Partial purification and characterization of cysteine proteinases from various developmental stages of Paragonimus westermani

Comp. Biochem. Physiol. Vol. 95B, No. 3, pp. 473-476, 1990 Printed in Great Britain

0305-04.91/90 $3.00 + 0.00 © 1990 Pergamon Press plc

PARTIAL PURIFICATION A N D CHARACTERIZATION OF CYSTEINE PROTEINASES FROM VARIOUS DEVELOPMENTAL STAGES OF P A R A G O N I M U S IVESTERMANI CHUL-YONG SONG* and MARC H. DRESDENt *Department of Biology, Chung-Ang University, Seoul, Korea; and tVerna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, st-Iouston, TX 77030, USA (Tel: 713 798-4527) (Recewed 20 June 1989) Abstract--1. During development of Paragonimus westermani, larvae develop during migration within the

host, and adult worms feed on pulmonary tissues, causing significant pathology in the mammalian host. In this report acidic extracts of various developmental stages (metacercariae and worms at one, two and three months of development) were examined for cysteine proteinase activity. 2. A soluble thiol-dependent proteinase activity with a native molecular weight of approximately 20,000 was isolated and partially purified. 3. The enzymes purified from the various developmental stages of the parasite had maximal activity at acidic pH and showed inhibitor susceptibilities similar to the vertebrate acidic cysteine proteinases. 4. Enzymatic activity was stable at pH 5.0 for at least two days when stored at 4°C. 5. It is suggested that these enzymes may be involved in the nutrition of these parasites and/or during penetration and lysis of the tissues.

INTRODUCTION

MATERIALS AND METHODS

Parasites The metacercariae of Paragonimus westermani were obtained from naturally infected crayfish (Cambaroides similis) collected in Wando-Gun, Korea. The mean size of the metacercariae were 285/~. Dogs were fed orally 200 metacercariae and were dissected sequentially on the first, second, and third month after the experimental infection. The worms were harvested from the organs in thoracic cavities and from the cysts in the lungs. The one-month worms were attached on the surface of the lung but had not penetrated to the parenchymal cell of the lung. The mean size was about 2.30 mm in length and 1.22 mm in width. The two-month worms were obtained from cysts in the lungs. The measured mean size was about 5.08 mm in length and 2.45 mm in width; immature eggs could be found in the uterus of these worms. The three-month worms (adult worms) have a short, stumpy form, and produce mature eggs. The measured mean size of adult worms was about 7.55mm in length, 4.25mm in width and 2.93 mm in thickness. The collected worms were washed several times with cold 0.85% saline and once with distilled water and lyophilized.

Paragonimus westermani is a trematode parasite that causes lung disease in its hosts. After oral infection by metacercariae, the parasite invades and migrates through a variety of host tissues. The pathologic lesions caused by these lung flukes are the result of the damage done during tissue migration and by the formation of cysts in the lungs. The role of proteolytic enzymes in the pathology of this disease remains to be determined. Proteolytic enzymes have been detected in many species and stages of parasitic helminths. Various proteinases have been detected in parasites, and the functional roles of the proteolytic enzymes are probably important to the physiology of the parasites (Auriault et al., 1982). The proteolytic enzymes of the adult worms of the trematode, Schistosoma mansoni, have been studied extensively (Grant and Senft, 1971; Deelder et al., 1977; Dresden and Deelder, 1979; Dresden et al., 1981; Chappell and Dresden, 1987). Proteolytic enzymes from the cercariae of S. mansoni and metacercariae of P. westermani able to hydrolyse connective tissue proteins have been reported (Dresden and Asch, 1972; Tzeng et aL, 1983; M c K e r r o w et al., 1985; Y a m a k a m i and Hamajima, 1987), These proteinases may play a significant role in facilitating tissue invasion by these parasites. In order to obtain better understanding of the possible role of these enzymes in the host-parasite relationship, the present study was undertaken to describe the partial purification and characterization of cysteine proteinases in various developmental stages of P. westermani.

Extraction of proteinase from metacercariae and worms of P. westermani All procedures were carried out at 4°C unless otherwise stated. For the preparation of proteolytic enzymes, lyophilized Paragonimus westermani metacercariae and worms (10mg/ml buffer) were homogenized with a tissue grinder on ice in 0.1 M sodium citrate buffer, pH 4.9, which had been shown to be optimal for solubilization of proteinase activity. Homogenates were centrifuged at 15,000rpm for 15min at 4°C, and the supernatant was removed and subjected to further purification. 473

474

CHUL-YONG SONG a n d MARC H. DRESDEN

Purification of cysteine proteinase using AcA 54 gel filtration chromatography The crude supernatant was subjected to molecular sieve chromatography on AcA54 Ultrogel (1.6 × 40cm; LKB, Sweden). Fractions (l.6ml), collected at a flow rate of 10.0ml/hr, were assayed for activity and protein content by spectrophotometric measurement at 280 nm. Fractions of high activities were pooled and concentrated using PEG 20,000 (mol. wt) (WAKO, Japan). The AcA54 column was calibrated with proteins of known tool. wt, including bovine serum albumin (66,000), ovalbumin (43,000), chymotrypsinogen (25,000), and ribonuclease (13,700).

