
Inhibitors Iodacetic acid (1 mM) E-64 (5 x 10-8 M) Leupeptin (4/tg/ml) Pepstatin (100 ttg/ml) TPCK (1 mM) TLCK (1 mM) DFP (1 mM) PMSF (1 mM) 1,10-phenanthroline (1 mM) EDTA (2 mM)
0 0 0 94 16 0 69 75 64 87
0 0 0 89 0 0 106 84 83 100
3-month
0 0 0 93 0 0 98 98 87 127
0 0 0 100 0 0 65 72 66 83
The concentration of inhibitors tested is shown in parentheses• *The activity against CBZ-phe-arg-AFC substrate in the absence of inhibitors, but in the presence of 5 mM DTT, was taken as 100%.
The effect of pH on activity against CBZ-phe-argAFC was studied as shown in Fig. 4. Maximal activity was obtained at or around pH 5.5 in 0.1 M sodium citrate with DTT. Very little activity was seen above pH 7.0 or below pH 3.0. Enzyme activity showed about 40% loss of activity when stored at 4°C for 2 days. No activity was lost after 1 month at -20°C. DISCUSSION
We have partially purified and characterized cysteine proteinase activity from four developmental stages (metacercariae, 1-month, 2-month and 3month worms) of Clonorchis sinensis. These enzymes belong to the cysteine proteinase class. Thus, they hydrolyze a substrate normally used to assay cysteine proteinases, such as CBZ-phe-arg-AFC, are thiol activated, and have an acidic pH optimum. Also, the proteinase activity is significantly blocked by inhibitors of cysteine proteinases, including E-64, iodoacetic acid, leupeptin, TPCK, and TLCK. The molecular weights of the enzymes, determined by AcA54 gel filtration chromatography, were 32,000 with metacercariae and 20,000 + 1414 with 1-month, 2-month, and 3-month (or adult) worms. Thus, a
different enzyme seems to be activated at least in metacercariae, suggesting developmental regulation of cysteine proteinase expression in C. sinensis. The molecular weight of the major cysteine proteinase activity in developing (1-month and 2-month old) worms was very similar to that of the major cysteine proteinase in the 3-month old (adult) worms of C. sinensis (Song et al., 1990). Another clue that the cysteine proteinase activity is developmentally regulated in C. sinensis came from the observation that metacercariae had the highest specific activity using both macromolecular and peptide substrates, suggesting that a different set of proteinases is active in metacercariae. Developmental regulation of cysteine proteinase activity has been previously reported in other trematode parasites. Thus, using molecular biological techniques, Davis et al. (1987) showed developmental regulation of expression of a hemoglobinase in Schistosoma mansoni. Similar conclusions were obtained for the overall cysteine proteinase activity in early developing schistosomula by Zerda et al. (1988). Yamakami and Hamajima (1987) reported the presence of neutral thiol proteinase activities in extracts of Paragonimus westermani metacercariae.
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Fig. 2. Molecular sieve chromatography of cysteine proteinases from C. sinensis metacereariae. Metacercariae were homogenized in 0.1 M sodium citrate, pH 4.9. The homogenate was centrifuged and the supernatant fraction was applied to an AcA~ ultragel column (1.6 x 45.0 cm). The column was eluted with the above buffer, and 1.4 ml fractions were assayed for activity on CBZ-phe-arg-AFC (©). Fractions were also monitored for protein content (0) at 280 nm. The inset shows a calibration of the column using proteins of known molecular weight (bovine serum albumin, ovalbumin, chymotrypsinogen A and ribonuclease).
