Identification of a fourth opioid core sequence in a prodynorphin cDNA cloned from the brain of the amphibian, Bufo marinus: deciphering the evolution of prodynorphin and proenkephalin.
In mammals, prodynorphin codes for three C-terminally extended forms of leu-enkephalin. This is not the case for the anuran amphibian, Bufo marinus. A combination of 3'RACE, RT-PCR and 5'RACE protocols was used to clone and characterize a prodynorphin cDNA from the brain of this amphibian that contained two met-enkephalin sequences. One met-enkephalin sequence was located at the N-terminal of Met(5)-dynorphin A(1-17), and the other met-enkephalin sequence was located in the N-terminal region of B. marinus prodynorphin in a position that aligned with a pentapeptide met-enkephalin site in mammalian proenkephalin. The latter B. marinus met-enkephalin sequence is flanked by sets of paired basic proteolytic cleavage sites. In addition to the extra met-enkephalin sequence and the Met(5)-dynorphin A(1-17) sequence, the B. marinus prodynorphin contained two C-terminally extended forms of leu-enkephalin [alpha-neo-endorphin and dynorphin B(1-13)]. In the toad precursor the alpha-neo-endorphin sequence is identical to human alpha-neo-endorphin. The B. marinus dynorphin B(1-13) sequence differs from human dynorphin B(1-13) by one amino acid (Thr(12) vs. Val(12)). Steady-state analysis suggests that dynorphin B(1-13) and possibly alpha-neo-endorphin may be cleaved to yield leu-enkephalin as an end-product in the amphibian brain. Finally, the alignment of the extra met-enkephalin sequence in the N-terminal of B. marinus prodynorphin with the corresponding met-enkephalin site in mammalian proenkephalin adds support to the hypothesis that the prodynorphin gene arose as a duplication of the proenkephalin gene.
Danielson
Phillip
P
Department of Biological Sciences, University of Denver, Denver, CO 80210, USA.
Walker
David
D
Alrubaian
Jasem
J
Dores
Robert M
RM
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002024:Bufo marinus / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D004723:Endorphins / Q000737:chemistry / Q000235:genetics; D004745:Enkephalins / Q000737:chemistry / Q000235:genetics*; D019143:Evolution, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D018076:DNA, Complementary; D004723:Endorphins; D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; C025681:alpha-neoendorphin; C033660:preproenkephalin