Characterization and sequence analysis of manganese superoxide dismutases from Brachyura (Crustacea: Decapoda): Hydrothermal Bythograeidae versus littoral crabs J. Marchand ⁎, V. Leignel, B. Moreau, B. Chénais EA 2160 Mer, Molécule, Santé Laboratoire de Biologie moléculaire et Génétique Evolutive, Université du Maine, UFR Sciences et Techniques, Avenue Olivier Messiaen, 72085 Le Mans Cedex 9, France abstract article info Article history: Received 17 October 2008 Received in revised form 26 February 2009 Accepted 28 February 2009 Available online 13 March 2009 Keywords: Bythograeidae Brachyura cDNA Characterization Coastal Hydrothermal Manganese superoxide dismutase mRNA expression Real-time PCR Hydrothermal vent conditions are particular and organisms living in these environments may have developed detoxification mechanisms and/or genetic adaptations. In particular, physico-chemical conditions are thought to generate reactive oxygen species, highly toxic for organisms. The enzyme superoxide dismutase constitutes the first line of defense against oxidative damage. To improve our understanding of the environmental impacts exerted on the vent organisms, we have characterized the two manganese superoxide dismutase cDNAs (mitochondrial: mMnSOD and cytoplasmic: cMnSOD) of three members of the Bythograeidae (Bythograea thermydron, Cyanagraea praedator and Segonzacia mesatlantica), the only endemic crab family living in hydrothermal vents. In comparison, the isolation of manganese superoxide dismutase cDNAs was also carried out in several littoral crab families. MnSOD signatures were found in both sequences from each species studied, as well as different residues involved in metal coordination and protein activity. The phylogenetic analysis performed confirms the probable ancient duplication that gave rise to the two MnSODs (cMnSOD and mMnSOD). This study describes two potential distinct mMnSOD isoforms presenting particular peptide signals. Nevertheless, no sequence particularity that could support the hypothesis of a genetic adaptation was found in Bythograeidae's MnSODs compared to the other sequences. The mRNA expression analysis performed by real-time PCR on B. thermydron and S. mesatlantica compared to Cancer pagurus and Necora puber revealed a higher cMnSOD and mMnSOD mRNA expression in hydrothermal crabs compared to littoral crabs. © 2009 Elsevier Inc. All rights reserved. 1. Introduction Since their discovery in 1977 (Corliss et al., 1979), populations of invertebrates from hydrothermal vents have attracted attention due to their capacity to live in one of the most particular environments on earth. In addition to the elevated hydrostatic pressure and the complete absence of sunlight, this environment is mainly characterized by high variations of temperature, low pH, and high levels of potentially toxic compounds such as hydrogen sulphide and heavy metals that would be lethal to non-vent marine species (Blum and Fridovich, 1984; Geret et al., 1998; Desbruyères et al., 2001). Blum and Fridovich (1984) suggested that several toxic compounds of the fluid, in contact with the oxygen of deep-sea water, could produce reactive oxygen species (ROS) such as superoxide anions (O 2 S - ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH S ), highly toxic for organisms. Metals in particular are known to enhance the production of ROS (Halliwell and Gutteridge, 1985; Stohs and Bagchi, 1995; Fridovich, 1998). The abundance of hydrogen sulphide (H 2 S) and O 2 near vents led to the oxidation of H 2 S in seawater and the production of both oxygen- and sulphur-centered radicals (Fridovich, 1998; Tapley et al., 1999). The occurrence of radionuclides (De Oliveira et al., 2001), high temperatures (Issels et al., 1986) and periodic alternants of oxic and anoxic conditions found in hydrothermal vents are also stimuli for the production of ROS (Abele-Oeschger et al., 1994; Warner et al., 2004; Buja, 2005). Cells contain a variety of antioxidant enzymes (superoxide dis- mutase, catalase, glutathione peroxidase, glutathione reductase, glutar- eodoxin, thioredoxin-reductase…) that limit reactions brought about by endogenous and exogenous activated oxygen species and thus are important protective mechanism to minimize cell oxidative damage produced (Winston and Di Giulio, 1991; Lemaire and Livingstone, 1993; Livingstone, 2001). The enzyme superoxide dismutase (SOD) constitu- tes the first line of defense against oxidative damage by catalyzing the dismutation of superoxide to give oxygen and hydrogen peroxide (Fridovich, 1989). Hydrogen peroxide is then transformed to water and oxygen by catalase, resulting in innocuous compounds to the cell (Bendich, 1993). SODs require metal cofactors for catalysis, and currently, six types are known: copper/zinc SOD (CuZnSOD) (Halliwell and Gutteridge, 1985), nickel SOD (Youn et al., 1996), manganese/iron SOD (Amo et al., 2003), iron/zinc SOD (Kim et al., 1996), iron SOD and manganese SOD (MnSOD) (Bowler et al., 1992). Commonly, animal cells have two SODs: a cytoplasmic CuZnSOD and a mitochondrial MnSOD. The tetrameric MnSOD plays an essential role in oxidative stress protection as it is located in mitochondria, one of the major sources of Comparative Biochemistry and Physiology, Part B 153 (2009) 191–199 ⁎ Corresponding author. Tel.: +33 2 43 83 32 50; fax: +33 2 43 83 37 95. E-mail address: justine.marchand@univ-lemans.fr (J. Marchand). 1096-4959/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2009.02.019 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb