Elsevier

Journal of Chromatography B

Volumes 1068–1069, 15 November 2017, Pages 268-276
Journal of Chromatography B

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

https://doi.org/10.1016/j.jchromb.2017.10.049Get rights and content

Highlights

  • A stable 22-kDa cysteine- like protease was purified to homogeneity from the medium in which adult Fasciola hepatica were maintained.

  • The cysteine- like protease was purified 11-fold with a specific activity of 1893.9 U/mg and overall activity yields of 31.5%.

  • This purified protease was able to detect specific antibodies in sera of experimentally infected sheep as soon as 2 weeks post-infection.

  • Its stability over a broad range of pH 5-10 makes it very suitable for potential biochemical application, especially as antigen for fascioliasis serodiagnosis.

Abstract

This study describes the purification and characterization of a stable protease activity isolated from Fasciola hepatica adult worms maintained in vitro by employing acetone precipitation (40–60%) followed by a gel filtration through Sephadex G–100 and DEAE– cellulose ion exchange column. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9 U/mg and 31.5% recovery. After the final ultrafiltration step, the purification fold was increased up to 13.1 and the overall activity yield reached a rate of 18.8%. The MW of the purified protease was estimated by reducing SDS–PAGE to be 22 kDa while the proteolytic activity detection was carried out by zymography on non-denaturing SDS–PAGE containing the casein as substrate. Using this substrate, the protease showed extreme proteolytic activity at pH 5.5 and temperature 35–40 °C and was highly stable over a wide range of pH, from 5.0 to 10.0. In addition to its preference for the Z-Phe-Arg-AMC fluorogenic substrate resulting in maximum proteolytic activity (99.7%) at pH 7.0, the pure protease exhibited highest cleavage activity against hemoglobin and casein substrates at pH 5.5 (85.6% and 82.8%, respectively). The Km values obtained for this protease were 5.4, 13, 160 and approximately 1000 μM using respectively the fluorogenic substrate Z-Phe-Arg-AMC, hemoglobin, casein and albumin. The protease activity was completely inhibited either by E-64 inhibitor (5 mM) or iodoacetamide (10 mM), indicating its cysteine nature. The usefulness of the purified protease as an antigen was studied by immunoblotting. Thus, sera from sheep experimentally infected with F. hepatica recognized the protease band at 2 weeks post-infection (WPI) and strongly at 7 WPI. The early detection of antibodies anti- F. hepatica suggests the application of this molecule as a specific epitope for the serodiagnosis of fascioliasis disease.

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.

References (59)

  • A. Varghese et al.

    Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes

    Vet. Parasitol.

    (2012)
  • D.P. Knox et al.

    The contribution of molecular biology to the development of vaccines against nematode and trematode parasites of domestic ruminants

    Vet. Parasitol.

    (2001)
  • J.P. Dalton et al.

    Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines

    Int. J. Parasitol.

    (2003)
  • J.P. Dalton et al.

    Immunomodulatory molecules of Fasciola hepatica: candidates for both vaccine and immunotherapeutic development

    Vet. Parasitol.

    (2013)
  • M.M. Bradford

    A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal. Biochem.

    (1976)
  • S. Moore

    Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction

    J. Biol. Chem.

    (1968)
  • A.J. Dowd et al.

    Stability studies on the cathepsin L proteinase of the helminth parasite, Fasciola hepatica

    Enzyme Microb. Technol.

    (2000)
  • F.L. Garcia-Carreno et al.

    Substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors

    Anal. Biochem.

    (1993)
  • G.L. Zimmerman et al.

    Separation of parasite antigens by molecular exclusion anion exchange, and chromatofocusing utilizing FPLC protein fractionation systems

    Vet. Parasitol.

    (1986)
  • K.G. Simpkin et al.

    Fasciola hepatica: a proteolytic digestive enzyme

    Exp. Parasitol.

    (1980)
  • R.M. Morphew et al.

    Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host

    Mol. Cell. Proteomics

    (2007)
  • V.T. Heussler et al.

    Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

    Mol. Biochem. Parasitol.

    (1994)
  • D. Bernal et al.

    Identification of enolase as a plasminogen-binding protein in excretory-secretory products of Fasciola hepatica

    FEBS Lett.

    (2004)
  • A.A. Rege et al.

    Isolation and characterization of a cysteine proteinase from Fasciola hepatica adult worms

    Mol. Biochem. Parasitol.

    (1989)
  • I. Corvo et al.

    The major cathepsin L secreted by the invasive juvenile Fasciola hepatica prefers proline in the S2 subsite and can cleave collagen

    Mol. Biochem. Parasitol.

    (2009)
  • M.M. Harmsen et al.

    Identification of a novel Fasciola hepatica cathepsin L protease containing protective epitopes within the propeptide

    Int. J. Parasitol.

    (2004)
  • S.J. Hawthorne et al.

    Partial characterization of a novel cathepsin L- like protease from Fasciola hepatica

    Biochem. Biophys. Res. Commun.

    (2000)
  • K. Wada et al.

    N-terminal amino acid sequences of the heavy and light chains of chicken liver cathepsin L

    FEBS Lett.

    (1986)
  • T. Towatari et al.

    Amino acid sequence of rat liver cathepsin L

    FEBS Lett.

    (1988)
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