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PubMed2000

Immunohistochemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors.

Although chromogranin A (CgA) is widely distributed in neuroendocrine tumors, the distribution of chromogranin B (CgB) has not been elucidated. Hormones produced by tumors are sometimes prohormones and not necessarily bioactive hormones. Prohormones have to be processed into bioactive peptides by prohormone convertases (PCs), and some of them have to be amidated by peptidylglycine a-amidating monooxygenase (PGM). Whether PCs and PGM are present or not in tumors may explain why some tumors are functioning and some are nonfunctioning. We investigated 45 carcinoids and 16 pancreatic endocrine tumors. Of the carcinoids, CgA was expressed in most of the tumors, except for the rectal and ovarian carcinoids, which expressed CgB strongly. The expressions of PC2, PC3, and PGM were 31%, 100%, and 87%, respectively. In the pancreatic tumors, CgA was expressed in all tumors, whereas CgB was not expressed in any tumor. The expressions of PC2, PC3, and PGM were 63%, 88%, and 63%, respectively. PC3 was expressed in all of the functioning tumors but not in two of the four nonfunctioning tumors. PC2 and PGM were not expressed in three of the four nonfunctioning tumors. In conclusion, expression of CgA and CgB was different depending on the tumor location. High frequency of PCs and PGM may explain why even nonfunctioning tumors produce some inconspicuous peptides.

Adenoma, Islet Cell↗

PubMed1996

Identification of the thyrotropin-releasing hormone precursor, its processing products, and its coexpression with convertase 1 in primary cultures of hypothalamic neurons: anatomic distribution of PC1 and PC2.

The processing of pro-TRH, has been extensively studied in our laboratory using a corticotropic cell line, AtT20, transfected with the pro-TRH gene. We have also demonstrated that the convertases PC1 and PC2 process pro-TRH to cryptic peptides in vitro. However, although these processing pathways have been well characterized in vitro, little is known about the processing and subcellular distribution of pro-TRH and its derived peptides in hypothalamic neurons, an endogenous source of pro-TRH and PC enzymes. In this study we used multiple approaches to identify, both biochemically and anatomically, the presence and localization of pro-TRH (26 kDa) and its processing products. We also investigated the presence of PC1 and PC2 enzymes and the coexpression of pro-TRH and PC1 messenger RNAs. Identification of the TRH precursor was demonstrated by 1) Western blot analysis of cellular extracts, 2) immunoprecipitation of radiolabeled pro-TRH followed by analysis on acrylamide gel electrophoresis, 3) fluorescence immunocytochemistry, and 4) immunoelectron microscopy. The presence of the convertases PC1 and PC2 was determined by Western blot analysis of cellular extracts and fluorescence immunocytochemistry. The coexpression of pro-TRH with PC1 was shown by double in situ hybridization. Our findings support three main conclusions. First, this primary culture system of hypothalamic neurons is suitable for characterizing pro-TRH processing as well as identifying the anatomical location of its processing products. Second, prohormome processing takes place during axonal transport after removal of the signal peptide in the endoplasmic reticulum, and subsequent cleavages of the prohormone occur as intermediate peptides move down the axon toward the nerve terminal. This coupled transport-processing phenomenon may provide the necessary mechanism to ensure flexibility in differential processing of specific protein sequences that are determined by the secretory needs of cells. It appears that certain intermediate peptides differ in their subcompartmental distribution, suggesting the possibility of a differential processing and maturation of pro-TRH-derived peptides. Thirdly, the 87-kDa form of PC 1 may initiate the processing of pro-TRH at the Golgi complex level, which then continues to be processed by PC1 and PC2 in later stages of the secretory pathway.

Animals↗

PubMed1993

Differential processing of proenkephalin by prohormone convertases 1(3) and 2 and furin.

