Acidic Proregion Research Articles

Novel Antimicrobial Peptides from the Arctic Polychaeta Nicomache minor Provide New Molecular Insight into Biological Role of the BRICHOS Domain.

Endogenous antimicrobial peptides (AMPs) are among the earliest molecular factors in the evolution of animal innate immunity. In this study, novel AMPs named nicomicins were identified in the small marine polychaeta Nicomache minor in the Maldanidae family. Full-length mRNA sequences encoded 239-residue prepropeptides consisting of a putative signal sequence region, the BRICHOS domain within an acidic proregion, and 33-residue mature cationic peptides. Nicomicin-1 was expressed in the bacterial system, and its spatial structure was analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles the Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The peptide possessed cytotoxicity against cancer and normal adherent cells as well as toward human erythrocytes.

Molecular cloning of a novel cecropin-like peptide gene from the swallowtail butterfly, Papilio xuthus

A new cecropin-like antimicrobial peptide (Px-CLP) gene was isolated from the immunechallenged larvae of the swallowtail butterfly, Papilio xuthus, by employing annealing control primer (ACP)-based GeneFishing PCR. The full-length cDNA of Px-CLP is 310 nucleotides encoding a 70 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propeptide, a presumed 37-residue mature peptide, and an uncommon 7-residue acidic pro-region at the C-terminus. The deduced amino acid sequence of Px-CLP showed significant identities with other Lepidopteran cecropin D type peptides. RT-PCR revealed that the Px-CLP transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The peptides with or without C-terminal acidic sequence region were synthesized on-solid phage and submitted to antibacterial activity assay. The synthetic 37-mer peptide (Px-CLPa), which removed C-terminal acidic sequence region, was showed exclusively antibacterial activity against E. coli ML35; meanwhile, a 44-mer peptide (Px-CLPb) with C-terminal acidic peptide region was not active. This result suggests that Px-CLP is produced as a larger precursor containing a C-terminal pro-region that is subsequently removed by C-terminal modification.

Characterization of the hepcidin gene in eight species of bats

Hemochromatosis, or iron storage disease, has been associated with significant liver disease and mortality in captive Egyptian fruit bats (Rousettus aegyptiacus). The physiologic basis for this susceptibility has not been established. In humans, a deficiency or resistance to the iron regulatory hormone, hepcidin has been implicated in the development of hereditary hemochromatosis. In the present study, we compared the coding sequence of the hepcidin gene in eight species of bats representing three distinct taxonomic families with diverse life histories and dietary preferences. Bat hepcidin mRNA encoded a 23 amino acid signal peptide, a 34 or 35 amino acid pro-region, and a 25 amino acid mature peptide, similar to other mammalian species. Differences in the sequence of the portion of the hepcidin gene that encodes the mature peptide that might account for the increased susceptibility of the Egyptian fruit bat to iron storage disease were not identified. Variability in gene sequence corresponded to the taxonomic relationship amongst species.

Proconvertase proteolytic processing of an enzymatically active myeloperoxidase precursor

Optimal and efficient killing of ingested microbes by human neutrophils is mediated in large part by the action of hypochlorous acid produced by the myeloperoxidase–H2O2–chloride system in phagosomes. Myeloperoxidase gene transcription is limited to early myeloid precursors in the bone marrow, when myeloperoxidase is synthesized and stored in azurophilic granules for subsequent release from stimulated neutrophils. Promyeloperoxidase, the 90kDa myeloperoxidase precursor synthesized in the endoplasmic reticulum (ER), contains a 125-amino acid pro-region whose function and fate during myeloperoxidase biosynthesis are unknown. Promyeloperoxidase has two fates during myeloperoxidase biosynthesis; the majority undergoes proteolytic processing to generate mature myeloperoxidase, while the remainder is constitutively secreted from the cells in bone marrow. We used a promyelocytic cell line that produces endogenous myeloperoxidase as well as human embryonic kidney cells stably expressing normal and mutant forms of myeloperoxidase to examine proteolytic processing of promyeloperoxidase. We demonstrated that CMK-RVKR, an inhibitor of subtilisin-like proteinases, blocked cleavage of the pro-peptide of promyeloperoxidase in a post-ER compartment. Mutants with alanine substitution of basic residues in the predicted proteinase cleavage site failed to undergo maturation to normal myeloperoxidase subunits and were arrested at the promyeloperoxidase stage. Whereas specific mutants varied as to their stability, secreted promyeloperoxidase from the mutants retained the capacity to generate hypochlorous acid. Taken together, these studies demonstrate proconvertase-dependent cleavage of promyeloperoxidase as an essential step in normal proteolytic processing and granule targeting of myeloperoxidase. Furthermore, although mutations in the proteinase cleavage site reduced intracellular stability of the mutants, the integrity of the heme group was not compromised, as chlorinating activity was retained in the secreted promyeloperoxidase.

