Bioactivity studies on atypical natural opioid hexapeptides processed from proenkephalin (PENK) precursor polypeptides

Engin Bojnik; Patrycja Kleczkowska; Ezequiel Marron Fernandez de Velasco; Maïthé Corbani; Fruzsina Babos; Andrzej W Lipkowski; Anna Magyar; Sandor Benyhe

doi:10.1016/j.cbpb.2014.06.002

Bioactivity studies on atypical natural opioid hexapeptides processed from proenkephalin (PENK) precursor polypeptides

Comp Biochem Physiol B Biochem Mol Biol. 2014 Aug:174:29-35. doi: 10.1016/j.cbpb.2014.06.002. Epub 2014 Jun 16.

Authors

Engin Bojnik¹, Patrycja Kleczkowska², Ezequiel Marron Fernandez de Velasco³, Maïthé Corbani⁴, Fruzsina Babos⁵, Andrzej W Lipkowski², Anna Magyar⁵, Sandor Benyhe⁶

Affiliations

¹ Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, 6726 Szeged, Temesvari krt 62, Hungary.
² Mossakowski Medical Research Centre, Polish Academy of Sciences, Department of Neuropeptides, Warsaw, Poland.
³ Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA.
⁴ Institue of Functional Genomics, CNRS UMR 5203-INSERM U.661, University of Montpellier, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France.
⁵ MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Lorand University, Budapest, Hungary.
⁶ Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, 6726 Szeged, Temesvari krt 62, Hungary. Electronic address: benyhe@brc.hu.

PMID: 24947211
DOI: 10.1016/j.cbpb.2014.06.002

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.

Keywords: Enkephalin hexapeptides; Mu and delta opioid receptors; Natural chemical library; Proenkephalin evolution; Species differences.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Animals
Brain / metabolism
Enkephalins / chemistry
Enkephalins / metabolism*
Molecular Sequence Data
Opioid Peptides / chemistry
Opioid Peptides / metabolism*
Phylogeny
Protein Precursors / chemistry
Protein Precursors / metabolism*
Rats, Wistar
Receptors, Opioid / metabolism

Substances

Enkephalins
Opioid Peptides
Protein Precursors
Receptors, Opioid
proenkephalin