Original contributionA stress-responsive glutathione S-transferase confers resistance to oxidative stress in Caenorhabditis elegans
Introduction
The glutathione S-transferases (GSTs) are major cellular detoxification enzymes. This multifunctional enzyme family catalyzes the conjugation of various electrophiles with GSH, detoxifiing both exogenously and endogenously derived toxic compounds 2, 3, 4, 5. Organisms usually express multiple GSTs, each with a specialized function, determined by their substrate specificity and localization [5]. Seven species-independent soluble GST classes have been described (Alpha, Mu, Pi, Sigma, Theta, Zeta, and Omega) [6]. Additional species-specific classes have been found in plants, bacteria, and invertebrates 7, 8, 9. The C. elegans genome contains over 50 putative GSTs, based on database sequence comparisons (K. Henkle-Dührsen, unpublished data). Some belong to the known species-independent Alpha, Pi, and Sigma classes, but most are classified as nematode-specific GSTs [10].
One of these nematode-specific GSTs, Ce-GST-p24 (K08F4.7, formerly M-47), was initially identified by differential display RT-PCR experiments aimed at identifying genes that are induced in response to oxidative stress [1]. The upregulation of the Ce-GST-p24 at the steady state mRNA level in response to paraquat was subsequently confirmed by northern blot analysis [1]. Another study demonstrated that the Ce-GST-p24 promoter is induced by hyperbaric oxygen [11]. These observations led us to postulate that the Ce-GST-p24 is likely to deal with oxidatively damaged cellular components. We tested this hypothesis and examined the ability of this GST to protect the nematode against damage due to oxidative stress. Our results have allowed the identification of a single phase II detoxification enzyme—out of the large GST superfamily in C. elegans—capable of enhancing survival under oxidative stress conditions. Additionally, we gained insights into structural and functional characteristics of a member of the invertebrate and nematode-specific class of glutathione S-transferases.
Section snippets
Strains and culture of nematodes
Seven C. elegans strains were used in this study. The wild-type C. elegans strain N2 (var. Bristol) was obtained from the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN, USA). The strain CH1035 (rol-6, su1006) [12], which derives from Jim Kramer’s laboratory, was used as a control for the roller phenotype (which might affect scoring, etc.). CL2070 (dvls70) [13], CL2166 (dvIs19; gst(727 bp):GFP-NLS, integrated, 6x outcrossed), and CL3166 (dvIs20; gst(727 bp):GFP-NLS,
Induction of the Ce-GST-p24 promoter by oxidative stress in transgenic C. elegans
In C. elegans CL2166 and CL3166, the GFP expression is controlled by a 727 bp Ce-GST-p24 promoter region (Fig. 1A). In BL1 C. elegans, a 1000 bp Ce-GST-p24 promoter region controls the expression of the Ce-GST-p24-GFP fusion protein (Fig. 1A). In response to oxidative stress, a significant increase of the promoter activity in the CL2166 and BL1 C. elegans was observed by photometric measurement of the GFP fluorescence (Fig. 1B). In response to 200 μM plumbagin and 100 mM paraquat, the promoter
Discussion
Ce-GST-p24 is a candidate for the protection against oxidative stress in C. elegans [1]. We were interested in the effects of modulated levels of Ce-GST-p24 with respect to the resistance of the worms to oxidative stress. Adult BL1 C. elegans showed a significantly increased resistance to intracellularly generated ROS. The strongest effect was observed in response to the quinone juglone, where BL1 worms showed up to 50% higher resistance (Fig. 2). The BL1 were slightly more resistant than the
Acknowledgements
We are grateful to W. Tawe and O. Bossinger for helpful discussions. We also thank T. E. Volkmann for his technical assistance. The wild-type C. elegans strain was provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources of the National Institutes of Health. We wish to thank the C. elegans Gene Knockout Project team at OMRF for isolating a deletion mutant for K08F4.7 (Ce-GST-p24) and the A. Fire laboratory for providing the vector kits. The
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