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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.


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.


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.


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

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