Production, gene structure and characterization of two orthologs of leptin and a leptin receptor in tilapia Michal Shpilman a , Lian Hollander-Cohen a , Tomer Ventura b , Arieh Gertler c , Berta Levavi-Sivan a,⇑ a Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel b Faculty of Science, Health, Education and Engineering, GeneCology Research Centre, University of the Sunshine Coast, Queensland, Australia c Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel article info Article history: Available online 20 May 2014 Keywords: RNA-seq Synteny Recombinant leptin STAT3 abstract Full-length cDNA encoding two leptin sequences (tLepA and tLepB) and one leptin receptor sequence (tLepR) were identified in tilapia (Oreochromis niloticus). The full-length cDNA of tLepR was 3423 bp, encoding a protein of 1140 amino acid (aa) which contained all functionally important domains con- served among vertebrate leptin receptors. The cDNAs of tLepA and tLepB were 486 bp and 459 bp in length, encoding proteins of 161 aa and 152 aa, respectively. Modeling the three-dimensional structures of tLepA and tLepB predicted strong conservation of tertiary structure with that of human leptin, com- prised of four helixes. Using synteny, the tLeps were found near common genes, such as IMPDH1 and LLRC4. The cDNA for tLepA and tLepB was cloned and synthetic cDNA optimized for expression in Esch- erichia coli was prepared according to the cloned sequence. The tLepA- and tLepB-expressing plasmids were transformed into E. coli and expressed as recombinant proteins upon induction with nalidixic acid, found almost entirely in insoluble inclusion bodies (IBs). The proteins were solubilized, refolded and puri- fied to homogeneity by anion-exchange chromatography. In the case of tLepA, the fraction eluted con- tained a mixture of monomers and dimers. The purified tLepA and tLepB monomers and tLepA dimer showed a single band of 15 kDa on an SDS–polyacrylamide gel in the presence of reducing agent, whereas the tLepA dimer showed one band of 30 kDa in the absence of reducing agent, indicating its formation by S–S bonds. The three tLeps were biologically active in promoting proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor (hLepR), but their activity was four orders of magnitude lower than that of mammalian leptin. Furthermore, the three tLeps were biologically active in promoting STAT-LUC activation in COS7 cells transfected with the identified tLepR but not in cells transfected with hLepR. tLepA was more active than tLepB. Low or no activity likely resulted from low identity (9–22%) to mammalian leptins. In an in vivo experiment in which tilapia were fed ad libitum or fasted, there was no significant difference in the expressions of tLepA, tLepB or tLepR in the brain between the two groups examined both by real-time PCR and RNA next generation sequencing. In con- clusion, in the present report we show novel, previously unknown sequences of tilapia leptin receptor and two leptins and prepare two biologically active recombinant leptin proteins. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Leptin, a 16-kDa hormonal product of the obese (ob) gene, is a member of the class-I a-helical cytokines (Huising et al., 2006) secreted primarily by adipose tissue (Zhang et al., 1994). Leptin is a pleiotropic hormone that acts both centrally and peripherally. In mammals, it is known as an important hormone in the regula- tion of food intake and energy metabolism (Schwartz et al., 2000), reproductive function (Chehab et al., 1997; Ashworth et al., 2000) and immune responses (Gainsford et al., 1996; Peelman et al., 2004). However, it also has negative actions, such as enhancement of undesired immune responses in autoimmune diseases (Peelman et al., 2004), tumorigenesis (Gonzalez et al., 2006) and elevated blood pressure (Beltowski, 2006). In contrast, information regarding the role of leptin in nonmammalian animals, and in particular fish, is scarce and contradictory. On the one hand, researchers have shown that mammalian leptin reduces food intake in goldfish (Carassius auratus) (de Pedro et al., 2006) and striped bass (Morone saxatilis)(Won et al., 2012), and that leptin affects the expression and release of pituitary hormones in the European sea bass (Dicentrarchus labrax) http://dx.doi.org/10.1016/j.ygcen.2014.05.006 0016-6480/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +972 8 948 9307. E-mail address: berta.sivan@mail.huji.ac.il (B. Levavi-Sivan). General and Comparative Endocrinology 207 (2014) 74–85 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen