Elsevier

Aquaculture

Volumes 324–325, 12 January 2012, Pages 182-193
Aquaculture

Molecular cloning of glutathione peroxidase cDNAs from Seriola lalandi and analysis of changes in expression in cultured fibroblast-like cells in response to tert-butyl hydroquinone

https://doi.org/10.1016/j.aquaculture.2011.10.027Get rights and content

Abstract

Glutathione peroxidases (GPxs) protect cells against oxidative damage by catalyzing the reduction of hydroperoxides. In mammals, GPx1 is expressed in most tissues and has a wide substrate range, while GPx4 plays an important role in the reduction of complex lipid hydroperoxides in cell membranes and has been shown to be important for male fertility. These enzymes have not been well studied in marine teleosts, which accumulate large quantities of polyunsaturated fatty acids that are prone to oxidation. To better understand GPx expression and regulation in marine teleosts, we have cloned and characterized GPx cDNAs from yellowtail kingfish (YTK; Seriola lalandi) and investigated changes in gene expression in response to tert-butyl hydroquinone (t-BHQ), a redox-cycling metabolite of the common aquaculture feed additive, butylated hydroxyanisole. The putative GPx1 from YTK displayed a high degree of homology with GPx1 sequences from other vertebrates. Two putative GPx4 transcript variants were obtained from YTK liver. The GPx4 variants differed only in the amino terminal region of the predicted amino acid sequences, suggesting that the variants may be encoded from a single gene with alternative first exons. Computational analysis indicated a high probability of YTK GPx4 secretion. In cultured YTK fibroblast-like cells, t-BHQ treatment significantly induced the expression of GPx1 but not GPx4. The expression of glutamyl cysteine ligase, catalytic subunit (GCLC) was also significantly induced by t-BHQ, while the expression of the antioxidant enzymes peroxiredoxin 1 (Prx1) and Prx4 remained unchanged. The induction of GPx1 and GCLC by t-BHQ suggests that the conserved redox-sensing gene regulatory pathway known as the Keap1/Nrf2/ARE axis may be involved in regulating the expression of these genes in YTK. This work contributes towards a better understanding of the regulation of glutathione peroxidases in marine fish.

Introduction

The glutathione peroxidases (GPxs; EC 1.11.1.9) are a family of antioxidant enzymes that catalyze the reduction of hydrogen peroxide to water and organic hydroperoxides to the corresponding alcohols. In addition to the canonical glutathione-dependent, selenocysteine- (Sec-) containing GPxs, the discovery of a diverse array of closely related proteins across all orders of life has expanded the family to include forms with cysteine in the active site in place of Sec, as well as forms able to use thioredoxin as the reducing substrate (Maiorino et al., 2007, Toppo et al., 2008, Toppo et al., 2009). In mammals, there are four Sec-containing GPxs that differ in their subcellular localization, tissue distribution, and peroxide substrate preference (reviewed recently by Toppo et al., 2009). Glutathione peroxidase 1 (GPx1) is a cytosolic enzyme that displays a preference for hydrogen peroxide, but can efficiently reduce organic hydroperoxides and, less efficiently, free lipid hydroperoxides; it is expressed at relatively high levels in most tissues and cells, particularly liver, kidney, lungs and erythrocytes (Brigelius-Flohé, 1999, Mills, 1957). GPx2 is abundant in cells of the gastrointestinal epithelium, but is also inducible in lung cells (Singh et al., 2006); substrate specificity has not been determined conclusively, but gene knockout work in mice indicates that GPx2 at least partially compensates for the loss of GPx1 (Florian et al., 2010) suggesting a similar substrate range. GPx3 is a secreted form, with high levels present in plasma; like GPx1, GPx3 reduces hydrogen peroxide very efficiently, with respective kinetic rate constants for small organic hydroperoxides and complex lipid hydroperoxides about ten- and a hundred-fold less than that observed for hydrogen peroxide (Takebe et al., 2002). While GPx4 is also capable of reducing hydrogen peroxide and small organic hydroperoxides, it is unique among the mammalian GPx enzymes in its ability to efficiently reduce complex lipid hydroperoxides, including phospholipid hydroperoxides in cell membranes and intact liposomes (Ursini et al., 1982, Ursini et al., 1985); in mammals, GPx4 is expressed as three isoforms that localize to the cytosol, mitochondria and nucleus (Borchert et al., 2003, Imai et al., 2006, Maiorino et al., 2003) — the cytosolic form is present in most tissues at low levels and is essential for the survival of somatic cells from early embryo development (Liang et al., 2009, Schneider et al., 2009). Very high levels of mitochondrial GPx4 are expressed in the testes and sperm — this isoform has a possible structural role in the mitochondria of mature sperm (Ursini et al., 1999), with recent work confirming a vital role in male fertility in rodents (Liang et al., 2009, Schneider et al., 2009).

GPx tissue distribution, substrate preference, gene structure and gene regulation have not been well studied in fish, although a considerable number of piscine GPx cDNA sequences have been deposited in public databases. Paralogs of mammalian GPx1 and GPx4 appear to be present in all species of teleost fish studied so far. Two putative GPx4 cDNAs, encoded by separate genes, are expressed in zebrafish (Thisse et al., 2003) and carp (Hermesz and Ferencz, 2009). Recently, we reported the cloning and partial characterization of GPx1 and GPx4 cDNAs from the large marine species Thunnus maccoyii (southern bluefin tuna; SBT) (Thompson et al., 2010). As has been reported for goldfish GPx1 (Choi et al., 2007), and carp GPx4 (Hermesz and Ferencz, 2009), tissue distribution of mRNA for both genes was widespread in SBT, suggesting that in fish, as in mammals, GPx1 and GPx4 are expressed to some degree in most tissues. Previous work in our laboratory has also shown that GPx enzyme activity is present at high levels in liver and plasma, and at moderate levels in muscle tissue (Thompson et al., 2006). Presumably, as in mammals, GPx1 and GPx4 contribute to the control of physiological hydroperoxide levels in fish. Whether an additional role for GPx4 in male fertility exists in fish has yet to be determined. Knowledge of the mechanisms of gene regulation of GPx enzymes will contribute towards the elucidation of the physiological roles for these enzymes in fish.

To prevent the oxidative deterioration of long chain-polyunsaturated fatty acids (LC-PUFA) in manufactured feeds, synthetic antioxidant additives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are frequently used. In rodents, BHA is known to be metabolized to tert-butyl hydroquinone (t-BHQ) (Hirose et al., 1988), a redox-cycling phenolic antioxidant that displays pro-oxidative effects under certain conditions in vivo (Dinkova-Kostova and Wang, 2011). BHA, BHT and t-BHQ have long been known to alter the activity of liver detoxifying enzymes in rainbow trout (Eisele et al., 1983), and t-BHQ is a model inducer of antioxidant gene expression in mammals (Dinkova-Kostova and Wang, 2011).

Here we report the isolation of putative GPx1 and GPx4 cDNAs from the pelagic marine species Seriola lalandi (yellowtail kingfish; YTK), and the analysis of changes in the expression of these cDNAs in cultured fibroblast-like cells in response to t-BHQ.

Section snippets

Molecular cloning of putative GPx1 and GPx4 cDNAs from YTK liver

Full-length sequences for putative GPx1 and GPx4 cDNAs were obtained from YTK liver using rapid amplification of cDNA ends (RACE). Total RNA was prepared using the RNeasy® kit (QIAGEN) and RACE-ready cDNA was synthesized from 3 μg total RNA using the SMARTTM RACE cDNA Amplification Kit (Clontech/Takara Bio U.S.A.) according to the manufacturer's instructions, except that an RNase H-minus MMLV reverse transcriptase (Promega) was used in place of the reverse transcriptase supplied with the kit.

Cloning of and sequence analysis of GPx cDNAs from S.lalandi

A full-length putative GPx1 cDNA was obtained from YTK liver RNA using a combination of 3′- and 5′-RACE (GenBank accession no. JF914946.1). The 1006 base-pair (bp) cDNA (not including the poly-A tail) contained a predicted coding region of 567 bp, with a Sec codon (TGA) located 108 nucleotides from the start codon. The 3′ untranslated region (UTR) contained a predicted Sec insertion sequence (SECIS) element similar to that found in zebrafish GPx1a (Fig. 1). SECIS elements from zebrafish and

Discussion

The GPx family of antioxidant enzymes plays an important role in the detoxification of peroxides and are present in all known forms of life. This study describes two GPx enzymes isolated from the marine teleost, YTK. The analysis of the phylogeny of YTK GPx1 and GPx4 is in general agreement with previous studies, which propose a common ancestor for GPx1, 2 and 3, and a different progenitor for GPx4 (Toppo et al., 2008). This supports the notion that fish GPx4 enzymes, like mammalian GPx4, are

Acknowledgements

We are grateful to Dr. D.A.J. Stone for access to juvenile yellowtail kingfish specimens and to J. Bowyer and D. Fisher for assistance with sampling. N. Rout-Pitt designed the Prx4 primers used for quantitative PCR in the present study. This work was supported by a grant from the Fisheries Research and Development Corporation, Australia.

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    School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide 5001, Australia. Tel.: + 61 8 8201 5182; fax: + 61 8 8201 3015.

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