A cysteine protease of <i>Paragonimus westermani</i> eggs

, Kang, S Y; , Cho, M S; , Chung, Y B; , Kong, Y; , Cho, S Y

doi:1995.33.4.323

Korean J Parasito > Volume 33(4):1995 > Article

Original Article


Korean J Parasitol. 1995 Dec;33(4):323-330. English. Published online Dec 20, 1995. http://dx.doi.org/10.3347/kjp.1995.33.4.323
Copyright © 1995 by The Korean Society for Parasitology


A cysteine protease of Paragonimus westermani eggs
Shin-Yong Kang,¹Myung-Shin Cho,¹Young-Bae Chung,¹Yoon Kong,² and Seung-Yull Cho^*³
¹Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.
²Biomedical Research Center, Korea Institute of science and Technology, Seoul 136-791, Korea.
³Department of Parasitology, Catholic University Medical College, Seoul 173-701, Korea.

Received August 16, 1995; Accepted September 12, 1995.
Abstract
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-β-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.

Figures


	Fig. 1 Determination of optimal pH for proteolytic activity of the partially purified enzyme of P. westermani egg. The enzyme activity was detected in 0.1 M sodium acestate and 0.1 M sodium phosphate buffer. ●-●, activity against Cbz-phe-arg-MNA; ○-○, activity against Azocoll. See also the "Materials and Methods" for detailed description.


	Fig. 2 SDS-PAGE findings of the crude adult and egg extracts of P. westermani on 12.5% separating gel. 1 & 3, crude adult extracts in reducing (1) and non-reducing conditions (3); 2 & 4, crude egg extracts in reducing (2) and non-reducing (4) conditions. Coomassie blue stained. Mr, molecular mass in kDa.


	Fig. 3 Activity gel electrophoresis of the crude egg extracts of P. westermani showing two proteolytic zones. The 12.5% separation gel, copolymerized with 0.2% (w/v) gelatin was used for protein resolution. C, adult worm extracts of P. westermani; Egg, egg extracts. Proteolytic zones by the egg extracts are indicated(◂).


	Fig. 4 Partial purification of the cysteine protease from the egg extracts. The crude extracts were eluted through Sephacryl S-300 HR gel permeation column (1.6 × 70 cm). Aliquots of each fraction were assayed for Cbz-phe-arg-MNA degrading activity (○-○) and for protein (●-●). Pooled fractions with high enzyme activity are shown by a bar(-).


	Fig. 5 Electrophoretic analysis of crude egg extracts (1) and partially purified cysteine protease of P. westermani eggs (2) shown in 7.5-15% SDS-PAGE. The partially purified egg protease migrates as a single band at 35 kDa (Coomassie blue stained). Mr, molecular mass in kDa.

Tables


	Table 1 Substrate specificity of Paragonimus westermani egg extracts against synthetic and macromolecular substrates


	Table 2 Modulation of inhibitors on the proteolytic activity of the crude egg extracts of P. westermani

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