Purification and partial charcterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus

UMR 1112 INRA-UNSA, Laboratoire Réponse des Organismes aux Stress Environnementaux, Faculté des Sciences, Université de Nice-Sophia Antipolis, Nice, France.
European Journal of Biochemistry (Impact Factor: 3.58). 10/2002; 269(17):4359-66. DOI: 10.1046/j.1432-1033.2002.03141.x
Source: PubMed


This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, anti-mu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/m micro /pi GST from Fasciola hepatica.

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    • "They have been identified and characterized in such diverse species as Haliotis discus discus (Wan et al., 2008), Hyphantria cunea (Yamamoto et al., 2007), Anopheles gambiae (Ranson et al., 2001), Xenopus laevis (Carletti et al., 2003), and white leghorn chicks (Hsieh et al., 1999). In addition, a number of GSTs have been purified and biochemically characterized (Tomarev et al., 1993; Fitzpatrick et al., 1995; Hoarau et al., 2002; Yang et al., 2003). Though plenty of studies have demonstrated the xenobiotic detoxification activity of GSTs, studies on immunomodulatory properties of GST members are scarce. "
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    ABSTRACT: Glutathione S-transferases (GSTs) are versatile enzymes, act as primary intracellular detoxifiers and contribute to a broad range of physiological processes including cellular defense. In this study, a full-length cDNA representing a novel sigma-like GST was identified from Manila clam, Ruditapes philippinarum (RpGSTσ). RpGSTσ (884 bp) was found to possess an open reading frame of 609 bp. The encoded polypeptide (203 amino acids) had a predicted molecular mass of 23.21 kDa and an isoelectric point of 7.64. Sequence analysis revealed two conserved GST domain profiles in N- and C-termini. Alignment studies revealed that the identity between deduced peptides of RpGSTσ and known GSTσ members was relatively low (<35%), except a previously identified Manila clam GSTσ isoform (87.2%). Phylogenetic analysis indicated that RpGSTσ clustered together with molluscan GSTσ homologs, which were closely related to insect GSTσs. The RpGSTσ was subsequently cloned and expressed as recombinant protein, in order to characterize its biological activity. The recombinant RpGSTσ exhibited characteristic glutathione conjugating catalytic activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloronitrobenzene and ethacrynic acid. It had an optimal pH and temperature of 8.0 and 35 °C, respectively. Expression profiles under normal conditions and in response to lipopolysaccharide-, poly I:C- and Vibrio tapetis-challenges were also investigated. RpGSTσ demonstrated a differential tissue distribution with robust transcription in gills of normal animals. We explored potential association of GSTσ in cellular defense during bacterial infection and found that in challenged clams, RpGSTσ gene was significantly induced in internal and external tissues, in conjunction with manganese- as well as copper-zinc superoxide dismutase (MnSOD and CuZnSOD) genes. Moreover, the induction was remarkably higher in hemocytes than in gill. Collectively, our findings suggested that RpGSTσ could play a significant role in cellular defense against oxidative stress caused by bacteria, in conjunction with other antioxidant enzymes, such as SODs.
    Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 01/2012; 155(4):539-50. DOI:10.1016/j.cbpc.2012.01.001 · 2.30 Impact Factor
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    • "The antigen could differentiate between the F. gigantica positive and negative sera, as the antigen showed positive reactivity with animals infected with F. gigantica. Helminth mu-class GSTs, such as those seen in F. hepatica, are essentially internal, cytosolic proteins (Pemble et al., 1996; Hoarau et al., 2002). The release of GST into the host for antibody stimulation is most likely via tegumental shedding or degradation following death. "
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    ABSTRACT: Glutathione S-transferase from an Indian isolate of Fasciola gigantica of buffalo origin was isolated and characterized. Total RNA was transcribed to cDNA by reverse transcription and an amplicon of 657 bp glutathione S-transferase gene was obtained by polymerase chain reaction (PCR). The present isolate showed 99.1% sequence homology with the published sequence of the F. gigantica GST gene of cattle origin, with six nucleotide changes causing an overall change of four amino acids. Glutathione S-transferase protein was expressed in Escherichia coli using a prokaryotic expression vector pPROEXHTb. The recombinant protein was purified under non-denaturing and denaturing conditions by nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. Recombinant GST protein detected F. gigantica infection in naturally infected buffaloes by dot-ELISA.
    Journal of Helminthology 08/2009; 84(1):55-60. DOI:10.1017/S0022149X09990046 · 1.42 Impact Factor
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    • "However, information about marine invertebrate GSTs is quite scanty (Contreras-Vergara et al., 2004). A few of the aquatic invertebrates in which GST has been studied include, bivalve mollusks (Atactodea striata) (Yang et al., 2003), gastropods (Bulinus truncates) (Abdalla et al., 2006), marine shrimp (Litopenaeus vannamei) (Contreras-Vergara et al., 2004), crayfish (Macrobrachium vollenhovenii) (Adewale and Afolayan, 2005), clams (Raditapes decussates) (Hoarau et al., 2002), squids (Ommastrephes sloani pacificus) (Tomarev et al., 1993), octopi (Octopus vulgaris) (Chiou et al., 1995), and blue mussels (Mytilus edulis) (Fitzpatrick et al., 1995). Most of these studies focused on purification and/or biochemical measurement of total GST or different GST isoforms using in vivo organisms. "
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    ABSTRACT: We cloned and sequenced the full-length cDNA of a theta class glutathione S-transferase (GST-T) from the polychaete Neanthes succinea. The open reading frame of N. succinea GST-T cDNA was 678bp and encoded 226 amino acid residues. We generated recombinant N. succinea GST-T by expression in transformed Escherichia coli and studied the kinetic properties as well as the effects of inhibitors, pH, and temperature on N. succinea GST-T. GST-T expression was studied using real-time RT-PCR in response to exposure to the model oxidative stress-inducing agent, CuCl(2). Copper induced a concentration-dependant increase in the expression of GST-T. Moreover, polychaetes collected from a heavily contaminated lake near an industrial complex showed significantly higher levels of GST-T expression. Interestingly, the site-collected polychaetes with the highest GST-T mRNA expression levels also showed the highest metallothioneins levels. These results suggest that GST-T in polychaetes may have an antioxidant role and that N. succinea GST-T expression may be a useful biomarker for exposure to environmental contaminants such as copper. Our findings provide a better understanding of the biochemical characteristics of N. succinea GST-T, and elucidate the potential role of GST-T in heavy metal-induced oxidative stress and as a biomarker for environmental contamination.
    Aquatic Toxicology 07/2007; 83(2):104-15. DOI:10.1016/j.aquatox.2007.03.015 · 3.45 Impact Factor
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