DADP: the database of anuran defense peptides

Anuran tissues, and especially skin, are a rich source of bioactive peptides and their precursors. We here present a manually curated database of antimicrobial and other defense peptides with a total of 2571 entries, most of them in the precursor form with demarcated signal peptide (SP), acidic proregion(s) and bioactive moiety(s) corresponding to 1923 non-identical bioactive sequences. Search functions on the corresponding web server facilitate the extraction of six distinct SP classes. The more conserved of these can be used for searching cDNA and UniProtKB databases for potential bioactive peptides, for creating PROSITE search patterns, and for phylogenetic analysis.

Innate Immune Responses of a Scleractinian Coral to Vibriosis

Scleractinian corals are the most basal eumetazoan taxon and provide the biological and physical framework for coral reefs, which are among the most diverse of all ecosystems. Over the past three decades and coincident with climate change, these phototrophic symbiotic organisms have been subject to increasingly frequent and severe diseases, which are now geographically widespread and a major threat to these ecosystems. Although coral immunity has been the subject of increasing study, the available information remains fragmentary, especially with respect to coral antimicrobial responses. In this study, we characterized damicornin from Pocillopora damicornis, the first scleractinian antimicrobial peptide (AMP) to be reported. We found that its precursor has a segmented organization comprising a signal peptide, an acidic proregion, and the C-terminal AMP. The 40-residue AMP is cationic, C-terminally amidated, and characterized by the presence of six cysteine molecules joined by three intramolecular disulfide bridges. Its cysteine array is common to another AMP and toxins from cnidarians; this suggests a common ancestor, as has been proposed for AMPs and toxins from arthropods. Damicornin was active in vitro against Gram-positive bacteria and the fungus Fusarium oxysporum. Damicornin expression was studied using a combination of immunohistochemistry, reverse phase HPLC, and quantitative RT-PCR. Our data show that damicornin is constitutively transcribed in ectodermal granular cells, where it is stored, and further released in response to nonpathogenic immune challenge. Damicornin gene expression was repressed by the coral pathogen Vibrio coralliilyticus. This is the first evidence of AMP gene repression in a host-Vibrio interaction.

The NMR structure of protein-glutaminase from Chryseobacterium proteolyticum

Protein deamidation, the hydrolysis of side chain amido groups of protein-bound glutaminyl or asparaginyl residues to release ammonia, has received focused attention especially in food industries since protein deamidation is regarded as a promising method to improve functional properties of food proteins. Deamidation generally decreases an isoelectric point of proteins due to increase in number of negatively charged carboxyl groups and enhances protein solubility. In addition, deamidation leads to alteration of the tertiary structures of proteins with an improved amphiphilic character that is useful as an emulsifier or a foaming agent. Therefore, deamidation of food proteins have been investigated by various methods including mild acid treatment, anion-catalyzed deamidation, dry heating under mild alkaline conditions, and thermal treatment. Although deamidation by these treatments improved protein functionalities, there were undesired side-effects, such as concomitant peptide bond cleavages, that were unavoidably brought about by the chemical/ physical treatments. Therefore, enzymatic methods have advantages due to their selectivity and mild treatments. The possibilities of the use of transglutaminases, peptidoglutaminases, and proteases have been explored for this purpose. These enzymes, however, are not suitable because the primary catalytic reactions of transglutaminases and proteases are not deamidation itself, and primary substrates of peptidoglutaminases are not proteins. Protein-glutaminase (PG) is an enzyme produced from the microorganism Chryseobacterium proteolyticum strain 9670 (Yamaguchi et al. 2001). PG catalyzes only the deamidation of the side chain amido group of protein-bound glutaminyl residues to release ammonia without catalyzing the transglutamination and hydrolysis of asparaginyl residues or producing other undesirable changes in protein structures. PG is a monomeric single polypeptide with pI = 10.0 and a molecular weight of 19,860 and is synthesized as a prepro-form, containing a 21-amino-acid signal polypeptide, a 114-amino acid pro-region, and a sequence for the mature enzyme. PG with the pro-region (pro-PG) has no enzymatic activities, but when pro-PG is removed by an extracellular protease, an active enzyme is produced in C. proteolyticum. However, the amount of PG produced by C. proteolyticum is too small to be used for industrial application that limits the application of PG to deamidation of food proteins. Recently, we have constructed the high expression system of PG with Corynebacterium glutamicum, which enables us to prepare an amount of stable-isotope labeled PG for NMR experiments (Kikuchi et al. 2009). Here, we report the solution structure of mature PG determined by NMR and discuss the catalytic mechanism of PG on the structural basis.

Inhibition of bactericidal activity is maintained in a mouse α-defensin precursor with proregion truncations

α-Defensin biosynthesis requires the proteolytic conversion of inactive precursors to microbicidal forms. In mouse Paneth cell pro-α-defensin proCrp4(20–92), anionic amino acids positioned near the proregion N-terminus inhibit proCrp4 activity by an apparent charge neutralization mechanism. Because most pro-α-defensins contain proregions of highly conserved chain length, we tested whether decreasing the distance between the inhibitory acidic residues of the proregion and the α-defensin component of the precursor would alter proCrp4 inhibition. Accordingly, two proCrp4 deletion variants, (Δ44–53)-proCrp4 and (Δ44–58)-proCrp4, truncated in a manner corresponding to deletions between MMP-7 cleavage sites, were prepared and assayed for bactericidal peptide activity. Consistent with the properties of full-length proCrp4(20–92), (Δ44–53)-proCrp4 and (Δ44–58)-proCrp4 were processed effectively by MMP-7, lacked bactericidal activity at high peptide levels over a 3h exposure period, and failed to induce permeabilization of live Escherichia coliin vitro. Thus, bringing the inhibitory proregion domain into greater proximity with the Crp4 component of the precursor did not alter the activity of this pro-α-defensin. Therefore, the conserved distance that separates inhibitory acidic proregion residues from the Crp4 peptide is not critical to maintaining proCrp4(20–92) in an inactive state.

Suppression of Inhibin A Biological Activity by Alterations in the Binding Site for Betaglycan

Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin alpha-subunit. Homology modeling indicates that key alpha-subunit residues (Tyr(50), Val(108), Thr(111), Ser(112), Phe(118), Lys(119), and Tyr(120)) form a contiguous epitope in this region of the molecule. Disruption of betaglycan binding by the simultaneous substitution of Thr(111), Ser(112), and Tyr(120) to alanine yielded an inhibin A variant that was unable to suppress activin-induced follicle-stimulating hormone release by rat pituitary cells in culture. Together these results indicate that a high affinity interaction between betaglycan and residues Val(108)-Tyr(120) of the inhibin alpha-subunit mediate inhibin A biological activity.

In vitro activation of pro‐α‐defensins by neutrophil serine proteinases of rhesus macaque

α‐Defensins are abundant antimicrobial peptides in the azurophilic granules of phagocytic leukocytes or in the Paneth cells of small intestine. To investigate the mechanisms of myeloid pro‐α‐defensin processing, recombinant pro‐α‐defensin 4 (proRMAD4) was exposed to human neutrophil elastase (HNE), cathepsinG (cG) and proteinase 3 (P3) from azurophil granules and analyzed biochemically and in bacterical peptide assays. ProRMAD4 was converted in vitro to active RMAD4 by these proteases. Only HNE cleaved at the native RMAD4 N‐terminus. cG and P3 activated proRMAD4, but they cleaved at different sites within the proregion. Mutagenesis of acidic proregion amino acids (Asp,Glu) to charge neutral (Asn, Gln) did not significantly alter the bactericidal property of proRMAD4 suggesting H‐bonding interactions also participate in the peptide inhibition. Disulfide variants of proRMAD4 were extensively degraded by HNE, cG and P3 showing that the disulfide array protects the α‐defensin from proteolysis during conversion to the bactericidal form. These data support the hypothesis that azurophil serine proteinases activate macaque myeloid α‐defensins and convert them to active forms. This research was supported by NIH grants DK044632, AI059346, Human Frontiers Science Project