140
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Fig. 4. Effect of pH on C. sinensis cysteine proteinase activity. C. sinensis (metacercariae, 1 month-, 2 month-, and 3-month-old worms) cysteine proteinases partially purified by AcA54 molecular sieve chromatography were assayed in 0.I M sodium citrate buffer (pH 3.0, 4.0, 5.0 and 6.0), and in 0.1 M sodium phosphate buffer (pH 7.0) each in the presence of 5 mM DTT. CBZ-phe-arg-AFC was used as the substrate. Relative activity is shown (maximum set at 100%). Acknowledgement--This work was supported by the Research Fund from The Ministry of Education, Republic of Korea (1988-1989),
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Fig. 3. Molecular sieve chromatography of cysteine proteinases from developing worms of C. sinensis. C. sinensis worms of various developmental age were homogenized in 0.1 M sodium citrate, pH 4.9, and supernatant fractions were prepared as previously described. The supernatant fractions were subjected to AcA54 molecular sieve chromatography, and the fractions were monitored for protein content (0) and activity on CBZ-phe-arg-AFC ((3). Panel A shows the chromatographic profile for 1-month old worms, whereas Pane/B shows that for 3-month old (adult) worms. Chromatographic profile for 2-month old worms is not shown. Y a m a k a m i (1986) also reported an acidic cysteine proteinase in adult worms of the related parasite P. ohirai. Using gelatin-substrate S D S - P A G E to compare immature and mature flukes, Dalton and Hefferman (1989) showed that some of the cysteine proteinases released by Fasciola hepatica may be developmentally regulated. Recently, we (Song and Dresden, 1990) demonstrated an acidic cysteine proteinase in metacercariae as well as worms at various developmental stages of P. westermani and noted that the azocollytic activity of this proteinase in metacercariae was greater than that in the other stages of the parasite. The function of cysteine proteinases in C. sinensis is not known. It is possible that they are involved in pathologic changes associated with larval migration, including localized biliary epithelial destruction, or in parasite nutrition. Further studies on the tissue localization of these enzymes should provide clues to their function.
REFERENCES
Auriault C., Pierce R., Cesari 1. M. and Capron A. (1982) Neutral protease at different developmental stages of Schistosoma mansoni in mammalian hosts. Comp. Biochem. Physiol. 72B, 377-384. Bradford M. (1976) A rapid and sensitive method for the quantification of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248 254, Dalton J. P. and Hefferman M. (1989) Thiol proteases released in vitro by Fasciola hepatica. Mol. Biochem. Parasitol. 35, 161-166. Davis A. H., Nanduri J. and Watson D. (1987) Cloning and gene expression of Schistosoma mansoni protease. J. biol. Chem. 262, 12,851-12,855. Dresden M. H., Rege A. A. and Murrell K. D. (1985) Strongyloides ransomi: proteolytic enzymes from larvae. Exp. Parasitol. 59, 257-263. Song C.-Y. and Dresden M. H. (1990) Partial purification and characterization of cysteine proteases from various developmental stages of Paragonimus westermani. Comp. Biochem. Physiol. 95, 473~,76. Song C.-Y., Dresden M. H. and Rege A. A. (1990) Clonorchis sinensis: purification and characterization of a cysteine proteinase from adult worms. Comp. Biochem. Physiol. 97B, 825-829. Yamakami K. (1986) Purification and properties of thiol protease from lung fluke adult Paragonimus ohirai. Comp. Biochem. Physiol. 8313, 501 506. Yamakami K. and Hamajima F. (1987) Purification and properties of a neutral thiol protease form larval trematode parasite Paragonimus westermani metacercariae. Comp. Biochem. Physiol. 8713, 643648. Zerda K. S., Dresden M. H. and Chappel C. L. (1988) Schistosoma mansoni: expression and role of cysteine proteinases in developing schistosomula. Exp. Parasitol. 67, 238-246.
0305-0491/'91 $3.00 + 0.00 Pergamon Press plc
CYSTEINE PROTEINASE ACTIVITY IN VARIOUS DEVELOPMENTAL STAGES OF C L O N O R C H I S SINENSIS: A COMPARATIVE ANALYSIS CHUL YONG SONG* and AJAY A. REGEt~ *Department of Biology, Chung-Ang University, Seoul, Korea; and tVerna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA (Tel: 713-798-4531 (Received 9 October 1990) Abstract--1. Cysteine proteinase activity in acidic extracts of various developmental stages of Clonorchis sinensis (metacercariae, 1-, 2-, and 3-month old worms) was examined. All the activities were maximum at acidic pH and showed inhibitor susceptibilities similar to the vertebrate cysteine proteinases. 2. Specific activity of cysteine proteinase(s) was highest in metacercariae with either CBZ-phe-arg-AFC or Azocoll as the substrate. The immature and mature worms had similar (but less than metacercariae) levels of activity. 3. A soluble cysteine proteinase with a native molecular weight of approximately 20,000 + 1414 was partially purified from 1-, 2-, and 3-month worms. The molecular weight of similar activity in metacercariae was approximately 32,000. 4. Results suggest developmental regulation of cysteine proteinase activity in the life cycle of C. sinensis.
INTRODUCTION
Clonorchis sinensis is a trematode parasite that causes liver disease in its host. After oral infection with metacercariae, the parasites hatch out in the digestive tract and migrate up to the c o m m o n bile duct where they grow to maturity. During the development of Clonorchis sinensis, biliary epithelial hyperplasia and fibrosis develop around the worms, and dilation of the ducts into cyst-like cavities may occur. The biochemical mechanisms involved in both the nutrition of and the pathology caused by the parasite are not well understood. Proteolytic enzymes, including cysteine proteinases, are important to the host-parasite relationship and seem to play important roles in parasite physiology, pathogenesis, and evasion of host defense. Proteinases have been detected in various developmental stages of many trematode parasites. Thus, neutral proteases have been characterized in various developmental stages of Schistosoma mansoni (Auriault et al., 1982). Similarly, cysteine proteinases have been detected in the developmental stages of Fasciola hepatica (Dalton and Heffernan, 1989), and of Paragonimus westermani (Song and Dresden, 1990). However, very little is known about cysteine proteinase activity in C. sinensis. Earlier we reported the purification and characterization of a major cysteine proteinase from adult C. sinensis (Song et al., 1990). Here we present a comparative analysis of cysteine proteinase activity from various developmental stages of this parasite and demonstrate that some of these stages contain unique enzymes, suggesting their developmental regulation. ~:Author to whom correspondence should be addressed.
MATERIALS
AND METHODS
Parasites The metacercariae of Clonorchis sinensis were obtained from naturally infected Pseudorasbora parva collected in Kimhai-Gun, Korea. Rabbits were fed orally 300 metacercariae and were dissected sequentially on the first, second and third months after the experimental infection. The worms were harvested in the bile duct of the liver. The mean size of metacercariae was about 0.15 x 0.13 mm. The mean size of 1-month worms was 4.2 +0.16mm in length and 0.9 + 0.86 mm in width. The mean size of 2-month worms was 6.6+0.61 mm in length and 2.1 +0.17ram in width. The mean size of 3-month (adult) worms was 11.5 + 0.78 mm in length and 2.8 + 0.23 mm in width. The collected worms were washed several times with cold 0.85% saline, once again with distilled water, and lyophilized. Extraction of proteinases All procedures were carried out at 4°C unless otherwise stated. For the preparation of proteolytic enzymes, lyophilized C. sinensis metacercariae and worms (I0 mg/ml buffer) were homogenized in 0.1 M sodium citrate, pH 4.9, and processed as described previously (Song et al., 1990). The resulting supernatant fractions were subjected to further analysis. Assay of enzymatic activity Proteinase activities of the developmental stages (metacercariae, 1-month, 2-month, and 3-month worms) of C. sinensis were estimated using a low molecular weight, synthetic dipeptide substrate, carboxybenzoxyl-phenylalanine-arginine-7-amino-4-trifluoromethylcoumarin (CBZphe-arg-AFC; Enzyme Systems Products, Livermore, CA). The assay was done as previously described (Song et al., 1990). In some experiments, measurement of general proteolytic activity was done as previously described using Azocoll (Dresden et al., 1985). The assay mixtures contained 4 mg AzocoU, 5 mM DTT, and 50 #g of partially purified proteinase in a total volume of 1.0 ml buffer (0.1 M sodium citrate, 137
138
CHUL YONG SONGand AJAYA. REGE sulfonyl fluoride (PMSF), and 1,10-phenanthroline (all from Sigma), and ethylene diamine tetraacetate (EDTA, Yakuri Co., Japan).
100
RESULTS 5O
0-03 0.135
0.1 0.2 0.3 Molctriiy
0.4
0.5
Fig. 1. Effect of buffer molarity on cysteine proteinase activity in C. sinensis metacerceriae. C. sinensis metacercariae were used to prepare supernatant fractions by homogenization in 0.1 M sodium citrate, pH 4.9, and assayed for proteinase activity using CBZ-phe-arg-AFC in the presence of 5 mM DTT. Assay buffers were varied over a range of 0.014).5 M sodium citrate, pH 4.9, and relative activity was measured. Maximum activity is shown as 100%. pH 4.9). After incubating at 37°C for 5-14 hr, the unhydrolyzed substrate was removed by centrifugation, and the absorbance of the supernatant was measured at 540 nm. Purification of cysteine proteinase using AcA54 gel filtration chromatograph 3, The crude supernatant fractions were subjected to molecular sieve chromatography using AcA54 Ultrogel (1.6 x 40cm; LKB, Sweden), previously calibrated with proteins of known molecular weight. Fractionation was done with 0.1 M sodium acetate, pH 4.9, as previously described (Song et aL, 1990). Fractions (1.4 ml), collected at a flow rate of 10.0 ml/hr, were assayed for activity and protein content. Fractions with high activity were pooled and concentrated using polyethylene glycol (M r = 20,000) (WAKO, Japan). Quantification of protein content Protein content of each developmental stage was determined by the protein dye binding method of Bradford (1976). Bovine serum albumin (1-20 #g) was used as standard. Effects of proteinase inhibitors Partially purified proteinase samples (Peak I) were preincubated with inhibitors in 0.1 M sodium citrate, pH 4.9, containing 5 mM DTT, at 37°C for 30min. After incubation, the enzyme activity was measured using CBZ-phearg-AFC. The inhibitors tested were iodoacetic acid (1 mM; Wha Kwang Lab., Japan), t.-trans-epoxysuccinyl-leucylamido (4guanidino) butane (E-64; Enzyme Systems Products, Livermore, CA), leupeptin and pepstatin (both from Peninsula Lab., CA), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-c~-p-tosyl-L-lysine-chloromethyl ketone (TLCK), diisopropyl fluorophosphate (DFP), phenylmethyl
During preliminary studies of cysteine proteinases in the developmental stages of C. sinensis, the dependence of activity on ionic strength was tested with homogenates prepared in sodium citrate, pH 4.9. The ionic strength of assay buffers was varied over a range of 0.01~).5 M. As shown in Fig. 1, for metacercariae optimal activity against Z-phe-arg-AFC was obtained in 0.1 M sodium citrate, p H 4.9. Similar results were also obtained for the other three developmental stages studied. All of the activities were completely thiol dependent (data not shown). When specific activity of cysteine proteinases from crude extracts of the various developmental stages was assessed using CBZ-phe-arg-AFC, it was found that metacercariae had the highest specific activity (Table 1). When Azocoll was used as the substrate, the results were similar in that metacercariae had the highest specific activity, and the l-month, 2-month and 3-month old worms all had approximately similar specific activity. Further information about the nature of these proteolytic enzymes was obtained by testing activity on CBZ-phe-arg-AFC in the presence of various proteinase inhibitors. The results of these experiments are shown in Table 2. Out of the 10 inhibitors tested, iodoacetic acid, E-64, leupeptin, T P C K and T L C K showed significant inhibition of proteinase activity from all four developmental stages. N o significant inhibition was seen with D F P and P M S F (inhibitors of serine proteinase), pepstatin (aspartic proteinase) or 1,10-phenathroline and E D T A (inhibitors of metailo proteinases). For further characterization of cysteine proteinase activity, the supernatant fractions from the developmental stages (metacercariae, 1-month, 2-month, and 3-month worms) were subjected to molecular sieve chromatography on Ultrogel AcA54. The results for metacercariae are shown in Fig. 2. One major peak of activity was obtained with CBZ-phe-arg-AFC as the substrate. This peak had a molecular weight of approximately 32,000. Extracts from worms at 1, 2, and 3 months of maturation each yielded a single peak of activity. Figure 3 shows the chromatographic profiles for 1-month and 3-month-old worms. Each peak had an approximate molecular weight of 2 0 , 0 0 0 _ 1414. An elution profile of extracts from 2-month old worms was identical to those of 1-month and 3-month old worms (not shown).
Table 1. Specificactivity of cysteine proteinases in the developmentalstages of C. sinensis Specific activity (unit/mg) Metacercariae l-month 2-month 3-month Substrate CBZ-phe-arg-AFC 67.9 51.9 53.8 54.2 Azoeoll 0.896 0.609 0.454 0.787 Equivalent amounts of frozen tissue were extracted in 0.1 M sodium citrate, pH 4.9, and centrifuged. Supernatant fractions were assayed in the presence of 5 mM DTT. A unit is definedas nmoles AFC/hr for CBZ-phe-arg-AFC,and A540~m/hrfor Azocoll.
139
Cysteine proteinases in Clonorchis Table 2. Effects of inhibitors on proteinases from developmental stages of C. sinensis % Relative activity* Metacercariae 1-month 2-month
Inhibitors Iodacetic acid (1 mM) E-64 (5 x 10-8 M) Leupeptin (4/tg/ml) Pepstatin (100 ttg/ml) TPCK (1 mM) TLCK (1 mM) DFP (1 mM) PMSF (1 mM) 1,10-phenanthroline (1 mM) EDTA (2 mM)
0 0 0 94 16 0 69 75 64 87
0 0 0 89 0 0 106 84 83 100
3-month
0 0 0 93 0 0 98 98 87 127
0 0 0 100 0 0 65 72 66 83
The concentration of inhibitors tested is shown in parentheses• *The activity against CBZ-phe-arg-AFC substrate in the absence of inhibitors, but in the presence of 5 mM DTT, was taken as 100%.
The effect of pH on activity against CBZ-phe-argAFC was studied as shown in Fig. 4. Maximal activity was obtained at or around pH 5.5 in 0.1 M sodium citrate with DTT. Very little activity was seen above pH 7.0 or below pH 3.0. Enzyme activity showed about 40% loss of activity when stored at 4°C for 2 days. No activity was lost after 1 month at -20°C. DISCUSSION
We have partially purified and characterized cysteine proteinase activity from four developmental stages (metacercariae, 1-month, 2-month and 3month worms) of Clonorchis sinensis. These enzymes belong to the cysteine proteinase class. Thus, they hydrolyze a substrate normally used to assay cysteine proteinases, such as CBZ-phe-arg-AFC, are thiol activated, and have an acidic pH optimum. Also, the proteinase activity is significantly blocked by inhibitors of cysteine proteinases, including E-64, iodoacetic acid, leupeptin, TPCK, and TLCK. The molecular weights of the enzymes, determined by AcA54 gel filtration chromatography, were 32,000 with metacercariae and 20,000 + 1414 with 1-month, 2-month, and 3-month (or adult) worms. Thus, a
different enzyme seems to be activated at least in metacercariae, suggesting developmental regulation of cysteine proteinase expression in C. sinensis. The molecular weight of the major cysteine proteinase activity in developing (1-month and 2-month old) worms was very similar to that of the major cysteine proteinase in the 3-month old (adult) worms of C. sinensis (Song et al., 1990). Another clue that the cysteine proteinase activity is developmentally regulated in C. sinensis came from the observation that metacercariae had the highest specific activity using both macromolecular and peptide substrates, suggesting that a different set of proteinases is active in metacercariae. Developmental regulation of cysteine proteinase activity has been previously reported in other trematode parasites. Thus, using molecular biological techniques, Davis et al. (1987) showed developmental regulation of expression of a hemoglobinase in Schistosoma mansoni. Similar conclusions were obtained for the overall cysteine proteinase activity in early developing schistosomula by Zerda et al. (1988). Yamakami and Hamajima (1987) reported the presence of neutral thiol proteinase activities in extracts of Paragonimus westermani metacercariae.
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Fig. 2. Molecular sieve chromatography of cysteine proteinases from C. sinensis metacereariae. Metacercariae were homogenized in 0.1 M sodium citrate, pH 4.9. The homogenate was centrifuged and the supernatant fraction was applied to an AcA~ ultragel column (1.6 x 45.0 cm). The column was eluted with the above buffer, and 1.4 ml fractions were assayed for activity on CBZ-phe-arg-AFC (©). Fractions were also monitored for protein content (0) at 280 nm. The inset shows a calibration of the column using proteins of known molecular weight (bovine serum albumin, ovalbumin, chymotrypsinogen A and ribonuclease).
