Purified proteins resolved by 12?% SDS-PAGE/2-DE were stained with Coomassie brilliant G-250 (CBB) or further processed by immunoblotting using anti-rCsGSTo1 and 2 antibodies

Purified proteins resolved by 12?% SDS-PAGE/2-DE were stained with Coomassie brilliant G-250 (CBB) or further processed by immunoblotting using anti-rCsGSTo1 and 2 antibodies. Enzyme assay GST activity was spectrophotometrically determined employing a panel of substrates (Sigma-Aldrich); 1-chloro-2,4-dinitrobenzene (CDNB; pH?6.5, 340?nm), 1,2-dichloro-4-nitrobenzene (DCNB; pH?7.5, 345?nm), 4-nitrobenzyl chloride (4-NBC; pH?6.5, 310?nm), 4-nitrophenyl acetate (4-NPA; pH?7.0, 400?nm), 4-hydroxy nonenal (pH?7.5, 340?nm), cumene hydroperoxide (CHP; pH?6.5, 340?nm) and ethacrynic acid (pH?7.5, 340?nm). protective roles of TC-E 5002 CsGSTos in these organs under oxidative stress were investigated. Results The full-length cDNAs of and constituted 965?bp and 1,061?bp with open reading frames of 737?bp (246 amino acids) and 669?bp TC-E 5002 (223 amino acids). They harbored characteristic N-terminal thioredoxin-like and C-terminal -helical domains. A cysteine residue, which constituted omega-class specific active site, and the glutathione-binding amino acids, were recognized in appropriate positions. They shared 44?% sequence identity with each other and 14.8C44.8?% with orthologues/homologues from other organisms. Bacterially expressed recombinant proteins (rCsGSTo1 and 2) exhibited dehydroascorbate reductase (DHAR) and thioltransferase activities. DHAR activity was Rabbit Polyclonal to p300 higher than thioltransferase activity. They showed weak canonical GST activity toward 1-chloro-2,4-dinitrobenzene. worms and in response to oxidative conditions, thereby contributing to maintenance of parasite fecundity. Electronic supplementary material The online version TC-E 5002 of this article (doi:10.1186/s13071-016-1622-2) contains supplementary material, which is available to authorized users. causes one of the major fish-borne-zoonotic trematodiases. It is prevalent in several countries of Asia, especially where aquaculture systems associated with paddy field are widely used [1]. Approximately 35 million people are infected and another 600 million people are at risk worldwide [2]. Humans are infected by eating raw/undercooked freshwater fish infected with metacercariae. Light infections usually are asymptomatic. However, chronic cumulative infections invoke several hepatobiliary symptoms including cholecystitis, obstructive jaundice, cholangitis and ascites [3]. Pathological alterations like adenomatous hyperplasia and/or dysplasia of the biliary epithelium, mucin-secreting metaplasia and ductal dilatation with fibrosis are frequently observed in those patients [4]. Epidemiological evidence indicates that approximately 10?% of cholangiocarcinoma is associated with chronic infections [5, 6]. Long-standing inflammations accompanied by clonorchiasis might result in oxidative damage of the biliary ductal epithelium and malignant transformation. is classified as a Group 1 biocarcinogen [7]. survives more than ten years within the biliary lumen, where free oxygen radicals generated by lipid peroxidation and several hydrophobic substances derived from liver metabolism prevail [8]. In order to adapt to the hostile micromilieu, continuously produces diverse antioxidant enzymes, among which several species of glutathione transferases (GSTs: E.C. are the major components [9, 10]. At least eight proteoforms of mu- and sigma-class GST isozymes have been described. Some are intimately involved in protection of the worm during oxidative stress as well as in neutralization of cytopathic host bile [9]. Nucleotide sequences coding for kappa- (“type”:”entrez-protein”,”attrs”:”text”:”GAA51086″,”term_id”:”358332422″,”term_text”:”GAA51086″GAA51086) and zeta-type (“type”:”entrez-protein”,”attrs”:”text”:”GAA44819″,”term_id”:”358231327″,”term_text”:”GAA44819″GAA44819) GSTs have also been identified, but their protein identity and biological properties remain elusive. GSTs are ubiquitously expressed in almost all known organisms [11]. Typical catalytic activity of GSTs is refined by the conjugation of glutathione (GSH; -Glu-Cys-Gly) to a wide variety of nonpolar electrophilic, endogenous and exogenous toxic compounds [12]. GSTs play crucial roles against various toxicants, especially in helminth parasites that lack the cytochrome P-450 (CYP450) phase II detoxification enzyme. Most helminth GSTs can be classified into mu- and sigma-types [10, 13], although some GSTs demonstrate mosaic patterns of substrate/inhibitor specificity [14]. Omega-class GST (GSTo) is a relatively ancient cytosolic enzyme, but is the most recently characterized [11, 15]. A RNA polymerase-related protein designated stringent starvation protein A (SspA) represents a bacterial GST-like molecule due to its highly comparable structural property with GSTo, but lacks GST activity [16]. GSTo has interesting features compared with the other types of GSTs. GSTo has distinct enzymatic properties, e.g. GSH-dependent thioltransferase and dehydroascorbate reductase activity (DHAR), which might be attributable to its structural similarity to glutaredoxin [15]. GSTo shows high affinity toward and spp. and free-living [13, 20C22]. GSTo (induces resistance to oxidative stress [23]. Transgenic that overexpress GSTo (GSTO-1) exhibits increased resistance during oxidative injuries [24]. In our previous study involving proteome analysis of GSTs, we observed that CsGSTos were inducible during stimulation of the worm with bile juice [9]. This result prompted us to further characterize biochemical features and biological functions relevant to the CsGSTos in response to oxidative stress. In this study, we characterized biochemical properties of two species of GSTos. We demonstrated that expression profiles of the CsGSTos were spatiotemporally regulated in accordance with the maturation of the worms reproductive system. We subsequently investigated possible biological protective roles of CsGSTos in these organs under oxidative stressful conditions. Methods Parasites metacercariae were collected from naturally infected freshwater fish (omega-class GSTs Expressed sequence tags (ESTs) were constructed through a screening of randomly selected clones of an adult.