| Home | E-Submission | Sitemap | Contact us |  
top_img
Korean J Parasitol > Volume 32(4):1994 > Article

Original Article
Korean J Parasitol. 1994 Dec;32(4):231-241. English.
Published online Dec 20, 1994.  http://dx.doi.org/10.3347/kjp.1994.32.4.231
Copyright © 1994 by The Korean Society for Parasitology
Characterization of a cysteine proteinase from adult worms of Paragonimus westermani
C Y Song,*1 and T S Kim2
1Department of Biology, College of Science, Chung-Ang University, Seoul 156-756, Korea.
Received October 21, 1994; Accepted November 05, 1994.

Abstract

Paragonimus westermani, the lung fluke, is known to migrate to the pulmonary tissue of mammalian hosts and causes pathological changes in the lungs. An acidic thiol-dependent proteinase with a molecular weight of approximately 20,000 daltons was purified to homogeneity using ion-exchange chromatography and gel filtration chromatography. On SDS-PAGE, the molecular weight of the enzyme was 17,500 daltons. Isoelectric point was 6.45. The enzyme was similar to the acidic cysteine proteinase of vertebrates in the properties of pH 5.5 for at least two days when stored at 4℃. The cysteine proteinase was capable of degrading collagen and hemoglobin. Sera of patients with paragonimiasis and mice infected with P. westermani reacted in immunoblots with the partially purified proteinase. This result suggested that the cysteine proteinase of P. westermani may play a role in migration in tissues, and in acquisition of nutrients by parasites from the host. It is also potentially an antigen for the serodiagnosis of paragonimiasis.

Figures


Fig. 1
Elution profile of cysteine proteinase of P. westermani(A) The crude extracts were loaded on CM-trisacryl M cathion-exchange column. Fractions were assayed for activity on CBZ-phe-arg-AFC (•) and monitored for protein content (▪) at 280 nm. (B) Affinity chromatography with thiol-activated Sepharose 4B. The enzyme purified from CM-trisacryl column chromatography was loaded. 2 ml fractions were assayed for activity as described previously. Markings are the same as in A.


Fig. 2
Activity peaks purified from thiol-activated Sepharose 4B were loaded onto a column (1.6 × 40 cm) of sephacryl S-200 HR. 1.4 ml fractions were assayed for activity. Marking are the same as in A. Molecular weight determination of purified cysteine proteinase by Sephacryl S-200 HR gel filtration chromatography (inset). Purified enzyme was applied to Sephacryl S-200 column (1.6 × 40 cm) which had been precalibrated with marker proteinas. The markers were as follows. A: bovine serum albumin (66,000), B: ovalbumin (43,000), C: chymotrypsinogen (25,000), D: ribonuclease (13,700).


Fig. 3
7.5-15.0% gradient SDS-PAGE analysis of cysteine proteinase purified from P. westermani. Lane A, homogenate supermatan: Lane B, active peak from CM-Trisacryl M; Lane C, active peak from thiol-activated Sepharose 4B affinity gel; Lane D, active peak from Sephacryl S-200 HR. Molecular weight markers included the following proteins (Pharmacia LKB, Sweden): phosphorylase B (94,000), albumin (67,000), ovalbumin (43.000), carbonic anhydrase (30,000), trypsin inhibitor (20,100) and α-lactalbumin (14,400).


Fig. 4
Isoelectric focusing analysis (pH range 3.5-9.5) of the purified cysteine proteinase. Lane A, homogenate supernatant; Lane B, active peak from CM-Trisacryl M; Lane C, active peak from thiol-activated Sepharose 4B affinity gel; Lane D, active peak from Sephacryl S-200 HR. Isoelectric point markers included the following proteins (Pharmacia, Sweden): trypsinogen (pi 9.30). lentil lectin-basic band (pi 8.65), lentil lectin middles band (pi 8.45), lentil lectin-acidic band (pi 8.15), horse myoglobin-basic band (pi 7.35). horse myoglobin-acidic band (pi 6.85). human carbonic anhydrase B (pi 6.55), bovine carbonic anhydrase B (pi 5.85), β-lactoglobin (pi 5.20). soybean trypsin inhibitor (pi 4.55), and amyloglucosidase (pi 3.50).


Fig. 5
Degradation of collagen substrates by P. westermani cysteine proteinase. Proteinase purified from gel filtration chromatography was assayed for collagenolysis in the presence of 5 mM DTT. Collagen degradation is indicated by the disappearance of α- and β-chains (upper two arrow heads) from a 7.5-15.0% SDS-PAGE. Lane A, control collagen (Type I); Lane B-F, incubated for 30 min, 1, 2, 4, and 8 hr, respectively.


Fig. 6
Degradation of hemoglobin by P. westermani cysteine proteinase. Purified cysteine proteinase from gel filtration chromatography was assayed for hemoglobinolytic activity in the presence of 5 mM DTT (incubation time of lane B. C and D are 2, 4, and 8 hr, respectively). Disappearance of the monomeric and dimeric hemoglobin was analysed by a 7.5-15.0% SDS-PAGE. Lane A, control hemoglobin without enzyme.