A short proregion of trialysin, a pore‐forming protein of Triatoma infestans salivary glands, controls activity by folding the N‐terminal lytic motif

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect that transmits the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Its saliva contains trialysin, a protein that forms pores in membranes. Peptides based on the N-terminus of trialysin lyse cells and fold into alpha-helical amphipathic segments resembling antimicrobial peptides. Using a specific antiserum against trialysin, we show here that trialysin is synthesized as a precursor that is less active than the protein released after saliva secretion. A synthetic peptide flanked by a fluorophore and a quencher including the acidic proregion and the lytic N-terminus of the protein is also less active against cells and liposomes, increasing activity upon proteolysis. Activation changes the peptide conformation as observed by fluorescence increase and CD spectroscopy. This mechanism of activation could provide a way to impair the toxic effects of trialysin inside the salivary glands, thus restricting damaging lytic activity to the bite site.

Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin

Hepcidin is encoded as an 84 amino acid prepropeptide containing a typical N-terminal 24 amino acid endoplasmic reticulum targeting signal sequence, and a 35 amino acid proregion (pro) with a consensus furin cleavage site immediately followed by the C-terminal 25 amino acid bioactive iron-regulatory hormone (mature peptide). We performed pulse-chase studies of posttranslational processing of hepcidin in human hepatoma HepG2 cells and in primary human hepatocytes induced with bone morphogenic protein (BMP-9). In some experiments, the cells were treated with the furin protease inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) or furin siRNA. In the absence of furin inhibitor, hepcidin was found to be processed in less than 1 h and secreted as a 3 kDa form reactive with anti-mature but not anti-pro antibody. In the presence of furin inhibitors or furin siRNA, a 6 kDa form reactive with both anti-pro and anti-mature antibody was rapidly secreted into the medium. Processing was not affected by inhibitors of the hypoxia inducible factor (HIF) pathway, or by treatment with 30 μM holo- or apo-transferrin. In conclusion, the hepatic prohormone convertase furin mediates the posttranslational processing of hepcidin. The proteolytic cleavage of prohepcidin to hepcidin is not regulated by iron-transferrin or the HIF pathway.

Cecropin P1 and novel nematode cecropins: a bacteria-inducible antimicrobial peptide family in the nematode Ascaris suum

Cecropin P1 was first identified as a mammalian antimicrobial peptide isolated from the pig intestine. Much research aimed at characterizing this peptide has been reported. Recently, the workers who discovered the peptide corrected their original conclusion, and confirmed that this peptide originates in fact from the pig intestinal parasitic nematode, Ascaris suum. In the present study, we carried out a semi-exhaustive search for bacteria-inducible transcripts in A. suum by the cDNA subtraction method. The transcripts encoding cecropin P1 and novel Ascaris cecropins, designated cecropins P2, P3 and P4, were found to be positively induced factors. Chemically synthesized Ascaris cecropins were bactericidal against a wide range of microbes, i.e. Gram-positive (Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus) and Gram-negative (Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens and Esherichia coli) bacteria, and were weakly but detectably active against yeasts (Saccharomyces cerevisiae and Candida albicans). Cecropin P1-like sequences were also detected at least in two other species (Ascaris lumbricoides and Toxocara canis) of the Ascarididae. All Ascaris cecropin precursors contain an acidic pro-region connected by a tetra-basic cleavage site at the C-terminus. Such an acidic pro-region is also reported to be present in the tunicate cecropin-type antimicrobial peptide styelin. On the basis of the evolutionary position of nematodes and tunicates, the ancestral cecropin may have contained the acidic pro-region at the C-terminus.

Novel frenatins from the skin of the Australasian giant white-lipped tree frog, Litoria infrafrenata: Cloning of precursor cDNAs and identification in defensive skin secretion

The Australasian anuran amphibian genus Litoria, contains many phenotypically-diverse species as a result of radial evolution of an ancestral species into different biotopes much in the manner of the indigenous marsupial mammals. In common with members of the Central/South American genus Phyllomedusa, their specialized skin granular glands are factories for the production of a plethora of biologically-active peptides. Here we report a more detailed study of those present in the defensive skin secretion of the Australasian giant white-lipped tree frog, Litoria infrafrenata, and, for the first time, we have identified three novel frenatins by deduction of primary structures from cDNAs that were cloned from a library constructed from lyophilized skin secretion using a recently-developed technique. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of a frenatin peptide. Processed peptides corresponding in molecular mass to the deduced molecular masses of frenatins (named 1.1, 3, 3.1 and 4.1) were identified in the same secretion sample using HPLC and mass spectroscopy. The application of this technique thus permits parallel peptidomic and transcriptomic analyzes on the same lyophilized skin secretion sample circumventing sacrifice of specimens from endangered herpetofauna.

The structural organization of aurein precursor cDNAs from the skin secretion of the Australian green and golden bell frog, Litoria aurea

Aureins are a family of peptides (13–25 residues), some of which possess potent antimicrobial and anti-cancer properties, which have been classified into 5 subgroups based upon primary structural similarities. They were originally isolated from the defensive skin secretions of the closely related Australian bell frogs, Litoria aurea and Litoria raniformis, and of the 23 aurein peptides identified, 10 are common to both species. Using a recently developed technique, we have constructed a cDNA library from the defensive secretion of the green and golden bell frog, L. aurea, and successfully cloned a range of aurein precursor transcripts containing entire open-reading frames. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of aurein. The deduced precursor structures for the most active aureins (2.2 and 3.1) confirmed the presence of a C-terminal amidation motif whereas that of aurein 5.3 did not. Processed peptides corresponding in molecular mass to aureins 2.2, 2.3, 2.5, 3.1 and 5.3 were identified in the same secretion sample using LC/MS. The application of this technique thus permits parallel peptidomic and transcriptomic analyses on the same lyophilized skin secretion sample circumventing sacrifice of specimens of endangered herpetofauna.

Common structural properties specifically found in the CSαβ-type antimicrobial peptides in nematodes and mollusks: evidence for the same evolutionary origin?

The structural properties of the Ascaris suum antibacterial factor (ASABF)-type antimicrobial peptides, isolated from nematodes, were compared with the CSαβ-type antimicrobial peptides found in other organisms. The spacing of the half-cystine residues, cysteine pairings, and organization of the precursor were different from the ‘classical’ CSαβ-type antimicrobial peptides, such as drosomycin and plant defensins, and identical only to the MGD and myticin in mollusks. In addition, ABF-5, a member of the ASABF-type antimicrobial peptides in Caenorhabditis elegans, is predicted to contain a basic mature region and an acidic pro-region, similar to MGD and myticin. These results suggest that the ASABF-type antimicrobial peptides, MGD and myticin are similar in their structure.

Two energetically disparate folding pathways of alpha-lytic protease share a single transition state.

The Lysobacter enzymogenes alpha-lytic protease (alphaLP) is synthesized with a 166 amino acid pro region (Pro) that catalyzes the folding of the 198 amino acid protease into its native conformation. An extraordinary feature of this system is the very high energy barrier (DeltaG = 30 kcal mol-1) that effectively prevents alphaLP from folding in the absence of Pro (t1/2 = 1800 years). A pair of mutations has been isolated in the protease that completely suppresses the catalytic defect incurred in Pro by truncation of its last three amino acids. These mutations also accelerate the folding of alphaLP in the absence of Pro by 400-fold. An energetic analysis of the two folding reactions indicates that the mutations stabilize the transition states of both the catalyzed and uncatalyzed folding reactions by 3 kcal mol-1. This finding points to a single transition state for these two distinct and energetically disparate folding pathways, and raises the possibility that all alphaLP folding pathways share the same transition state.

Role of carbohydrate structures in the binding of beta1-latency-associated peptide to ligands.

Transforming growth factor beta1 (TGF-beta1) is a potent growth differentiation and morphogenesis factor. The amino-terminal 248 amino acid pro region of TGF-beta1, the beta1-latency-associated peptide (beta1-LAP), is noncovalently associated with TGF-beta1 in an inactive complex. Previous studies suggested that deglycosylated beta1-LAP can not form this latent complex with TGF-beta1. To study the role of the carbohydrate structures of beta1-LAP in its biological functions, we expressed simian beta1-LAP in Escherichia coli with a 10 histidine residue tag on the N-terminus. This polypeptide was solubilized from inclusion bodies with 6 M guanidine hydrochloride and purified by metal chelate affinity chromatography. Purified beta1-LAP was refolded to its dimeric form using a chaotrope-mediated folding procedure. The dimeric beta1-LAP forms 90 kDa complexes with TGF-beta1, TGF-beta2, and TGF-beta3, and reverses the inhibitory activity of TGF-beta1 on Mv1Lu cells. Solid phase binding assays demonstrate that refolded beta1-LAP binds to heparin and thrombospondin 1. FET cell adhesion promoted by refolded beta1-LAP was blocked by an RGD peptide. Purified beta1-LAP produced in Chinese hamster ovary cells, deglycosylated with N-glycosidase F, forms a 80-90 kDa complex with mature TGF-beta1. The carbohydrate structures of beta1-LAP are not required for binding to ligands or for its biological activity.

Inhibition of alpha-lytic protease by pro region C-terminal steric occlusion of the active site.

alpha-Lytic protease, a chymotrypsin-like serine protease, is synthesized with an N-terminal 166 amino acid pro region which is absolutely required for folding of the protease. The pro region is also the most potent inhibitor of the protease known with a Ki of approximately 10(-10) M. Compared to its role in the folding reaction, relatively little is known about the mechanism by which the pro region inhibits the mature protease. While proteinaceous protease inhibitors generally function by occluding the active sites of their respective targets [Bode, W., & Huber, R. (1992) Eur. J. Biochem. 204, 433-451], the pro region of alpha-lytic protease with its dual roles in folding and inhibition might be expected to show a novel mechanism of inhibition. However, experiments that probe both the structural and enzymatic consequences of pro region binding indicate that the pro region does not measurably distort the protease active site. Instead, the catalytic site is fully functional in the complex. Pro region inhibition of the protease is due to simple steric obstruction; the pro region C-terminus lies in the substrate binding sites of the protease. The implications of these results are discussed with regard to alpha-lytic protease maturation and folding. In addition, the proposed mechanism of alpha-lytic protease pro region inhibition is discussed with respect to data from other pro region families.

The NH2-terminal proregion of peptidylglycine alpha-amidating monooxygenase facilitates the secretion of soluble proteins.

A highly conserved ten amino acid proregion separates the peptidylglycine alpha-hydroxylating monooxygenase (PHM) domain of the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) protein from the NH2-terminal signal peptide; propeptides with amino acid sequences similar to the PAM proregion have been identified in other secreted proteins. In AtT-20 cells, but not in human embryonic kidney (hEK)-293 cells, an endogenous endoprotease acting at a site distal to the trans-Golgi network efficiently removes the propeptide from stably transfected monofunctional PHM (PHMs). We constructed a mutant PHM protein (delta ProPHMs) in which the proregion was deleted and the signal peptide joined directly to the monooxygenase domain. Newly synthesized, enzymatically active delta ProPHMs was secreted from both AtT-20 cells and hEK-293 cells more slowly than PHMs. In endocrine cells, the proregion was not required for storage in regulated secretory granules. We transferred the PAM proregion to prohormone convertase 2 (PC2), another soluble constituent of secretory granules, to determine whether the effect of the proregion were transferrable. In both AtT-20 cells and hEK-293 cells, the PAM/PC2 fusion molecule was able to exit the endoplasmic reticulum more rapidly than PC2.