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Fig. 1. pH profiles for P. westermani proteinases. Parilgonimus westermani (metacercariae, one-, two- and 3-month worms) proteinases prepared by AcA54 gel filtration chromatography were assayed in 0.1 M sodium citrate buffer with 5mM DTT (pH 3.0, 4.0, 5.0 and 6.0) and in 0.1M sodium phosphate buffer with 5 mM DTT (pH 7.0) using CBZ-phe-arg-AFC as substrate. Maximal activity was set at 100%.

Assay of enzymatic activity Proteinase activities of the developmental stages (metacercariae, one-, two- and three-month worms) of P. westermani were estimated by use of a low mol. wt synthetic dipeptide substrate with a fluorescent amino terminal leaving group, carboxybenzoyl-phenylalanine-arginine-7-amino-4-trifluoromethylcoumarin (CBZ-phe-arg-AFC; Enzyme Systems Products, Livermore, CA). The assay buffer contained 0.1 M sodium citrate with 5 mM DTT (pH 5.0) and 10/~g of CBZ-phe-arg-AFC dissolved in 10 ~tl of dimethyl sulfoxide in a total volume of 0.5 ml. After incubation for 3 hr at 37°C, the released 7-amino-4-trifluoromethylcoumarin (AFC) was measured in a Turner fluorometer (Model 111, Sequoia-Turner Corporation, CA) at excitatory and emission wavelengths of 400 and 505 rim, respectively. A standard curve was constructed with AFC to allow the presentation of the data as nmol of AFC produced.

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Effects of proteinase inhibitors The developmental stages of proteinase (peak 1) containing inhibitors in 0.1 M sodium citrate buffer (pH 5.0) were incubated at 37°C for 3 14 hr. After incubation, the released 7-amino-4-trifluoromethylcoumarin (AFC) was measured in a Turner 111 fluorometer. The tested inhibitors were iodoacetic acid (1 mM; Wha Kwang Lab., Japan), N-ethylmaleimide (1 mM; Sigma), L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (5 × 10 ~M; Taisho Pharmaceuticals, Japan), phenylmethyl sulfonyl fluoride ( l m M ; Sigma), 1,10-phenanthroline (1 raM; Sigma), and ethylene diamine tetraacetate (2 mM; Yakuri Co., Japan). Proteinase activity against Azocoll Azocoll (azo-dye-derivatized hide powder) was obtained from Sigma (USA). Measurement of general proteolytic activity was done as previously described using Azocoll (Dresden and Asch, 1972). The mixtures assayed contained 4 mg of Azocoll and 50 #g of purified proteinase (peak 1) in a total volume of 1.0ml buffer (0.1 M sodium citrate, pH 5.0). Samples assayed were incubated at 37°C for 5 14 hr. After incubation, the unhydrolysed substrate was removed by centrifugation (Eppendorf, FRG) for 3 min and absorbance measured at 540 nm. RESULTS

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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 20pg) was used as a standard.

60

70

80

Fig. 2. Molecular sieve chromatography of P. westermani acidic proteinases. Paragonimus westermani metacercariae were extracted in 0.1 M sodium citrate, pH 5.5. The extracts were centrifuged and the supernatant fractions were loaded onto a column of AcA54 Ultrogel. The column was eluted with 0.l M sodium citrate, pH 5.5, and 1.6-ml fractions (Fno) were assayed for activity on CBZ-phe-arg-AFC (O). Fractions were monitored for protein content (O) 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). The molecular weight of P. westermani proteinase activity (PI) is indicated by CP.