140
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Fig. 4. Effect of pH on C. sinensis cysteine proteinase activity. C. sinensis (metacercariae, 1 month-, 2 month-, and 3-month-old worms) cysteine proteinases partially purified by AcA54 molecular sieve chromatography were assayed in 0.I M sodium citrate buffer (pH 3.0, 4.0, 5.0 and 6.0), and in 0.1 M sodium phosphate buffer (pH 7.0) each in the presence of 5 mM DTT. CBZ-phe-arg-AFC was used as the substrate. Relative activity is shown (maximum set at 100%). Acknowledgement--This work was supported by the Research Fund from The Ministry of Education, Republic of Korea (1988-1989),
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Fig. 3. Molecular sieve chromatography of cysteine proteinases from developing worms of C. sinensis. C. sinensis worms of various developmental age were homogenized in 0.1 M sodium citrate, pH 4.9, and supernatant fractions were prepared as previously described. The supernatant fractions were subjected to AcA54 molecular sieve chromatography, and the fractions were monitored for protein content (0) and activity on CBZ-phe-arg-AFC ((3). Panel A shows the chromatographic profile for 1-month old worms, whereas Pane/B shows that for 3-month old (adult) worms. Chromatographic profile for 2-month old worms is not shown. Y a m a k a m i (1986) also reported an acidic cysteine proteinase in adult worms of the related parasite P. ohirai. Using gelatin-substrate S D S - P A G E to compare immature and mature flukes, Dalton and Hefferman (1989) showed that some of the cysteine proteinases released by Fasciola hepatica may be developmentally regulated. Recently, we (Song and Dresden, 1990) demonstrated an acidic cysteine proteinase in metacercariae as well as worms at various developmental stages of P. westermani and noted that the azocollytic activity of this proteinase in metacercariae was greater than that in the other stages of the parasite. The function of cysteine proteinases in C. sinensis is not known. It is possible that they are involved in pathologic changes associated with larval migration, including localized biliary epithelial destruction, or in parasite nutrition. Further studies on the tissue localization of these enzymes should provide clues to their function.
REFERENCES
Auriault C., Pierce R., Cesari 1. M. and Capron A. (1982) Neutral protease at different developmental stages of Schistosoma mansoni in mammalian hosts. Comp. Biochem. Physiol. 72B, 377-384. Bradford M. (1976) A rapid and sensitive method for the quantification of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248 254, Dalton J. P. and Hefferman M. (1989) Thiol proteases released in vitro by Fasciola hepatica. Mol. Biochem. Parasitol. 35, 161-166. Davis A. H., Nanduri J. and Watson D. (1987) Cloning and gene expression of Schistosoma mansoni protease. J. biol. Chem. 262, 12,851-12,855. Dresden M. H., Rege A. A. and Murrell K. D. (1985) Strongyloides ransomi: proteolytic enzymes from larvae. Exp. Parasitol. 59, 257-263. Song C.-Y. and Dresden M. H. (1990) Partial purification and characterization of cysteine proteases from various developmental stages of Paragonimus westermani. Comp. Biochem. Physiol. 95, 473~,76. Song C.-Y., Dresden M. H. and Rege A. A. (1990) Clonorchis sinensis: purification and characterization of a cysteine proteinase from adult worms. Comp. Biochem. Physiol. 97B, 825-829. Yamakami K. (1986) Purification and properties of thiol protease from lung fluke adult Paragonimus ohirai. Comp. Biochem. Physiol. 8313, 501 506. Yamakami K. and Hamajima F. (1987) Purification and properties of a neutral thiol protease form larval trematode parasite Paragonimus westermani metacercariae. Comp. Biochem. Physiol. 8713, 643648. Zerda K. S., Dresden M. H. and Chappel C. L. (1988) Schistosoma mansoni: expression and role of cysteine proteinases in developing schistosomula. Exp. Parasitol. 67, 238-246.