Recombinant vaccinia virus vectors were used to coexpress mouse prohormone convertase 1 (mPC1), mPC2, or human furin together with human proenkephalin in GH4C1 cells (rat pituitary somatomammotrophs) to examine the proteolytic processing of proenkephalin by these enzymes. Radioimmunoassays performed on high pressure gel permeation size-fractionated extracts obtained from GH4C1 cells and corresponding conditioned media revealed distinct profiles of immunoreactivity for products generated by each enzyme. PC1 produced intermediate sized processing products (3-10 kDa); the major immunoreactive enkephalin-containing species observed eluted at the positions of peptide B, the 5.3-kDa fragment, and free Leu5-enkephalin. PC2 exhibited a more complete processing profile. The major immunoreactive enkephalins produced were free Met5-enkephalin-Arg-Phe, free Met5-enkephalin-Arg-Gly-Leu, free Leu5-enkephalin, and free Met5-enkephalin. Thus PC2 appears to be more capable of generating active opioid units from proenkephalin than is PC1. Finally, furin cleaved proenkephalin to generate peptide B, an unidentified peak between the 18- and 5.3-kDa fragments, and a small amount of the 5.3-kDa fragment. Radiosequencing data verified that the production of the 5.3-kDa fragment by PC1 occurred as a result of a Lys-Lys cleavage. The ability of PC1 to cleave proenkephalin (but not proopiomelanocortin) at a Lys-Lys site implies that the structural context of the paired basic cleavage site may be more important in the determination of cleavage specificity than the particular pair of basic residues at the site.

Amino Acid Sequence↗

PubMed2000

The SAAS granin exhibits structural and functional homology to 7B2 and contains a highly potent hexapeptide inhibitor of PC1.

Prohormone convertases (PCs) 1 and 2 are thought to mediate the proteolytic cleavage of many peptide precursors. Endogenous inhibitors of both PC1 and PC2 have now been identified; the 7B2 protein is a nanomolar inhibitor of PC2, while the novel protein proSAAS was recently reported to be a micromolar inhibitor of PC1 [Fricker et al. (2000) J. Neurosci. 20, 639-648]. We here report evidence that 7B2 and proSAAS exhibit several elements of structural and functional homology. Firstly, 26 kDa human, mouse and rat proSAAS, like all vertebrate 7B2s, contain a proline-rich sequence within the first half of the molecule and also contain a C-terminal 40 residue peptide (SAAS CT peptide) separated from the remainder of the protein by a furin consensus sequence. The SAAS CT peptide contains the precise sequence of a hexapeptide previously identified by combinatorial peptide library screening as a potent inhibitor of PC1, and the vast majority of the inhibitory potency of proSAAS can be attributed to this hexapeptide. Further, like the 7B2 CT peptide, SAAS CT-derived peptides represent tight-binding competitive convertase inhibitors with nanomolar potencies. Lastly, recombinant PC1 is able to cleave the proSAAS CT peptide to a product with a mass consistent with cleavage following the inhibitory hexapeptide. Taken together, our results indicate that proSAAS and 7B2 may comprise two members of a functionally homologous family of convertase inhibitor proteins.

Amino Acid Sequence↗

PubMed2000

Immunohistochemical evidence for the involvement of protein convertases 5A and 2 in the processing of pro-neurotensin in rat brain.

The neuropeptides/neurotransmitters neurotensin (NT) and neuromedin (NN) are synthesized by endoproteolytic cleavage of a common inactive precursor, pro-NT/NN. In vitro studies have suggested that the prohormone convertases PC5A and PC2 might both be involved in this process. In the present study, we used dual immunohistochemical techniques to determine whether either one or both of these two convertases were co-localized with pro-NT/NN maturation products and could therefore be involved in the physiological processing of this propeptide in rat brain. PC2-immunoreactive neurons were present in all regions immunopositive for NT. All but three regions expressing NT were also immunopositive for PC5A. Dual localization of NT with either convertase revealed that NT was extensively co-localized with both PC5A and PC2, albeit with regional differences. These results strongly suggest that PC5A and PC2 may play a key role in the maturation of pro-NT/NN in mammalian brain. The regional variability in NT/PC co-localization patterns may account for the region-specific maturation profiles previously reported for pro-NT/NN. The high degree of overlap between PC5A and PC2 in most NT-rich areas further suggests that these two convertases may act jointly to process pro-NT/NN. At the subcellular level, PC5A was largely co-localized with the mid-cisternae Golgi marker MG-160. By contrast, PC2 was almost completely excluded from MG-160-immunoreactive compartments. These results suggest that PC5A, which is particularly efficient at cleaving the two C-terminal-most dibasics of pro-NT/NN, may be acting as early as in the Golgi apparatus to release NT, whereas PC2, which is considerably more active than PC5A in cleaving the third C-terminal doublet, may be predominantly involved further distally along the secretory pathway to release NN.

Animals↗

PubMed2000

Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro.

Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. Processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.

Amyloid↗

PubMed1999

Proglucagon processing profile in canine L cells expressing endogenous prohormone convertase 1/3 and prohormone convertase 2.

The tissue-specific differential processing of proglucagon (Pg) yields glucagon in pancreatic A cells and glucagon-like peptide-1 (GLP-1), GLP-2, and glicentin in intestinal L cells. It has been suggested that the difference in Pg cleavage in A and L cells is due to the presence of distinct prohormone convertases (PC) in the two cell types, PC1/3 in the L cell and PC2 in the A cell. PC2 has been shown to cleave the N-terminal part of Pg, being essential for glucagon formation and PC1/3 to cleave the C-terminal part of Pg, leading to the formation of GLP-1. However, some of the cleavage sites in Pg have not proven to be substrates exclusively for either PC2 or PC1/3, and the cleavage profile of Pg in a primary cultured L cell has not yet been correlated with the actual presence of PC2 and PC1/3 in the L cell. We demonstrate here the presence of PC1/3, PC2, and the PC2 chaperone 7b2, in L cells using light immunohistochemistry on sections from canine ileum and on a canine intestinal cell culture enriched for L cells. Analysis of the cultured L cells, using gel chromatography and RIA, confirms the classical intestinal cleavage profile of Pg, resulting in mainly glicentin, oxyntomodulin, GLP-1-(7-37), and GLP-2. Despite the presence of 7b2 and mature PC2, as demonstrated by Western blot, absolute minimal amounts of glucagon were detected. These data show that the presence of intracellular PC2 and 7b2 in a primary cell possessing Pg does not have to lead to the formation of glucagon. This formation must then require an additional element to occur, or alternatively, the results could be explained by a canine specific organization of PC2 and Pg into separate compartments, which would prevent interaction.

Animals↗

PubMed1999

Prohormone convertases 1 and 2 process ProPACAP and generate matured, bioactive PACAP38 and PACAP27 in transfected rat pituitary GH4C1 cells.

Pituitary adenylate cyclase-activating polypetide (PACAP) exists in two amidated forms, PACAP38 and PACAP27, which are expressed in the magnocellular and parvocellular neurons of the paraventricular nucleus (PVN) and the magnocellular neurons of the supraoptic nucleus (SON) of the hypothalamus. The prohormone convertases PC1 and PC2, subtilisin-like PCs of the Kex2 family, are expressed in neuroendocrine cells. Immunocytochemistry and in situ hybridization of PC1 and PC2 in the hypothalamus have shown that PC1 and PC2 are also present in the PVN and SON. Therefore, it is possible that the precursor of PACAP is processed by PC1 and/or PC2 in the hypothalamic nuclei and then converted to its mature forms. To test this hypothesis, rat pituitary GH4C1 cells were supertransfected with human PACAP cDNA and either rat PC1 or PC2 cDNA. The acid extracts of these cells were analyzed by reversed-phase HPLC for proPACAP, PACAP38 and/or PACAP27 radioimmunoassays using three antibodies with different recognition sites, and then bioassayed for the ability to stimulate adenylate cyclase. The cells transfected with PACAP cDNA alone yielded PACAP-like immunoreactivity (PACAP-li) corresponding to molecular weights between 15 and 20 kDa without PACAP bioactivity. Cotransfection of these cells with PC1 or PC2 generated PACAP-li, which coeluted with synthetic PACAP38 and PACAP27, respectively. Western blot also revealed 4.5- and 3.0-kDa PACAP-li bands, which correspond to the molecular weights of PACAP38 and PACAP27, respectively. The HPLC fractions containing PACAP-li, which were coeluted with synthetic PACAP38 and PACAP27, showed marked bioactivities. These findings suggest that the precursor of PACAP expressed in the PVN and SON of the hypothalamus could be efficiently processed by PC1 and PC2, and then converted to mature, bioactive PACAP38 and PACAP27.

Animals↗

PubMed1998

Identification of inhibitors of prohormone convertases 1 and 2 using a peptide combinatorial library.

A positional scanning synthetic peptide combinatorial library containing approximately 52 million hexapeptides was used to identify potential inhibitory peptides for recombinant mouse prohormone convertase 1 (PC1) and PC2 and to provide information on the specificity of these enzymes. The library surveys revealed that a P6 Leu, a P4 Arg, a P2 Lys, and a P1 Arg were most inhibitory against PC1, and a P6 Ile and a P4 Arg were most inhibitory against PC2. Using information derived from the library surveys, hexapeptide sets were synthesized and screened for inhibition of PC1 and PC2. The data obtained revealed the preference of both enzymes for a P3 Val. At P5, many substitutions were well tolerated. PC1 and PC2 proved to differ mainly in the selectivity of their S6 subsites. In PC1, this subsite displayed a strong preference toward occupation by Leu; the Ki value for peptide Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 28 times lower than that for peptide Ac-Ile-Ile-Arg-Val-Lys-Arg-NH2. In contrast, PC2 discriminated little between Leu and Ile at P6, as evidenced by the small (1.5-fold) difference in Ki values for these two peptides. Several hexapeptides synthesized as a result of the screen were found to represent potent inhibitors of PC2 (with Ki values in the submicromolar range) and, particularly, of PC1 (with Ki values in the low nanomolar range). The most potent inhibitor, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2, proved to be the same peptide for both enzymes and inhibited PC1 and PC2 in a competitive, fast-binding manner with Ki values of 3.2 and 360 nM, respectively. The four most potent peptide inhibitors of PC1 and PC2 were also tested against soluble human furin and found to exhibit a different rank order of inhibition; for example, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 440-fold less potent against furin than against PC1, with a Ki of 1400 nM.

Amino Acid Sequence↗

PubMed1993

Processing of two homologous precursors, pro-neuropeptide Y and pro-pancreatic polypeptide, in transfected cell lines expressing different precursor convertases.

The processing of two homologous precursors, pro-neuropeptide Y (pro-NPY) and pro-pancreatic poly-peptide (pro-PP), was studied in four neuroendocrine cell lines after transfection: CA-77 medullary thyroid carcinoma cells, AtT-20 corticotrope pituitary cells, RIN2A-19 pancreatic endocrine cells, and NB1 neuroblastoma cells. Northern blot analysis indicated that the AtT-20 cells only expressed precursor convertase 3; in contrast, NB1 cells only expressed precursor convertase 2, whereas the RIN2A-19 and CA-77 cells expressed both enzymes. Despite these differences in expression pattern of precursor convertases the four cell lines were, surprisingly, indistinguishable in respect to their processing of pro-PP and pro-NPY. In all four cell lines, pro-NPY was almost completely converted to NPY, and, in all four cell lines, only around 50% of the PP precursor was converted to PP. The relatively poor processing efficiency of pro-PP was rather similar to the processing efficiency of the endogenously produced precursors in the respective cell lines, pro-calcitonin (CA-77), proopiomelanocortin (AtT-20), proinsulin (RIN2A-19), and pro-vasoactive intestinal polypeptide (NB1). At least in the CA-77 cells, NPY and PP were apparently sorted to the regulated secretory pathway, as upon stimulation with secretagogue the release of the transfected peptides increased in parallel with the endogenously expressed peptide, calcitonin gene-related peptide. Mutagenesis studies showed that on the N-terminal side of the di-basic processing site, the otherwise important difference in structure between PP and NPY, a proline for glutamine in position 34, was not responsible for the difference in processing efficiency. On the C-terminal side of the processing site, the efficient processing of pro-NPY could not be transferred to pro-PP by exchanging the whole C-terminal domains of the precursors. It is concluded that pro-NPY is processed more efficiently than pro-PP in all neuroendocrine cell lines tested independent on their expression of the two main precursor convertases and that mutagenesis data indicate that the structural element responsible for the efficient processing of pro-NPY is not located on the N-terminal side of the dibasic processing site.

