Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues ☆ S. Pierre, S. Coupé, N. Prévot-d'Alvise, S. Gaillard, S. Richard, E. Gouze, J. Aubert, J.P. Grillasca ⁎ Équipe de Biologie Moléculaire Marine—PROTEE, Université du Sud Toulon-Var, BP 20132, Avenue de l'Université, 83957 La Garde Cedex, France abstract article info Article history: Received 21 July 2009 Received in revised form 6 January 2010 Accepted 7 January 2010 Available online 18 January 2010 Keywords: Cloning Dicentrarchus labrax Sparus aurata Expression RACE RNA Wap65 Warm Temperature Acclimation-related 65 kDa Protein The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis. © 2010 Elsevier Inc. All rights reserved. 1. Introduction Water temperature is one of the most important environmental factors that directly affect ectotherm fishes. The physiological responses of fishes to seasonal temperature changes, that span over weeks to months, are well known (Hazel and Prosser, 1974), and are to be distinguished from short term adjustment in which heat shock proteins are mainly involved (Kikuchi et al., 1995). Acclimation response is most significant in eurythermal fishes, such as goldfish and carp, which are able to live over a wide range of temperatures, from near zero to over 30 °C (Kinoshita et al., 2001). Long term temperature variations, from warm to cold or reversely, trigger many physiological responses that are now being studied at both cellular and molecular levels (Lemoine et al., 2008; Käkelä et al., 2008; Sardella et al., 2008; Castilho et al., 2009; Fangue et al., 2009; Mitrovic and Perry, 2009; Shi et al., 2010). Such physiological responses likely tend to maintain homeostasis and keep an efficient and optimal behaviour in new environmental conditions. Among the physiological responses consecutive to sustained warm temperature acclimation, the synthesis of WAP65 protein has been clearly demonstrated. WAP65 protein was originally found in a warm acclimated goldfish (Watabe et al., 1993), and further characterized in various tissues of goldfish and carp. This cytosolic protein is named WAP65 for “Warm temperature Acclimation-related 65 kDa Protein”. Specific immunoblotting assays performed on goldfish muscle tissues showed that Wap65 could not be detected in crude extracts of muscle tissues of 10 °C- or 20 °C-acclimated goldfishes, but was highly detectable within 5 days and for at least 9 days in muscle tissues of warm acclimated goldfishes (Kikuchi et al., 1995). These results confirmed that Wap65 was specifically involved in warm acclimation physiological responses. To date, the structure–function relationship of Wap65 is not clearly defined, and its exact role in the mechanism of acclimation to high temperatures remains unclear. First, it is important to note that the amino acid sequence analysis of WAP65 revealed that the 10 first N-terminal amino acids are different from the sequence of HSP70, suggesting that WAP65 and HSP have different functions and/or pathways in response to water temperature raise (Kikuchi et al., 1995). Also, WAP65 interestingly presents about 30% identity at the protein level with the primary structure of the hemopexin (HPX) of mammals that is involved in the transport of heme to the liver, which led some authors consider WAP65 as a counterpart of mammal hemopexins (Delanghe and Langlois, 2001; Tolosano and Altruda, 2002 Aliza et al., 2008). In fishes, WAP65 may also have other central functions such as the modulation of the immune system (Picard and Schulte, 2006). It is Comparative Biochemistry and Physiology, Part B 155 (2010) 396–402 ☆ Sequence data cited in this article have been deposited at GenBank under accession numbers EF136379 for Dicentrarchus labrax and FJ664124 for Sparus aurata. ⁎ Corresponding author. Tel.: + 33 4 94142401; fax: + 33 4 94142045. E-mail address: grillasca@univ-tln.fr (J.P. Grillasca). URL: http://eb2m.univ-tln.fr (J.P. Grillasca). 1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2010.01.002 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb