Purification and biochemical characterization of a 22-kDa stable cysteine- like protease from the excretory-secretory product of the liver fluke Fasciola hepatica by using conventional techniques
Introduction
The parasitic trematode Fasciola hepatica, also known as the common liver fluke, is the causative agent of fascioliasis, a worldwide-distributed disease that can causes economic losses and mortality in domestic ruminants such as cattle and sheep [1], [2], [3]. Humans may acquire the infection by ingestion of raw vegetables or contaminated water with the parasite’s metacercariae [4], [5].
F. hepatica liver flukes secrete several proteolytic enzymes that belong to the cathepsin L group of the cysteine proteases family [6], [7] previously identified and characterized as papain-like cysteine proteases [8]. Many of which resemble mammalian liver cathepsin L (CL) proteases both in amino acids sequence and substrate specificity [9]. They are expressed in newly excysted juvenile (NEJ), immature and adult stages of parasite with different enzymatic properties and timing of expression [10].
F. hepatica cathepsin B (FhCB) has been reported as the predominant cathepsin protease released by newly excysted juveniles (NEJs), contrasting to the situation in adult flukes where F. hepatica cathepsin L (FhCL) are the main secretory enzymes [6], [11]. cathepsin L proteases from the liver fluke have been found to play critical roles in the different host-parasite interactions. A number of studies have shown that adult F. hepatica, when maintained in vitro, secretes a battery of cysteine proteases capable of cleaving host immunoglobulins in a papaine- like manner [12], [13]. Thus, secreted proteases assist the parasite to evade host antibody-mediated immune mechanisms either by the suppression of immune cell proliferation or by reducing the expression of CD4 on the surface of the host T-cells [14]. Moreover, many of cathepsin proteases have found to be exhibit proteolytic activities against extracellular matrix and basement membrane components, which would facilitate the penetration and migration of the parasite through the intestinal wall and hepatic mass [15], [16], [17].
Two cysteine proteases of molecular weights 25 and 26 kDa have been previously purified from the excreted-secreted products of F. hepatica and their primary structures were determined [18]. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L1- like [19] and cathepsin L2- like [20]. The two proteases, CL1 and CL2 have shown to be immunodominant antigens in human fascioliasis and other susceptible hosts [21], and are reported to be useful in diagnostic test of F. hepatica infection in animal and human populations [22], [23], [24]. In addition, vaccine trials in both cattle and sheep with purified native FhCL1 and FhCL2 proteases induced protection to experimental challenge with metacercariae of F. hepatica and gave a significant reduction in embryonation/hatch rate effects, liver damage, fluke burden and production of viable eggs [25], [26], [27].
In this study, we report the purification of a cysteine- like protease activity released into the excretory-secretory product of adult F. hepatica parasitic trematode by employing the principles of liquid chromatography and electrophoresis to provide basic information about its main biochemical and kinetic characteristics. In addition to these aspects, an immunoblotting analysis was conducted using experimentally infected sheep anti-serum in order to evaluate the potential use of this protease as immunodiagnostic agent for the early detection of F. hepatica infection in cattle.
Section snippets
Source of crude enzyme
Adult F. hepatica parasites were removed from the bile ducts of naturally infected bovine livers at local abattoir. After they have washed several times with Nacl 9‰ to remove all traces of blood and bile, the intact worms were incubated at 37 °C for 3 h in phosphate-buffered saline PBS, pH 7.3 containing 2% d- glucose, 30 mM HEPES and 0.02% Sodium azide (NaN3). Following incubation period, the culture medium was centrifuged at 12,000×g, 4 °C for 30 min, filtered through 0.22 mm Millipore filter and
Enzyme purification
Until recent decades, liquid chromatography has received much attention because of its high feasibility as a fundamental method for the separation and purification of many diversified components from either the somatic or metabolic products of F. hepatica for the development of pure and well-defined molecules that can be used as antigenic fractions in immunodiagnostic and immunization trials [35], [36], [37].
In the current study, a proteolytic activity was isolated from the excretory-secretory
Conclusion
Proteolytic enzymes are the important molecules released in the excretory-secretory product of the parasitic pathogen F. hepatica because of their role in numerous vital processes, especially feeding and tissue migration. Using the principles of acetone precipitation, molecular exclusion and ion exchange chromatography, a stable protease activity was purified to homogeneity and then characterized to be a 22-kDa cysteine- like protease. The high enzyme stability over a broad pH range makes it a
Acknowledgements
This work was supported by the Biology, Water and Environment Laboratory, University 8 May 1945, Guelma. We would like to thank Prof. M. Nasri and the post-graduate students of the LGEM laboratory (ENIS, Tunisia) for their technical assistance. We are also very grateful to Prof. A. Benakhla (Department of Veterinary Sciences, El-Tarf) for kindly providing sheep sera and Dr. Abdi Sabrina (slaughterhouse of seybousse, Annaba) for his help in the collect of parasites.
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