Amino Acid Sequence↗

PubMed2001

Can melatonin regulate the expression of prohormone convertase 1 and 2 genes via monomeric and dimeric forms of RZR/ROR nuclear receptor, and can melatonin influence the processes of embryogenesis or carcinogenesis by disturbing the proportion of cAMP and cGMP concentrations? Theoretic model of controlled apoptosis.

The presented model of controlled apoptosis has been based on the assumption that correct information exchange between an organism as a whole, and each of its cells is conditioned by mutual proportions of cAMP and cGMP concentrations (CcAMP, CcGMP), according to the formula CcAMP x CcGMP = 'a' (constant). The regulation of balance of these 'second messengers' in a cell and an extracellular space would depend on the mutual proportions of concentrations of Melatonin and monomers of Melanin. These indoloderived compounds could be the activators of the transcription factors i.e. RZR and NFkappa-B, regulating the expression of Prohormone Convertase (PC) gen and Nitric Oxide Synthase (NOS) gen, respectively. Additionally, maternal Melatonin and Nitric Oxide (NO), being able to pass through trophoblast or placenta freely, would play decisive role in the synchronization of embryogenesis and intrauterine development of the fetus. In case of an embryo or a fetus, the result of CcAMP and CcGMP multiplication, different from the proper constant 'a'-value, would mean occurrence of disorders in the structure and functioning of the cellular tensegrity system and, in consequence, disturbances in the intercellular information exchange. It would lead to deviation in cellular metabolism, oriented cell movement, uncontrolled apoptosis, and as a consequence, would lead to the development of fetal defects. In case of a child or an adult, a sudden occurrence and prolongation of such disturbances in CcAMP-CcGMP proportions would induce a process of apoptosis of normal cells and an initiation of a cancerogenesis. On the other hand, the recovery of equilibrium in the information exchange system would initiate apoptosis of neoplastic cells, and simultaneously, proliferation of connective tissue cells. According to the presented hypothesis, a decrease in CcAMP and destabilization of the CcAMP-CcGMP balance in an embryo or a fetus would result from relatively excessive amounts of maternal Melatonin (monomers) in fetal circulation, while a decrease of CcAMP and destabilization of the CcAMP-CcGMP balance in a child or an adult would be a consequence of relatively insufficient amounts of Melatonin (dimers) in an organism. It seems possible, that determination of both CcAMP and CcGMP would enable an early detection of high risk of developmental defects occurrence in an embryo or a fetus and neoplastic processes in a child or an adult. This method might also be considerably useful in monitoring a safe substitutional hormonotherapy.

Apoptosis↗

PubMed2001

IDA-1, a Caenorhabditis elegans homolog of the diabetic autoantigens IA-2 and phogrin, is expressed in peptidergic neurons in the worm.

