Purification and characterization of two-domain glutaredoxin in the parasitic helminth Fasciola gigantica Ankita Gupta a , Banchob Sripa b , Timir Tripathi a, ⁎ a Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India b Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand abstract article info Article history: Received 3 August 2015 Received in revised form 28 February 2016 Accepted 10 May 2016 Available online 14 May 2016 Glutaredoxins (Grxs) are small thiol-dependent proteins and key elements of redox signaling as they regulate the redox state of important cellular proteins. In the present study, the complete sequence of a glutaredoxin protein, obtained from the liver fluke Fasciola gigantica, was PCR-amplified and cloned. The 690-bp open reading frame (ORF) encodes a 230-amino acid protein with two conserved domains (FgGrxD1 and FgGrxD2) and has similar- ities with two monothiol Grxs of Saccharomyces cerevisiae, i.e., ScGrx3 and ScGrx4. The full-length FgGrx along with its two constituent domains were overexpressed in Escherichia coli as hexahistidyl-tagged proteins. The af- finity chromatography resulted in almost pure and soluble proteins. The full-length FgGrx and the FgGrxD2 showed reddish-brown color, indicating the presence of bound iron \\in the second domain. In the insulin based reduction assay, both FgGrx and FgGrxD2 containing the active site motif CGFS exhibited a weak reducing activity, whereas FgGrxD1 was inactive. Additionally, FgGrx did not show any GSH-disulfide transhydrogenase activity when 2-hydroxyethyl disulfide (HED) or de-hydroascorbate (DHA) were taken as substrates. These re- sults indicated the probable role of FgGrx in cellular iron-sulfur homeostasis. FgGrx was found to be reversibly S-glutathionylated, suggesting a potential redox regulation that is likely to take place at the active site Cys158. Since there is only one Cys in FgGrxD2, the Cys158 might be involved in Fe \\ S binding. This study is the first re- port on the presence of Grx in platyhelminthic parasites and provides a starting point for further characterization of the redox network in liver flukes. © 2016 Elsevier Ireland Ltd. All rights reserved. Keywords: Redox Thiol Glutathionylation Liver fluke Cysteine Thioredoxin 1. Result and discussion Tropical infectious diseases caused by helminth parasites pose a se- rious threat to human health and can lead to large economic losses [1]. Fascioliasis, caused by the trematodes of the genus Fasciola, belongs to a group of neglected tropical diseases. WHO has recommended the use of triclabendazole drug for the treatment of fascioliasis; the drug is active against both the parasitic stages, i.e., metacercariae and adult stages. However, recent studies have suggested that these helminth parasites have gained resistance to triclabendazole in several countries [2]. F. gigantica is challenged by reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by the activated immune re- sponse system of the host. Both ROS and RNS initiate lipid peroxidation and reduce the functionality of proteins, cell membranes, and nucleic acids, ultimately resulting in the death of the parasite. To cope up with the oxidative stress, parasites depend on the action of antioxidant en- zymes and low molecular weight antioxidants. Thus, the proteins asso- ciated with the redox balance serve as interesting target molecules for novel antiparasitic strategies [3,4]. Glutaredoxins (Grxs) are small ubiquitous proteins that are con- served in organisms throughout evolution. They are not only involved in the glutathione-dependent reduction of proteins, antioxidant de- fense, and redox regulation but also in other essential cellular functions, such as cellular differentiation, prevention of apoptosis and Fe \\ S ho- meostasis. Two isoforms of Grxs, monothiol ( \\ CXXS \\ ) and dithiol Grxs ( \\ CXXC \\ ), have been identified on the basis of the number of ac- tive site cysteine residues. The oxidoreductase reactions of monothiol and dithiol Grxs are different. The monothiol mechanism only requires the N-terminal Cys residue of the active site to reduce the mixed disul- fide between GSH and the target proteins while the dithiol mechanism requires both the Cys residues to reduce the target proteins [5,6]. It has been observed that certain monothiol Grxs, such as Saccharomyces cerevisiae Grx5 (ScGrx5), form an intramolecular disulfide bond be- tween the additional conserved Cys residue and the N-terminal active site Cys residue by operating on the basis of the a dithiol mechanism. Monothiol Grxs are further classified into single- and multi-domain monothiol Grxs with an N-terminal Trx-like domain or multi-Grx-do- main [7–9]. Many monothiol Grxs have been reported to bind iron-sul- fur clusters (Fe \\ S) in the presence of GSH, both in vivo and in vitro. Studies have also revealed that the amino acids near the active site play an important role in the binding process of Fe \\ S clusters [10]. Parasitology International 66 (2017) 432–435 ⁎ Corresponding author. E-mail addresses: timir.tripathi@gmail.com, ttripathi@nehu.ac.in (T. Tripathi). http://dx.doi.org/10.1016/j.parint.2016.05.005 1383-5769/© 2016 Elsevier Ireland Ltd. All rights reserved. Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint