Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Expression of mouse proopiomelanocortin in an insulinoma cell line. Requirements for beta-endorphin processing

Proopiomelanocortin (POMC) is a neuroendocrine precursor protein which is processed at paired basic amino acids in a tissue-specific manner. To study this phenomenon, a vaccinia virus recombinant, which directs the synthesis of mouse POMC (VV:mPOMC) was constructed and used to infect epithelial (BSC-40) and endocrine (Rin m5F) cell lines. Bona fide mPOMC was produced in both cell types and beta-endorphin immunoreactivity was secreted in a nonregulated manner from BSC-40 cells and in a regulated manner from Rin m5F cells. Although the precursor was not cleaved to smaller beta-MSH or beta-endorphin immunoreactive peptides in BSC-40 cell extracts, Rin m5F cells produced primarily authentic gamma-lipotropin and des-acetyl beta-endorphin. Furthermore, production of these peptides was restricted to the regulated secretory pathway in Rin m5F cells. Site-directed mutagenesis was then used to change the inefficiently recognized Lys-Lys potential cleavage site near the carboxyl terminus of beta-endorphin to Lys-Arg. Expression of the mutant precursor in Rin m5F cells resulted in the synthesis of both des-acetyl beta-endorphin and beta-endorphin.     

Tissue distribution and processing of proSAAS by proprotein convertases

The conversion of inactive precursor proteins into bioactive neuropeptides and peptide hormones involves regulated secretory proteins such as prohormone convertases PC1 and PC2. The neuroendocrine protein 7B2 represents a specific binding protein for PC2, and the protein proSAAS, which interacts with PC1, exhibits certain structural and functional homologies with 7B2. With the intention of better understanding the physiological role of proSAAS and its derived peptides, we investigated its tissue localization using a new radioimmunoassay (RIA) to a C-terminal proSAAS-derived peptide. Immunoreactivity corresponding to this SAAS-derived peptide is mostly localized to the brain and gut. Analysis of the brain distribution of the proSAAS-derived peptides indicates that the hypothalamus and pituitary are the two richest areas, consistent with the previously described high expression of PC1 in these two areas. In order to investigate the cleavage of proSAAS by prohormone convertases, we incubated recombinant His-tagged proSAAS with recombinant mouse proPC2 or furin, separated the cleavage products using high-pressure gel permeation chromatography and analyzed the products by RIA. Our results indicate that either PC2 or furin can accomplish in vitro rapid removal and efficient internal processing of the C-terminal peptide, exposing the inhibitory hexapeptide to possible further digestion by carboxypeptidases. Finally, we also studied proSAAS processing in the brains of wild-type and PC2 null mice and found that proSAAS is efficiently processed in vivo. Whereas the C-terminal peptide is mostly internally cleaved in wild-type mouse brain, it is not processed as efficiently in the brain of PC2 null mice, suggesting that PC2 is partially responsible for this cleavage in vivo.     

Cleavage of recombinant proenkephalin and blockade mutants by prohormone convertases 1 and 2: an in vitro specificity study

Proenkephalin (PE) derived-peptides are thought to be generated predominantly through endoproteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). In order to compare cleavage site preferences of these convertases, we studied the processing of recombinant wild-type rat PE and of two mutant PEs by recombinant purified mouse PC1 and PC2. Western blot analyses of timed digestions showed that both mouse PC1 and PC2 were able to produce a variety of large and intermediate sized-peptides from wild-type PE as well as from the precursors mutated at initial blockade sites. PC2 exhibited a broader specificity against PE than PC1, generating a much greater number of peptide products. Mass spectrometric identification of cleavage products showed that PC2 appeared to be the principal enzyme involved in the generation of smaller active opioids. Both enzymes were able to cleave various KR- and KK-containing sites, but PC2 was also able to cleave efficiently at an RR-V site and a KK-M site not cleaved by PC1, suggesting the exclusion of large aliphatic residues at the P1' position in PC1 cleavage. Alternative cleavage sites were readily chosen by convertases in blockade mutants, confirming in vivo results that cleavages do not follow an obligatory order. Furthermore, glycosylated PE was less efficiently processed by PC2, indicating that glycosylation may serve as a mechanism to hinder processing.     

Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.     

Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3.

Previous studies using selectively modified pro-ocytocin/neurophysin substrate analogues and the purified metalloprotease, pro-ocytocin/neurophysin convertase (magnolysin; EC 3.4 24.62), have shown that dibasic cleavage site processing is associated with a prohormone sequence organized in a beta-turn structure. We have used various peptide analogues of the pro-ocytocin-neurophysin processing domain, and recombinant prohormone convertase 1/3, to test the validity of this property towards this member of the family of prohormone convertases (PCs). The enzymatic cleavage analysis and kinetics showed that: (a) with methyl amide (N-Met) modification, a secondary structure beta-turn breaker, the enzyme substrate interaction was abolished; (b) cleavage was favoured when the dibasic substrate side-chains were oriented in opposite directions; (c) the amino acid present at the P'1 position is important in the enzyme-substrate interaction; (d) the flexibility of the peptide substrate is necessary for the interaction; (e) Addition of dimethylsulfoxide to the cleavage assay favoured the cleavage of the pro-ocytocin/neurophysin large substrate over that of the smaller one pGlu-Arg-Thr-Lys-Arg-methyl coumarin amide. These data allowed us to conclude that proteolytic processing of pro-ocytocin-related peptide substrates by PC1/3 as well as by the metalloenzyme, magnolysin, involves selective recognition of precise cleavage site local secondary structure by the processing enzyme. It is hypothesized that this may represent a general property of peptide precursor proteolytic processing systems.     

Expression and processing of mouse proopiomelanocortin in bovine adrenal chromaffin cells. A model system to study tissue-specific prohormone processing.

Many neuroendocrine precursor proteins, such as proopiomelanocortin (POMC), are cleaved in a tissue specific manner at distinct pairs of basic amino acids. Elucidating the specificity of the prohormone endoprotease(s) is essential to understanding cleavage specificity. However, isolation of these enzymes has been difficult, due to the inability to distinguish authentic maturation enzyme from the many other trypsin-like activities present in tissue homogenates. Recently, a "signature" of the insulin cell endoprotease(s) was defined in vivo by assessing the processing of a series of mutant cleavage sites in a model prohormone, mouse POMC (mPOMC) (Thorne, B. A., and Thomas, G. (1990) J. Biol. Chem. 265, 8436-8443. To investigate mechanisms of tissue-specific processing, we sought to identify the endoprotease signature of a cell having a processing phenotype distinct from insulinoma cells. In this report, the cleavage site specificity of the endoprotease(s) expressed in bovine adrenal chromaffin cells is examined. High levels of mPOMC (1.6 pmol/10(6) cells) were expressed in these cells using a vaccinia virus vector, and the precursor was targeted to the regulated secretory pathway. Analysis of POMC-derived peptides revealed that chromaffin cells processed the prohormone to a set of peptides highly similar to anterior pituitary corticotrophs, including adrenocorticotropin hormone (ACTH) and beta-lipotropin, gamma-lipotropin, and beta-endorphin. This processing contrasted with the pattern of cleavage site utilization in Rin m5F insulinoma cells, which more closely resembled that of the intermediate pituitary melanotrophs. However, the processing preference for the sequences of pairs of basic amino acids (as tested using the entire series of mutant cleavage sites; -LysArg- (native), -ArgArg-, -ArgLys-, -LysLys-, -HisArg-, -MetArg- at the ACTH/beta-lipotropin junction and -LysLys- (native), -LysArg-, -ArgArg-, -ArgLys- in beta-endorphin) was the same in both insulinoma and adrenal chromaffin cells, suggesting recognition and cleavage by similar enzymes in both cell types. The cell-specific processing of mPOMC may thus result from expression of a common core set of processing enzymes and factors unique to each cell type affecting the enzyme accessibility to precursor cleavage sites.     

Mammalian neural and endocrine pro-protein and pro-hormone convertases belonging to the subtilisin family of serine proteinases.

Conversion of pro-hormones and precursor proteins into biologically active peptides and proteins involves the concerted action of a number of convertases and post-translation modification enzymes. The identification of the yeast convertase kexin as a prototype processing enzyme led to the discovery of the mammalian convertase designated furin, PC1 and PC2. Whereas furin is ubiquitously expressed, PC1 and PC2 are found only in endocrine and neural tissues and cell lines. In man and mouse, the genes coding for furin, PC1 and PC2 reside on three different chromosomes. The analysis of the intracellular processing of PC1 and PC2 and the removal of their pro-segment is presented, together with a summary of the cleavage specificity of these enzymes for precursors such as pro-opiomelanocortin (POMC) and human pro-renin. The distinct tissue distribution of PC1 and PC2 and their coregulation with POMC in the pituitary neurointermediate lobe adds credence to their physiological role as convertases involved in the tissue-specific processing of precursor proteins.     

Proteolytic processing of chromogranin A by the prohormone convertase PC2

The neuroendocrine secretory protein chromogranin A (CgA) is a precursor for various biologically active peptides. Several single and paired basic residues are present within its primary amino acid sequence comprising cleavage sites for prohormone convertases. In this study, SH-SY5Y human neuroblastoma cells were stably transfected with the prohormone convertase PC2 to analyse the proteolytic processing of endogenous chromogranin A and, in particular, the formation of the chromogranin-A-derived peptide GE-25. Our analyses revealed a significant change in the pattern of proteolytic conversion of chromogranin A in cells expressing PC2. Mock-transfected control cells contained mainly the intact chromogranin A molecule and hardly any shorter products were found. On the other hand, PC2-transfected cells showed extensive processing of chromogranin A, resulting in significantly lower amounts of the intact precursor and especially high levels of the free peptide GE-25.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.     

Neuropeptide processing profile in mice lacking prohormone convertase-1

Prohormone convertase 1 (PC1; also known as PC3) is believed to be responsible for the processing of many neuropeptide precursors. To look at the role PC1 plays in neuropeptide processing in brain and pituitary, we used radioimmunoassays (RIA) as well as quantitative peptidomic methods and examined changes in the levels of multiple neuropeptide products in PC1 knockout (KO) mice. The processing of proenkephalin was impaired in PC1 KO mouse brains with a decrease in the level of Met-Enkephalin immunoreactivity (ir-Met-Enk) and an accumulation of higher molecular weight processing intermediates containing ir-Met-Enk. Processing of the neuropeptide precursor VGF was also affected in PC1 KO mouse brains with a decrease in the level of an endogenous 3 kDa C-terminal peptide. In contrast, the processing of proSAAS into PEN was not altered in PC1 KO mouse brains. Quantitative mass spectrometry was used to analyze a number of peptides derived from proopiomelanocortin (POMC), provasopressin, prooxytocin, chromogranin A, chromogranin B, and secretogranin II. Among them, the levels of oxytocin and peptides derived from chromogranin A and B dramatically decreased in the PC1 KO mouse pituitaries, while the levels of peptides derived from proopiomelanocortin and provasopressin did not show substantial changes. In conclusion, these results support the notion that PC1 plays a key role in the processing of multiple neuroendocrine peptide precursors and also reveal the presence of a redundant system in the processing of a number of physiologically important bioactive peptides.     

Rapid Increases in Peptide Processing Enzyme Expression in Hippocampal Neurons

Abstract: Recent studies have demonstrated that seizure activity causes a dramatic increase in neuropeptide expression in specific regions of the rat hippocampus. In this study we investigated the effect of electroconvulsive treatment (ECT) on the expression of three posttranslational processing enzymes involved in the production of many bioactive peptides from their inactive precursors. Peptidylglycine α-amidating monooxygenase (PAM) converts peptidylglycine substrates into α-amidated products and prohormone convertases 1 and 2 perform the tissue-specific endoproteolytic cleavage of many prohormones. After a single ECT, in situ hybridization demonstrated a rapid increase in the level of PAM mRNA in the dentate granule cells of the hippocampus, reaching peak levels between 1 and 4 h and then returning to near baseline levels within 24 h. Northern blot analysis confirmed the changes in PAM mRNA expression seen by using in situ hybridization. Similar rapid changes in PAM mRNA expression were seen after repeated ECT, suggesting that chronic ECT did not affect the regulation of PAM expression in the hippocampus. Immunohistochemical staining demonstrated an increase in PAM protein in the molecular layer of the dentate gyrus at 4 and 8 h after a single ECT. Based on in situ hybridization, levels of mRNA for the prohormone convertases 1 and 2 were also increased in dentate granule cells after a single ECT. Prohormone convertase 2 mRNA levels exhibited a slower response to ECT, not reaching maximal levels until 8 h after ECT. The response of the dentate granule cells of the hippocampus to ECT provides a model system for studying the rapid, coordinate regulation of peptide-processing enzymes.     

Spatiotemporal expression, distribution, and processing of POMC and POMC-derived peptides in murine skin.

In murine skin, after depilation-induced anagen, there was a differential spatial and temporal expression of pro-opiomelanocortin (POMC) mRNA, of the POMC-derived peptides beta-endorphin, ACTH, beta-MSH, and alpha-MSH, and of the prohormone convertases PC1 and PC2 in epidermal and hair follicle keratinocytes and in the cells of sebaceous units. Using a combination of in situ hybridization histochemistry and immunohistochemistry, we found cell-specific variations in the expression of POMC mRNA that were consistent with immunoreactivities for POMC-derived peptides. Cells that contained POMC peptide immunoreactivity (IR) also expressed POMC mRNA, and where the IR increased there was a parallel increase in mRNA. The levels of PC1-IR and PC2-IR also showed cell-specific variations and were present in the same cells that contained the POMC peptides. Based on the cleavage specificities of these convertases and on the spatial and temporal expression of the convertases and of ACTH, beta-endorphin, beta-MSH, and alpha-MSH, we can infer that the activities of PC1 and PC2 are responsible for the cell-specific differential processing of POMC in murine skin.     

Yeast KEX1 protease cleaves a prohormone processing intermediate in mammalian cells

A vaccinia virus vector was used to express the yeast KEX1 gene, which encodes a prohormone carboxypeptidase specific for the removal of basic amino acids from prohormone processing intermediates, in mammalian cells. When produced in BSC-40 cells, Kex1p was localized to the perinuclear region and conferred a large increase in enzymatic activity characteristic of this carboxypeptidase. Expression of the KEX1 gene together with the yeast KEX2 gene, which encodes a prohormone endopeptidase specific for cleavage at pairs of basic amino acids, and the mouse proopiomelanocortin (mPOMC) cDNA in BSC-40 cells resulted in the full conversion of mPOMC to mature peptides including gamma-lipotropin. This in vivo processing of mPOMC to mature peptides by the KEX2/KEX1 gene products demonstrates a significant functional homology of the basic prohormone processing machinery in yeast and neuroendocrine cells.     

Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides

A new approach using targeted sequence collections has been developed for identifying endogenous peptides. This approach enables a fast, specific, and sensitive identification of endogenous peptides. Three different sequence collections were constituted in this study to mimic the peptidomic samples: SwePep precursors, SwePep peptides, and SwePep predicted. The searches for neuropeptides performed against these three sequence collections were compared with searches performed against the entire mouse proteome, which is commonly used to identify neuropeptides. These four sequence collections were searched with both Mascot and X! Tandem. Evaluation of the sequence collections was achieved using a set of manually identified and previously verified peptides. By using the three new sequence collections, which more accurately mimic the sample, 3 times as many peptides were significantly identified, with a false-positive rate below 1%, in comparison with the mouse proteome. The new sequence collections were also used to identify previously uncharacterized peptides from brain tissue; 27 previously uncharacterized peptides and potentially bioactive neuropeptides were identified. These novel peptides are cleaved from the peptide precursors at sites that are characteristic for prohormone convertases, and some of them have post-translational modifications that are characteristic for neuropeptides. The targeted protein sequence collections for different species are publicly available for download from SwePep.     

Comparative aspects of intracellular proteolytic processing of peptide hormone precursors: studies of proopiomelanocortin processing

In this review, the mechanisms underlying the intracellular processing of peptide hormone precursors, with a focus on proopiomelanocortin (POMC), were discussed on the basis of recent information. POMC as well as other prohormones is processed to active peptides through proteolytic cleavage by prohormone convertases PC1 and/or PC2. However, the cleavage-specificity of PC1 and PC2 in mammals is somewhat different from that in amphibians. From the comparative endocrinological point of view, expression and tissue distribution of PC1 and PC2 were discussed here. In mammals, proteolytic processing of POMC occurs coordinately with the maturation of secretory granules. Studies using immunoelectron microscopy with DAMP (3-[2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as a pH probe revealed that the acidic pH in the secretory granules, generated by vacular type-H+-ATPase, provides a favorable environment for activating PC1 in AtT-20 cells, a mouse corticotrope tumor cell line. Recent data indicate that the 7B2 protein serves as a chaperone in the regulation of PC2 activation and to control the timing for activating the convertase. Together, secretory granules in endocrine and neuroendocrine cells provide proper sites for biosynthesizing hormones in addition to serving as storage sites and vehicles for the transport of peptide hormones.     

The prohormone proenkephalin possesses differential conformational features of subdomains revealed by rapid H-D exchange mass spectrometry

Active enkephalin and related peptide hormones or neurotransmitters are generated by proteolytic processing of inactive prohormone precursors. Little is known about the relative accessibilities of prohormone cleavage sites and conformations of subdomains that undergo proteolytic processing. Therefore, this study investigated the conformational features of the prohormone proenkephalin (PE) by rapid hydrogen-deuterium exchange mass spectrometry (DXMS). DXMS analyzes rates of hydrogen exchange of the polypeptide backbone of PE with deuterium from D(2) O (heavy water) by mass spectrometry, accomplished at sub-second and multisecond time periods. Results showed differential accessibilities of cleavage sites and adjacent subdomains of PE to the aqueous environment. Importantly, protease cleavage sites of PE with greater relative accessibilities correspond to sites most readily cleaved by processing proteases to generate active peptide neurotransmitters. For comparison, peptides derived from PE (by pepsin digestion) displayed greater accessibility to the solvent environment, illustrated by their higher rates of H-D exchange compared to that of intact PE protein. The more limited H-D exchange accessibilities of PE protein, compared to peptides derived from PE, indicate that PE possesses tertiary conformation. These results demonstrate that differential tertiary conformations of PE subdomains undergo ordered proteolytic processing to generate active enkephalin peptides for cell-cell communication in the nervous and endocrine systems.