As a n initial step in assessing the role o f cysteine proteinases in the d e v e l o p m e n t of P. westermani, the presence of these enzymes was tested for in extracts of various life stages of the parasite. To optimize the activity of these proteinases, assays were carried out at 0.1 M sodium citrate, p H 4.9. The effect of p H o n activity against CBZ-phe-argA F C is s h o w n in Fig. 1. These proteinases were purified by AcA54 gel filtration c h r o m a t o g r a p h y from the four life stages of the parasite. M a x i m u m activity was o b t a i n e d at p H 6.0 in 0.1 M sodium citrate with DTT. N o activity was seen below p H 3.0 in sodium citrate with DTT. Enzyme activity was stable when stored at 4°C for two days; no activity was lost after three weeks at - 2 0 ° C (data not shown). Preliminary characterization of P. westermani proteinases from various developmental stages (metacercariae, one-, two- a n d t h r e e - m o n t h worms) was performed using molecular sieve c h r o m a t o g r a p h y on Ultrogel AcA54. The results of these experiments are s h o w n in Fig. 2. Fractions obtained by

475

C y s t e i n e p r o t e i n a s e s in Paragonimus Table 1. Summary of purification from the developmental stages of P. westermani cysteine proteinases Developmental stage Crude extract Metacercariae One-month worms Two-month worms Three-month worms AcA54 Ultrogel Metacercariae One-month worms Two-month worms Three-month worms

Total protein (mg)

Total activity (units)*

Specific activity (unit/mg)

Purification (fold)

1.08 1.57 5.74 6.56

168 158 151 197

156 101 26 30

-----

0.72 0.77 0.72 0.77

179 175 120 100

249 227 167 130

1.6 2.3 6.4 4.3

*nmol A F C hr i mg enzyme- ~. Equivalent amounts of frozen developmental stages of the worms were extracted in 0.1 M sodium citrate, pH 5.5, and centrifuged. Supernatant fractions were loaded onto a column (1.6 × 40 cm) of AcA54 Ultrogel in sodium citrate, pH 4.9.

chromatography from sodium citrate (pH4.9) extracts of metacercariae exhibited one major peak of activity when assayed against CBZ-phe-arg-AFC. Similarly, extracts from worms at one, two and three months of maturation yielded similar peaks of activity at the same elution volume (data not shown). Calibration of these columns with proteins of known molecular weight (Fig. 2, inset) indicated approximate molecular weights of 20,250 ___1750. Efficiency of purification and specific activity of the developmental stages of the parasite were assessed. The results of these experiments are shown in Table 1. Crude extracts and partially purified enzyme activity of metacercariae clearly have the highest specific activity among the stages of the worms studied. Azocollytic activities were assessed in these partially purified proteinase preparations from the four developmental stages of the parasite. The results of these experiments also indicate that crude extracts and partially purified enzymes of metacercariae have the greatest hydrolytic activity among the stages of the parasites tested. These results suggest that cysteine proteinase activity decreases with development of the parasite. Further information about the nature of these proteolytic enzymes was obtained by testing various proteinase inhibitors. Aliquots of partially purified proteinase of each developmental stage of the parasite were adjusted to give equivalent activity against CBZ-phe-arg-AFC, and the activity in the absence of inhibitor was taken as 100%. The results of these experiments are shown in Table 2. Inhibitors of cysteine proteinases, including iodoacetic acid, NEM, and E-64, showed the greatest inhibitory effect among the inhibitors tested, for all four developmental

stages. No significant inhibition was observed with PMSF and DFP (inhibitors of serine proteinases) or with 1,10-phenanthroline and EDTA (inhibitors of metallo proteinases). DISCUSSION

We have isolated and partially characterized proteolytic enzymes from four developmental stages (metacercariae, one-, two- and three-month worms) of Paragonimus westermani. These enzymes share properties with the mammalian cathepsins B, H, and L (Barrett, 1979) and, as such, belong to the cysteine proteinase class. Recently, Yamakami and Hamajima (1987) reported the presence of neutral thiol proteinase activity in extracts of P. westermani cercariae. Furthermore, Yamakami (1986) demonstrated an acidic cysteine proteinase in adult worms of the related parasite, Paragonimus ohirai. In this report, we confirm the presence of cysteine proteinases in both metacercariae and adult worms at various stages of development of P. westermani. These enzymes hydrolyze substrates normally used to assay cysteine proteinases, such as CBZ-phe-arg-AFC and CBZarg-arg-AFC (Dresden et al., 1985) and are thiolactivated and have an acidic pH optimum. The molecular weights of the enzymes, determined by AcA54 gel filtration chromatography, was 20,250 for all four stages of the parasite. These results are similar to those reported for other lung parasite cysteine proteinases (Rege et al., 1989) and for P. westermani metacercariae (Yamakami and Hamajima, 1987). The effects on these enzymes of various proteinase inhibitors, particularly E-64

Table 2. Effects of inhibitors on partially purified proteinases from developmental stages of P. westermani % Relative activity* Inhibitors lodoacetic acid (1 mM) NEM (I mM) E-64 (5 x l0 -8 M) PMSF (1 mM) D F P (1 mM) 1,10-Phenanthroline (1 mM) EDTA (2 mM)

Metacercariae 0 0 0 99 98 96 102

One-month

Two-month

Three-month

0 0 0 98 106 83 114

0 0 0 84 98 87 75

0 0 0 75 83 66 83

The concentration of inhibitors tested is shown in parentheses. *The activity against CBZ-phe-arg-AFC substrate in the absence of inhibitors was taken as 100%. NEM: N-ethylmaleimide; E-64: L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane; PMSF: phenylmethyl sulfonyl fluoride; DFP: diisopropyl fluorophosphate; EDTA: ethylenediamine tetraacetate.

