title: Bioactivity studies on atypical natural opioid hexapeptides processed from proenkephalin (PENK) precursor polypeptides.
abstract: Endogenous opioids are derived from four related polypeptide precursors: proenkephalin (PENK), prodynorphin (PDYN), pronociceptin (PNOC) and proopiomelanocortin (POMC). In mammals PENK encodes for four copy of Met-enkephalin, one octapeptide Met-enkephalin-Arg-Gly-Leu, one heptapeptide Met-enkephalin-Arg-Phe and a single copy of Leu-enkephalin. Our detailed bioinformatic search on the existing PENK sequences revealed several atypical hexapeptide Met-enkephalins in different vertebrate animals. They are located either in the second enkephalin unit or in the seventh enkephalin core position at the C-terminus. Altogether four different hexapeptide sequences were obtained representing eleven animal species: Met-enkephalin-Arg(6) (YGGFMR) in the bird zebra finch, Met-enkephalin-Asp(6) (YGGFMD), Met-enkephalin-Ile(6) (YGGFMI) in zebrafish; and Met-enkephalin-Ser(6) (YGGFMS) in two pufferfish species. All novel peptides were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. The four novel enkephalins were equipotent in stimulating G-proteins. Affinities of the peptides determined by equilibrium competition assays in receptor binding experiments were statistically different. At the MOP receptors the highest affinity (Ki 4nM) was obtained with the zebra finch peptide Met-enkephalin-Arg(6). The pufferfish Met-enkephalin-Ser(6) exhibited the highest affinity (Ki 6.7nM) at the DOP receptor. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library concept is expected.
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, 6726 Szeged, Temesvari krt 62, Hungary.
authors.forename: Engin
authors.initials: E
authors.lastname: Bojnik
authors.affiliation: Mossakowski Medical Research Centre, Polish Academy of Sciences, Department of Neuropeptides, Warsaw, Poland.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Kleczkowska
authors.affiliation: Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Marron Fernandez de Velasco
authors.affiliation: Institue of Functional Genomics, CNRS UMR 5203-INSERM U.661, University of Montpellier, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France.
authors.forename: Maïthé
authors.initials: M
authors.lastname: Corbani
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Lorand University, Budapest, Hungary.
authors.forename: Fruzsina
authors.initials: F
authors.lastname: Babos
authors.affiliation: Mossakowski Medical Research Centre, Polish Academy of Sciences, Department of Neuropeptides, Warsaw, Poland.
authors.forename: Andrzej W
authors.initials: AW
authors.lastname: Lipkowski
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Lorand University, Budapest, Hungary.
authors.forename: Anna
authors.initials: A
authors.lastname: Magyar
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, 6726 Szeged, Temesvari krt 62, Hungary. Electronic address: benyhe@brc.hu.
authors.forename: Sandor
authors.initials: S
authors.lastname: Benyhe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D004745:Enkephalins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D018847:Opioid Peptides / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D017208:Rats, Wistar; D011957:Receptors, Opioid / Q000378:metabolism
chemical_list: D004745:Enkephalins; D018847:Opioid Peptides; D011498:Protein Precursors; D011957:Receptors, Opioid; C029992:proenkephalin
keywords: Enkephalin hexapeptides; Mu and delta opioid receptors; Natural chemical library; Proenkephalin evolution; Species differences