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

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


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-Arg6 (YGGFMR) in the bird zebra finch, Met-enkephalin-Asp6 (YGGFMD), Met-enkephalin-Ile6 (YGGFMI) in zebrafish; and Met-enkephalin-Ser6 (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-Arg6. The pufferfish Met-enkephalin-Ser6 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.

Original languageEnglish (US)
Pages (from-to)29-35
Number of pages7
JournalComparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology
Issue number1
StatePublished - Aug 2014

Bibliographical note

Funding Information:
This work was supported by TÁMOP — 4.2.2A-11/1KONV-2012-0024 , 2012-052 and OTKA 108518 grants.


  • Enkephalin hexapeptides
  • Mu and delta opioid receptors
  • Natural chemical library
  • Proenkephalin evolution
  • Species differences


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