Evolution of gnathostome prodynorphin and proenkephalin: Characterization of a shark proenkephalin and prodynorphin cDNAs Leanne K. Komorowski, Stephanie G. Lecaude, Christian G. Westring, Phillip B. Danielson, Robert M. Dores ⇑ University of Denver, Department of Biological Sciences, Denver, CO 80210, USA article info Article history: Available online 22 December 2011 Keywords: Prodynorphin Proenkephalin Shark Evolution abstract Analyses of prodynorphin and proenkephalin cDNAs cloned from the central nervous system of the shark, Heterodontus portusjacksoni, provided additional evidence that these two opioid precursor-coding genes were most likely directly derived from a common ancestral gene. The two cDNAs could be aligned by inserting only seven gaps. The prodynorphin cDNA encodes five opioid sequences which could be aligned to opioid positions B through F in the proenkephalin cDNA. The sequence identity within the opioid positions was 59% at the amino acid level. Shark a-neo-endorphin, dynorphin A, and dynorphin B have amino acid motifs in common with shark met-enkephalin-8, and shark proenkephalin opioid positions E and F, respectively, which have not been observed in other gnathostome prodynorphin and proenkeph- alin precursor sequences. Shark prodynorphin encodes both kappa (a-neo-endorphin, dynorphin A, and dynorphin B) and delta (met-enkephalin and leu-enkephalin) opioid sequences. Mixed function prodynorphin precursors (encoding both enkephalins and dynorphins) are also found in representatives of the teleost fishes, lungfishes, and amphibians. It appears that only mammals evolved a prodynorphin precursor that exclusively encodes kappa opioid agonists (dynorphins). Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Within the opioid networks in the central nervous system of mammals there are distinct enkephalinergic and dynorphinergic neurons [17]. The enkephalins (met-enkephalin, C-terminally extended forms of met-enkephalin, and leu-enkephalin) are de- rived from the proenkephalin gene, whereas, the dynorphin-related ligands (dynorphin A, dynorphin B, and a-neo-endorphin) are derived from the prodynorphin gene [11]. In birds and reptiles, the enkephalins and the dynorphins also appear to be expressed in separate neurons [34]. However, the distinction between ‘‘enkephalinergic’’ and ‘‘dynorphinergic’’ neurons in other mem- bers of Class Sarcopterygii (lungfishes and anamniote tetrapods) is more complex. In amphibians and lungfishes the proenkephalin gene only encodes met-enkephalin-related ligands (for review see [18]), while the prodynorphin gene encodes both dynorphin- related ligands (i.e., a-neo-endorphin, dynorphin A and dynorphin B) and either at least one met-enkephalin sequence [5] or one leu-enkephalin sequence [10,31]. Hence in these organisms, ‘‘dynorphinergic’’ neurons release both kappa opioid agonists (the dynorphins) and delta opioid agonists (the enkephalins) [30]. The prodynorphin gene and the proenkephalin gene are mem- bers, along with the proopiomelanocortin (pomc) gene and the proorphanin gene, of the opioid/orphanin gene family [4,7,9]. There is general agreement that this family of proprotein-coding genes was derived from a proto-opioid-like gene [9] that is predicted to have been present in the genome of an ancestral chordate, and that the gene family proliferated as a result of the two genome duplica- tion events (2R hypothesis) that have occurred in phylum Chordata [15,22,29]. From studies that have been done on the bony jawed vertebrates (i.e., ray-finned fishes, lobe-finned fishes, and tetra- pods) it is clear that the proenkephalin, prodynorphin, pomc, and proorphanin genes are all present in these extant vertebrates (for review see: [39]). Hence, it would be reasonable to propose that at the time of the second chordate genome duplication event, which coincides with the emergence of the jawed vertebrates (sub- phylum Gnathostomata), a proto-enkephalin/dynorphin gene was duplicated to give rise to distinct proenkepahlin and prodynorphin genes. If the proposed timing of this duplication is correct, then it should be possible to identify distinct proenkephalin and prody- norphin genes in the genome of cartilaginous fish that may reveal features of the ancestral proto-enkephalin/dynorphin gene. The extant cartilaginous fish can be grouped into two subclasses, the Elasmobranchii and the Holocephali [27]. The Elasmobranchii can be further separated into three extant orders: Batoidea (skates and rays), Squalomorpha (squaloid sharks), and Galeomorpha 0016-6480/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2011.12.016 ⇑ Corresponding author. E-mail address: rdores@du.edu (R.M. Dores). General and Comparative Endocrinology 177 (2012) 353–364 Contents lists available at SciVerse ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen