Production of bioactive peptides in an in vitro system

Abstract

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.

Figure 1. The process of in vitro preparation of bioactive peptides

Figure 2. Time course of liberation of α-MSH, Met-RGL, and Met-enk from their precursors

Two micrograms of mPOMC and rPE were sequentially digested with mPC2 and hCPE, respectively. In additionally, hPAM reactions were performed using ACTH- and POMC-derived peptides to generate des-acetyl α-MSH. The details of each reaction were described in Materials and Methods. Peptide products were measured by specific RIAs. Panel A, Met-enk; panel B, Met-enk-RGL; panels C and D, des-acetyl α-MSH derived from ACTH and POMC, respectively. Each sample was tested in duplicate at each time point. Results are shown as the percentage of maximal conversion.

Figure 3. α-MSH generated by in vitro processing can efficiently activate the MC4-receptor

HEK-293 cells expressing MC4-receptors were treated with different concentrations of des-acetyl α-MSH produced by our method. After 2 h, the levels of intracellular cAMP were measured using the Parameter™ cyclic AMP immunoassay kit. Each sample was tested in duplicate at each concentration.

Figure 4. Proghrelin is cleaved by multiple PCs, but not by PC2

Panel A: Alignments of proghrelin. Pro; prosequence. Panel B: Digestion of His-tagged proghrelin (2 μg) was carried out with indicated enzymes. All samples were electrophoresed on the same gel; some lanes are omitted for clarity. The cleavage products are indicated with asterisks.

Figure 5. PC cleavage of proghrelin produces ghrelin, but not obestatin

(A) Tandem mass spectrum of [Ghrelin 1-31]⁴⁺ at m/z 877.45 (predicted average m/z 877.02): (B) Tandem mass spectrum of [Ghrelin 32-97]⁵⁺ at m/z 1458.6 (predicted average m/z 1458.4). All major peaks correspond to the peptide bond cleavage; b ions are fragments containing peptide N-termini while y ions are those containing peptide C-termini. Tandem mass spectra confirm that PCs cleave proghrelin between Arginine 31 and Alanine 32, and yield a smaller fragment of 3505.1 Da and a larger fragment of 7290.5 Da.