Fig. 7
Immunoblot analysis of P. westermani cysteine proteinase under denaturing conditions . 7.5-15.0% polyacrylamide gel electrophoresis and electrophoretic transfer of purified proteinase onto nitrocellulose membranes were performed as described in materials and methods. Lane A-F, human antisera (paragonimiasis); Lane G, mouse antiserum (3 months after experimental infection): Lane H, normal human serum. Molecular weight standards are indicated.

Tables


Table 1
Activity of cysteine proteinase from Paragonimus westermani against synthetic substrate and thiol dependence of enzymea)


Table 2
Summary for the purification of cysteine proteinase from P. westermani adult worms


Table 3
Stability of cysteine proteinase purified from P. westermani


Table 4
Effects of Inhibitors on cysteine proteinase purified from P. westermani

References
1. Aoki T. Jap J Parasitol 1980;29:325–332.
2. Auriault C, Pierce R, Cesari IM, Capron A. Neutral protease activities at different developmental stages of Schistosoma mansoni in mammalian hosts. Comp Biochem Physiol B 1982;72(3):377–384.
  
3. Chappell CL, Dresden MH. Schistosoma mansoni: proteinase activity of "hemoglobinase" from the digestive tract of adult worms. Exp Parasitol 1986;61(2):160–167.
  
4. Chappell CL, Dresden MH. Purification of cysteine proteinases from adult Schistosoma mansoni. Arch Biochem Biophys 1987;256(2):560–568.
  
5. Dresden MH, Rege AA, Murrell KD. Strongyloides ransomi: proteolytic enzymes from larvae. Exp Parasitol 1985;59(2):257–263.
  
6. Dresden MH, Deelder AM. Schistosoma mansoni: thiol proteinase properties of adult worm "hemoglobinase". Exp Parasitol 1979;48(2):190–197.
  
7. Dresden MH, Rutledge ML, Chappell CL. Properties of the acid thiol proteinase from Schistosoma mansoni adults. Mol Biochem Parasitol 1981;4(1-2):61–66.
  
8. Halton DW. Observations on the nutrition of digenetic trematodes. Parasitology 1967;57(4):639–660.
  
9. Hamajima F, Yamakami K. Jap J Parasitol 1981;30:127–134.
10. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.
  
11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265–275.
 
12. McKerrow JH. Parasite proteases. Exp Parasitol 1989;68(1):111–115.
  
13. McKerrow JH, Doenhoff MJ. Schistosome proteases. Parasitol Today 1988;4(12):334–340.
  
14. Rege AA, Dresden MH. Strongyloides spp.: demonstration and partial characterization of acidic collagenolytic activity from infective larvae. Exp Parasitol 1987;64(3):275–280.
  
15. Rege AA, Herrera PR, Lopez M, Dresden MH. Isolation and characterization of a cysteine proteinase from Fasciola hepatica adult worms. Mol Biochem Parasitol 1989;35(1):89–95.
  
16. Rege AA, Song CY, Bos HJ, Dresden MH. Isolation and partial characterisation of a potentially pathogenic cysteine proteinase from adult Dictyocaulus viviparus. Vet Parasitol 1989;34(1-2):95–102.
  
17. Ruppel A, Diesfeld HJ, Rother U. Immunoblot analysis of Schistosoma mansoni antigens with sera of schistosomiasis patients: diagnostic potential of an adult schistosome polypeptide. Clin Exp Immunol 1985;62(3):499–506.
 
18. Simpkin KG, Chapman CR, Coles GC. Fasciola hepatica: a proteolytic digestive enzyme. Exp Parasitol 1980;49(2):281–287.
  
19. Song CY, Choi DH, Kim TS, Lee SH. Isolation and partial characterization of cysteine proteinase from sparganum. Korean J Parasitol 1992;30(3):191–199.
 
20. Song CY, Rege AA. Cysteine proteinase activity in various developmental stages of Clonorchis sinensis: a comparative analysis. Comp Biochem Physiol B 1991;99(1):137–140.
  
21. Song CY, Dresden MH. Partial purification and characterization of cysteine proteinases from various developmental stages of Paragonimus westermani. Comp Biochem Physiol B 1990;95(3):473–476.
  
22. Tsang VC, Peralta JM, Simons AR. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol 1983;92:377–391.
  
23. Yamakami K. Purification and properties of a thiol protease from lung fluke adult Paragonimus ohirai. Comp Biochem Physiol B 1986;83(3):501–506.
  
24. Yamakami K, Hamajima F. Purification and properties of a neutral thiol protease from larval trematode parasite Paragonimus westermani metacercariae. Comp Biochem Physiol B 1987;87(3):643–648.
  
25. Yamasaki H, Aoki T, Oya M. Jap J Parasitol 1989;38:373–384.
Editorial Office
c/o Department of Medical Environmental Biology
Chung-AngUniversity College of Medicine, Dongjak-gu, Seoul 06974, Korea
Tel: +82-2-820-5683   Fax: +82-2-826-1123   E-mail: kjp.editor@gmail.com
About |  Browse Articles |  Current Issue |  For Authors and Reviewers
Copyright © 2019 by The Korean Society for Parasitology and Tropical Medicine. All rights reserved.     powerd by m2community