Production of bioactive peptides in an in vitro system.
An in vitro system for the preparation of bioactive peptides is described. This system couples three different posttranslational modification enzymes, prohormone convertases (PCs), carboxypeptidase E, and peptidyl alpha-amidating enzyme, to transform recombinant precursors into bioactive peptides. Three different precursors, mouse proopiomelanocortin (mPOMC), rat proenkephalin (rPE), and human proghrelin, were used as model systems. The conversion of mPOMC and rPE to smaller peptide products was measured by radioimmunoassay. After optimization of the system, excellent efficiency was obtained: about 85% of starting mPOMC was converted to des-acetyl alpha-melanocyte-stimulating hormone (alpha-MSH). For proenkephalin, 75 and 96% yields were obtained for the opioid peptides Met-RGL and Met-enk, respectively. Cell-based assays demonstrated that in-vitro-generated des-acetyl alpha-MSH successfully activated the melanocortin 4 receptor. Proghrelin digestion was used to screen the specificity of PC cleavage and to confirm the cleavage site by mass spectroscopy. Mature ghrelin was produced by human furin, mouse prohormone convertase 1, and human prohormone convertase 7 but not by mouse prohormone convertase 2. These results demonstrate that our in vitro system (1) can produce peptides in quantities sufficient to carry out functional analyses, (2) can be used to determine the specificity of proprotein convertases on recombinant precursors, and (3) has the potential to identify novel peptide functions on both known and orphan G-protein-coupled receptors.
Ozawa
Akihiko
A
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Cai
Yang
Y
Lindberg
Iris
I
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D043423:Carboxypeptidase H / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004745:Enkephalins / Q000378:metabolism; D004798:Enzymes / Q000378:metabolism*; D054439:Ghrelin; D006801:Humans; D051379:Mice; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D036361:Peptide Hormones / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000378:metabolism; D045664:Proprotein Convertase 1 / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D051381:Rats; D044105:Receptor, Melanocortin, Type 4 / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D000521:alpha-MSH / Q000378:metabolism / Q000502:physiology
D004745:Enkephalins; D004798:Enzymes; D054439:Ghrelin; D009097:Multienzyme Complexes; D036361:Peptide Hormones; D010455:Peptides; D011498:Protein Precursors; D044105:Receptor, Melanocortin, Type 4; C029992:proenkephalin; D000521:alpha-MSH; D011333:Pro-Opiomelanocortin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D043423:Carboxypeptidase H; D045664:Proprotein Convertase 1