476

CHUL-YONGSONG and MARC H. DRESDEN

(Barrett et al., 1982; Dresden et al., 1985), iodoacetic acid, and N E M (Dresden et al., 1985) also confirm the suggestion that the 0.1 M sodium citrate, pH 5.5, extract contained cysteine proteinases. The proteinase specific activity with CBZ-phe-argA F C and with Azocoll of extracts and partially purified enzymes of metacercariae was greater than that of the other stages of the parasite. However, total cysteine proteinase activity was relatively constant during development. The cysteine proteinases of P. westermani may be involved in pathologic changes associated with larval migration, including localized tissue destruction. Further studies on the purification and characterization of cysteine proteinases should provide information about their function. Acknowledgement--This work was supported by a grant from the Korea Science and Engineering Foundation (08620412-006-2). REFERENCES

Auriault C., Pierce R., Cesari I. M. and Capron A. (1982) Neutral protease activities at different developmental stages of Schistosoma mansoni in mammalian hosts. Comp. Biochem. Physiol. 72B, 377-384. Barrett A. J. (1979) Cathepsin B and other thiol proteinases. In Proteinases in Mammalian Cells and Tissues (Edited by Barrett A. J.), pp. 181-248. North Holland, Amsterdam. Barrett A. J., Kembhavi A. A., Brown M. A., Kirschke H., Knight C. G., Tamai M. and Hanada K. (1982) L-Transepoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteinyl proteinases including cathepsins B, H, and L. Bioehem. J. 201, 189 198. Bradford M. (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of proteindye binding. Analyt. Biochem. 72, 248-254.

Chappell C. L. and Dresden M. H. (1987) Purification of cysteine proteinases from adult Schistosoma mansoni. Arch. Biochem. Biophys. 256, 560-568. Deelder A. M., Reinders P. N. and Rotmans J. P. (1977) Purification studies on an acidic protease from adult Schistosoma mansoni. Acta Leidensia 45, 91-103. Dresden M. H. and Asch H. L. (1972) Proteolytic enzymes in extracts of Schistosoma mansoni cercariae. Biochim. biophys. Aeta 289, 378 384. Dresden M. H. and Deelder A. M. (1979) Schistosoma mansoni: Thiol proteinase properties of adult worm "hemoglobinase'. Exp. Parasitol. 48, 190-197. Dresden M. H., Rutledge M. L. and Chappell C. L. (1981) Properties of the acidic thiol proteinase from Schistosoma mansoni adults. Biochem. Molec. Parasitol. 4, 61~6. Dresden M. H., Rege A. A. and Murrell K. D. (1985) Strongyloides ransomi: Partial characterization of proteolytic enzymes. Exp. ParasitoL 59, 257--263. Grant C. T. and Senft A. W, (1971) Schistosoma proteolytic enzyme. Comp. Biochem. Physiol. 38B, 663-678. McKerrow J. H., Pino-Heiss S., Lindquist R. and Werb Z. (1985) Purification and characterization of an elastinolytic proteinase secreted by cercariae of Sehistosoma mansoni. J. biol. Chem. 260, 3703-3707. Rege A. A., Song C.-Y., Bos H. J. and Dresden M. H. (1989) Isolation and partial characterization of a potentially pathogenic cysteine proteinase from adult Dictyocaulus viviparus. Vet. Parasitol. (in press). Tzeng S., McKerrow J. H., Fukuyama K., Jeong K. and Epstein W. L. (1983) Degradation of purified skin keratin by a proteinase secreted from Schistosoma mansoni cercariae. J. Parasitol. 69, 992-994. Yamakami K. (1986) Purification and properties of thiol protease from lung fluke adult Paragonimus ohirai, Comp. Biochem. Physiol. 83B, 501-506. Yarnakami K. and Hamajima F. (1987) Purification and properties of a neutral thiol protease from larval trematode parasite earagonimus westermani metacercariae. Comp. Biochem. Physiol. 87B, 643~648.