The closely related mammalian proteins IA-2 and phogrin are protein tyrosine phosphatase-like receptor proteins spanning the membrane of dense core vesicles of neuroendocrine tissues. They are of interest as molecular components of the secretory machinery and as major targets of autoimmunity in type I diabetes mellitus. The Caenorhabditis elegans genome has a single copy of an IA-2/phogrin homolog ida-1 III (islet cell diabetic autoantigen), which encodes the ida-1 (B0244.2) gene product as a series of 12 exons over a 10-kb region of chromosome III. The full-length sequence of the ida-1 cDNA encoded a 767-amino acid type 1 transmembrane protein of 87 kDa. The PTP catalytic site consensus sequence of IDA-1, like IA-2 and phogrin, diverged and would not be active. Expression of green fluorescent protein (GFP) under the ida-1 gene promoter showed activity in a subset of around 30 neurons with sensory functions and the uv1 cells of the vulva in hermaphrodites. Males showed additional expression in male-specific neurons. In situ experiments in rat brain showing the distribution of IA-2 and phogrin suggested a complimentary and overlapping pattern compared with the proprotein convertases PC1 and PC2. In C. elegans, IDA-1-expressing cells comprised a subset of those expressing the PC2 homolog KPC-2 (C51E3. 7), consistent with IDA-1 being a component of neuropeptide-containing dense core vesicles. The results support the hypothesis that C. elegans IDA-1 is the functional homolog of IA-2 and phogrin in mammals. Analysis of the function of IDA-1 should contribute to our understanding of the function of these proteins in signal transduction, vesicle locomotion, and exocytosis.

Amino Acid Sequence↗

PubMed2003

Distribution and colocalization of cholecystokinin with the prohormone convertase enzymes PC1, PC2, and PC5 in rat brain.

During posttranslational processing to generate CCK 8, pro-cholecystokinin (CCK) undergoes endoproteolytic cleavage at three sites. Several studies using endocrine and neuronal tumor cells in culture and recombinant enzymes and synthetic substrates in vitro have pointed to the subtilisin/kexin-like enzymes prohormone convertase (PC) 1, PC2, and PC5 as potential candidates for these endoproteolytic cleavages. In these experimental models, they all appear to be able to cleave pro-CCK to make the correct products. One rodent model has provided information about the role of PC2. PC2 knockout mouse brains had less CCK 8 than wild-type, although a substantial amount of CCK was still present. The degree to which CCK levels were reduced in these mice was regionally specific. These data indicated that PC2 is important for normal production of CCK but that it is not the only endoprotease that is involved in CCK processing. To evaluate whether PC1 and PC5 are possible candidates for the other enzymes involved in CCK processing, the distribution of PC1, PC2, and PC5 mRNA was studied in rat brain. Their colocalization with CCK mRNA was examined using double-label in situ hybridization. PC2 was the most abundant of these enzymes in terms of the intensity and number of cells labeled. It was widely colocalized with CCK. PC1 and PC5 mRNA-positive cells were less abundant, but they were also widely distributed and strongly colocalized with CCK in the cerebral cortex, hippocampus, amygdala, ventral tegmental area, and substantia nigra zona compacta. The degree of colocalization of the enzymes with CCK was regionally specific. It is clear that PC1 and PC5 are extensively colocalized with CCK and could be participating in CCK processing in the rat brain and may be able to substitute for PC2 in its absence. These three enzymes may represent a redundant system to ensure production of biologically active CCK.

Amino Acid Sequence↗

PubMed1995

In vitro processing of anthrax toxin protective antigen by recombinant PC1 (SPC3) and bovine intermediate lobe secretory vesicle membranes.

Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell intoxicated. Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage. In this study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA. Recombinant PC1, partially purified from the medium of stably transfected L-cells, cleaved PA to a 63-kDa fragment (PA63) and a 20-kDa fragment (PA20). Amino-terminal sequence analysis of the 63 kDa product demonstrated that cleavage occurred between Arg167 and Ser168. The pH optimum for in vitro PA cleavage was 6.0 and the enzymatic activity was calcium-dependent. Medium from untransfected L-cells did not cleave PA. Site-directed mutagenesis of the tetrabasic cleavage site revealed that PC1 preferred to cleave sequences containing basic residues at positions -1 and -4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substrates at a monobasic residue site in vitro. Substrates having basic residues at the -1 and -2 positions were cleaved with approximately twofold less efficiency than wild-type PA. Mutants of PA containing basic residues in positions -1 and either -2 or -4 of the cleavage site were predicted to be substrates for PC1 and were more toxic to L-cells expressing PC1 than to untransfected L-cells. These results demonstrate that PA is cleaved by PC1 in vivo. Membranes from bovine intermediate lobe secretory vesicles which contain both prohormone convertases, PC1 and PC2, also cleaved PA to PA63 with a pH optimum of 5.5. Immunodepletion studies using antisera against PC1 and PC2 showed that these are the enzymes primarily responsible for the cleavage of PA in the membrane preparation. Thus, both recombinant PC1 and a membrane preparation containing endogenous PC1 can activate PA.

Amino Acid Sequence↗

PubMed1996

The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates.

Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.

Animals↗

PubMed2000

Synthesis of peptidyl methylcoumarin esters as substrates and active-site titrants for the prohormone processing proteases Kex2 and PC2.

Peptidyl methylcoumarin amides are well established as model substrates for understanding protease specificity, but the corresponding methylcoumarin esters have attracted scant attention despite their potential utility in active-site titration and mechanistic characterization. We have devised techniques for the synthesis and deprotection of extended peptidyl methylcoumarin esters in good to moderate yields, and we have demonstrated their suitability for steady-state characterization and active-site titration of the Saccharomyces cerevisiae processing protease Kex2. Additionally, we have used one of these compounds to active-site titrate the homologous enzyme PC2, which had not previously been feasible using other types of substrates. These compounds should thus prove widely suitable for use as substrates and active-site titrants not only for proteases of the prohormone processing family but also for a wide range of other serine proteases.

Binding, Competitive↗

PubMed1995

The role of prohormone convertases PC1 (PC3) and PC2 in the cell-specific processing of proglucagon.

To elucidate the mechanism of the differential processing of proglucagon, we analyzed the processing products of proglucagon in three types of rodent endocrine cells and their relation to prohormone convertases PC1 (PC3) and PC2. Proglucagon gene was transfected into AtT-20 cells and GH3 cells, which are derived from pituitary tumors. InR1-G9 cells, which are insulinoma-derived cells, express an endogenous proglucagon gene. Oxyntomodulin was the predominant processing product in AtT-20 cells, which contained abundant PC1 mRNA. In contrast, glucagon was the major product in GH3 cells, which expressed PC2 mRNA. Oxyntomodulin and glucagon were produced in equal amounts in InR1-G9 cells, which expressed both PC1 and PC2 mRNAs. These findings suggest that PC1 and PC2 preferentially cleave proglucagon into oxyntomodulin and glucagon, respectively, thus contributing to the cell-specific processing of proglucagon.

Animals↗

PubMed1998

Pro-neurotensin/neuromedin N expression and processing in human colon cancer cell lines.

The regulatory peptide neurotensin NT has been proposed to exert an autocrine trophic effect on human colon cancers. In the present study, pro-neurotensin/neuromedin N (proNT/NN) expression and processing were investigated in 13 human colon cancer cell lines using a combination of radioimmunoassay and HPLC techniques. All 13 cell lines displayed low to moderate levels of proNT/NN ranging from 10 to 250 fmol/mg protein. However, only 6 (HCT8, LoVo, HT29, C119A, LS174T, and coloDM320) processed the precursor. Three of the latter (HCT8, LS174T, and coloDM320) were analysed in detail with regard to proNT/NN processing pattern and were found to produce NT and large precursor fragments ending with the NT or NN sequence. They had no detectable level of NN. Such a processing pattern resembles that generated by the prohormone convertase PC5. Northern and Western blot analysis of prohormone convertase expression in the 3 cell lines revealed that they were devoid of PC1 and PC2, whereas they all expressed PC5. These data indicate that proNT/NN is a good marker of human colon cancer cell lines while NT is found in only about half of the cell lines. They also suggest that, in addition to NT, several proNT/NN-derived products, possibly generated by PC5, might exert an autocrine positive effect on human colon cancer growth.

Aspartic Acid Endopeptidases↗