Cytotoxicity of a cysteine proteinase of adult <i>Clonorchis sinensis</i>

, Park, H; , Ko, M Y; , Paik, M K; , Soh, C T; , Seo, J H; , Im, K I

doi:1995.33.3.211

Korean J Parasito > Volume 33(3):1995 > Article

Original Article


Korean J Parasitol. 1995 Sep;33(3):211-218. English. Published online Sep 20, 1995. http://dx.doi.org/10.3347/kjp.1995.33.3.211
Copyright © 1995 by The Korean Society for Parasitology


Cytotoxicity of a cysteine proteinase of adult Clonorchis sinensis
H Park,^*¹M Y Ko,²M K Paik,²C T Soh,³J H Seo,⁴ and K I Im⁵
¹Biomedical Research Center, Korea Institute of Science and Technology (KIST), Seoul 130-650, Korea.

Received May 23, 1995; Accepted June 23, 1995.
Abstract
To clarify the correlation of the proteinase activity with pathogenicity of Clonorchis sinensis, the proteinase activity either in excretory-secretory products (ESP) or in crude extracts of adult C. sinensis was examined. Substrate gel electrophoresis of the ESP and crude extracts revealed four distinct enzyme bands, which were differently inhibited by the specific proteinase inhibitors. The proteinase of the ESP with molecular mass of 24 kDa, was purified 23- fold with 14.5% yield by spectra gel ACA 44 gel filtration. It exhibited optimal pH at 7.5 in sodium phosphate (0.1 M). Its activity was inhibited specifically by N-ethylmaleimide (NEM) and antipain whereas potentiated 1.9 folds in the presence of 5 mM dithiothreitol (DTT). Cytotoxicity of the proteinase increased in a dose-dependent manner up to 120 µg/ml while reduced by NEM and antipain, indicating that cysteine proteinase was responsible for the cytotoxicity. This result shows that the 24 kDa cysteine proteinase is deeply correlated with the pathogenicity of C. sinensis infection.

Figures

Fig. 1
The pH optimum for proteinase activity in C. sinensis worm extracts and ESP. The optimal pHs of the proteinase activities were ditermined by replacing the buffer sued in the respective enzyme assays in 50 mM glycine buffer for pH 3, in 50 mM sodium acetate buffer at pH 4, 4.5, 5 and 5.5, in 50 mM sodium phosphate buffer for pH values between 6.0 and 8.5 and in 50 mM Trisaminomethane buffer for pH values between 9 and 11. ○, worm extract; •, ESP proteinase.


	Fig. 2 Identification of proteinase activities of adult C. sinensis is shown by substrate gel electrophoresis (each lane contained the 20 µg protein, 5 mM inhibitor). (A) Effect of NEM and TPCK on the worm proteinase. Rf values at a, 0.04; b, 0.61; c, 0.71; d, 0.78. (B) Effect of NEM on ESP enzymes Rf values are A, 0.11; B, 0.46; C, 0.62; D,0.92.


	Fig. 3 Cytotoxic activity of C. sinensis on CHO cells, Error bars represent the mean ± standard error, n=6. –●–, worm extract; –○–, ESP.

Fig. 4
Elution profile and cytotoxic activity of partially purified enzyme by spectra/ Gel AcA 44 gel filtration. Each fractions are assayed for cytotoxicity (——) and protein content (……), Inset also shows the determination of molecular weight in kDa. a, phosphorylase b (97.4); b, bovine albumin (66); c, egg albumin (45); d, carbonic anhydrase (31); e, trysin inhibitor (21.5); f, lysozyme (14.5).

Tables


	Table 1 Purification of proteinase from C. sinensis cultured media


	Table 2 Modulation of inhibitors on proteinases of C. sinensis worm extract and ESP


	Table 3 The effect of various inhibitors on partially purified proteinase from ESP against CHO cell

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