Initial processing of human proenkephalin in bovine chromaffin cells.
The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide, PPE undergoes processing at dibasic amino acid sites to yield its final opioid products-Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.
Liu
F
F
Department of Pharmacology, University of South Carolina School of Medicine, Columbia 29208, USA.
Housley
P R
PR
Wilson
S P
SP
D000302:Adrenal Cortex / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004745:Enkephalins / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D004745:Enkephalins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C029992:proenkephalin; C033660:preproenkephalin