Processing site blockade results in more efficient conversion of proenkephalin to active opioid peptides.
Prohormones are known to be processed at various cleavage sites in a defined temporal order, suggesting the possibility of sequential unfolding of processing sites. In order to investigate whether sequential processing at predefined sites is in fact required for proper processing, site-directed mutagenesis was performed to block known initial cleavage sites within proenkephalin. Pulse-chase/immunoprecipitation experiments were employed to analyze the fate of mutant and native proenkephalins in stably transfected AtT-20 cells. While processing did not occur at blockaded sites, surprisingly, overall processing of mutant proenkephalins proceeded efficiently, and alternative sites were chosen. When compared with native proenkephalin, processing of mutant proenkephalins occurred more slowly at early stages and more quickly at later stages. Experiments employing endoglycosidase H indicated that the early slow processing of mutant proenkephalins may be due to delays in intracellular transport. Metabolic labeling studies showed that more efficient production of bioactive opioids occurred in all processing site blockade mutants examined; these results were confirmed using several different radioimmunoassays of stored peptide products. We conclude that efficient processing of prohormone precursors does not require a specific temporal order of processing events. The fact that mutant proenkephalins were more fully processed than native proenkephalin may provide a route for more efficient production of opioid peptides in applications for chronic pain treatment.
Johanning
K
K
Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine, New Orleans, Louisiana 70112, USA. ilindb@sumc.edu
Mathis
J P
JP
Lindberg
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004744:Enkephalin, Methionine / Q000096:biosynthesis / Q000302:isolation & purification; D004745:Enkephalins / Q000096:biosynthesis / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010911:Pituitary Neoplasms; D017354:Point Mutation; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D004745:Enkephalins; D011498:Protein Precursors; D011994:Recombinant Proteins; C029992:proenkephalin; D004744:Enkephalin, Methionine; C033660:preproenkephalin