Deciphering the origin of Met-enkephalin and Leu-enkephalin in Lobe-finned fish: cloning of Australian lungfish proenkephalin

Abstract

The previous detection of Met-enkephalin and Leu-enkephalin in the CNS of the Australian lungfish, Neoceratodus forsteri, in a molar ratio comparable to mammals suggested that the lungfish proenkephalin precursor should contain the sequences of both Met-enkephalin and Leu-enkephalin as seen for mammalian proenkephalin. However, the cloning of a full-length proenkephalin cDNA from the CNS of the Australian lungfish indicates that the organization of this precursor is more similar to amphibian proenkephalin than mammalian proenkephalin. The Australian lungfish cDNA is 1284 nucleotides in length and the open reading frame (267 amino acids) contains seven opioid sequences (GenBank #AF232671). There are five copies of the Met-enkephalin sequence flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended forms of Met-enkephalin: YGGFMRSL and YGGFMGY. As seen for amphibians, no Leu-enkephalin sequence was detected in the Australian lungfish proenkephalin cDNA. The fact that Leu-enkephalin has been identified by radioimmunoassay and HPLC analysis in the CNS of the Australian lungfish indicates that a Leu-enkephalin-coding gene, distinct from proenkephalin, must be expressed in lungfish. Potential candidates may include a prodynorphin- or other opioid-like gene. Furthermore, the absence of a Leu-enkephalin sequence in lungfish and amphibian proenkephalin would suggest that the mutations that yielded this opioid sequence in tetrapod proenkephalin occurred at some point in the radiation of the amniote vertebrates.