Purification and characterization of a paired basic residue-specific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles

Pro-opiomelanocortin (adrenocorticotropin/endorphin prohormone) is processed to yield active hormones by cleavages at paired basic amino acid residues. In this study, an enzyme that specifically cleaves at the paired basic residues of this prohormone has been purified from bovine pituitary intermediate lobe secretory vesicles, the intracellular processing site of proopiomelanocortin. This enzyme, named pro-opiomelanocortin converting enzyme, has been characterized as a glycoprotein of Mr approximately 70,000. It has an apparent isoelectric point between 3.5 and 4.0. The pH optimum of the pro-opiomelanocortin converting enzyme is between 4 and 5, but the enzyme is highly active at the intravesicular pH of 5.1-5.6. The enzyme specifically cleaved the Lys-Arg pairs of pro-opiomelanocortin to yield Mr = to 21,000-23,000 ACTH, beta-lipotropin, Mr 13,000 and 4,500 ACTH, beta-endorphin, and a Mr = 16,000 NH2-terminal glycopeptide, the products synthesized by the pituitary intermediate lobe in situ. NH2- and COOH-terminal analysis of the products indicated that the pro-opiomelanocortin converting enzyme cleaves the peptide bond either between the Lys and Arg or on the carboxyl side of the Arg at Lys-Arg pairs of pro-opiomelanocortin. The intracellular localization, pH optimum, and cleavage specificity of the enzyme suggest that it may function as a pro-opiomelanocortin processing enzyme in the pituitary intermediate lobe in vivo.     

Chromogranin A can act as a reversible processing enzyme inhibitor. Evidence from the inhibition of the IRCM-serine protease 1 cleavage of pro-enkephalin and ACTH at pairs of basic amino acids

Bovine parathyroid chromogranin A inhibits the cleavage of Z-Ala-Lys-Arg-AMC by either trypsin or IRCM-serine protease 1 (IRCM-SP1), a putative novel processing enzyme originally isolated from porcine pituitary anterior and neurointermediate lobes. On larger substrates, chromogranin A is a reversible competitive inhibitor of the cleavage at pairs of basic amino acids by IRCM-SP1. The substrates tested included pituitary ACTH and adrenal medulla pro-enkephalin-derived peptides such as the 8.6 kDa synenkephalin-containing precursor and peptide B. Chromogranin A is itself selectively processed by IRCM-SP1, and ACTH was shown to compete for such cleavage. These data suggest that chromogranins as a class of acidic proteins could participate in the tissue-specific processing of pro-hormones.     

Pro-opiocortin processing in the pituitary: A model for neuropeptide biosynthesis

The biosynthesis of α-MSH, β-endorphin and ACTH in the pituitary is reviewed. These neuropeptides are synthesized from a common pro-protein, pro-opiomelanocortin. The pro-protein is cleaved intragranularly, at pairs of basic residues in the molecule to yield the respective peptide products. An unique, thiol protease (pro-opiocortin converting enzyme) and a carboxypeptidase B-like enzyme, both localized within pituitary secretory granules and having a pH optimum of 5–6, appear to be involved in the proteolytic processing of pro-opiomelanocortin.     

Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. A distinct acidic, tetrabasic residue-specific calcium-activated serine protease and a PC2-like enzyme.

Adrenocorticotropin (ACTH) is cleaved at the tetrabasic residue site, in pituitary intermediate lobe secretory vesicles, to yield ACTH1-17 and corticotropin-like intermediate lobe peptide (CLIP). ACTH1-17 is then converted to alpha-melanocyte-stimulating hormone (N-AcACTH1-13NH2) by first removing the Lys15-Lys16-Arg17 residues, followed by amidation of the COOH terminus and acetylation of the NH2 terminus. Bovine intermediate lobe secretory vesicle membranes were screened for proteolytic enzyme activity that will cleave the tetrabasic residues of ACTH. Two activities with pH optima of 5.0-6.0 and 7.5-8.0 were detected. The acidic, ACTH-converting enzyme cleaved ACTH1-39 at the tetrabasic residues between the Arg17-Arg18 bond to yield ACTH1-17 and CLIP, but did not cleave paired basic residues of pro-opiomelanocortin. This enzyme activity was characterized as a Ca(2+)-activated serine protease with unique specificity for the tetrabasic residues of ACTH1-39. The neutral activity preferentially generated ACTH1-17 and to a small extent ACTH1-16 from ACTH1-39 and ACTH1-24. This enzyme activity was Ca(2+)-dependent but was not inhibited by serine or aspartic protease inhibitors. The neutral activity was significantly immunodepleted by antiserum raised against bovine PC2/PC3, and together with specificity studies, suggests that the enzyme is a PC2-like serine protease. The pH optimum, distinct specificity for tetrabasic residues, and subcellular localization of the acidic ACTH-converting enzyme indicate a function of this enzyme in the in vivo conversion of ACTH1-39 to alpha-melanocyte-stimulating hormone in intermediate lobe secretory vesicles which have an acidic internal pH.     

A novel serine protease (IRCM-serine protease 1) from porcine neurointermediate and anterior pituitary lobes. Isolation, polypeptide chain structure, inhibitor sensitivity, and substrate specificity with fluorogenic peptide substrates

A novel serine protease, which we have called IRCM-serine protease 1, was purified from both porcine neurointermediate and anterior pituitary lobes. The enzyme was inhibited by soybean trypsin inhibitor, pancreatic trypsin inhibitor, benzamidine, phenylmethyl-sulfonyl fluoride, and thiol reagents including HgCl2, p-chloromercuribenzoate, and 5,5'-dithiobis-(2-nitrobenzoic acid) and was resistant to lima bean trypsin inhibitor, alpha 2-macroglobulin, alpha 1-antitrypsin, and C1-esterase inhibitor. IRCM-serine protease 1 displayed "trypsin-like" specificity toward a number of tripeptide coumarin-containing substrates, with kcat/km values ranging from 10(4) to 10(6) M-1 S-1. The best substrate was benzyloxycarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide with a kcat/Km value of 2.27 X 10(6) M-1 S-1. IRCM-serine protease 1, Mr = 169,000-190,000 determined by gradient gel electrophoresis and gel filtration, respectively, appears to be a homologous dimer. The monomeric subunits of the enzyme are composed of an Mr = 38,000 polypeptide chain which is modifiable by 125I-D-Tyr-Glu-Phe-Lys-Arg-CH2Cl, disulfide-linked to another polypeptide resulting in a subunit molecular weight of 88,000.     

Structural and immunological homology of human and porcine pituitary and plasma IRCM-serine protease 1 to plasma kallikrein: marked selectivity for pairs of basic residues suggests a widespread role in pro-hormone and pro-enzyme processing

IRCM-serine protease 1 (SP1), originally isolated from porcine pituitaries and exhibiting preference for cleavage at pairs of basic residues has now been isolated in sufficient quantities to be structurally characterized from both porcine and human pituitaries and plasmas. Whereas the porcine protease shows a high degree of amino acid sequence homology to human plasma pre-kallikrein, the human homologue exhibits an identity of sequence in the first 25 residues of each chain (regulatory and catalytic chains). In addition, human plasma and pituitary IRCM-SP1 and human plasma pre-kallikrein show virtually identical immunological and molecular properties. These data strongly suggest that IRCM-SP1 and plasma pre-kallikrein originate from the same gene product. Purified extracts from perfused rat pituitaries show that 32% of the IRCM-SP1 activity found in normal rat pituitaries, still remain. These data together with the demonstrated association of IRCM-SP1 with particulate fractions of the pituitary suggest that IRCM-SP1 represents a tissue form of plasma pre-kallikrein. The characterization of the digestion products obtained upon reaction of IRCM-SP1 with pro-insulin, ACTH1-39, pro-dynorphin and pro-enkephalin-derived peptides, somatostatin-28, and a pro-renin-like peptide confirmed the high degree of cleavage selectivity of this enzyme for pairs of basic residues.     

Localization of a 16,000-dalton fragment of the common precursor of adrenocorticotropin and beta-lipotropin in the rat and human pituitary gland

To clearly identify cells and organelles containing the common precursor (31,000 dalton) for both adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), an immunohistochemical localization of a fragment (16,000 dalton) of the precursor that is not common to beta-LPH and ACTH was conducted in rat and human pituitary glands. With the help of specific antibodies that do not cross-react with beta-LPH and ACTH, the 16,000-dalton fragment was localized in the cells that also produce ACTH and beta-LPH in both the pars distalis and pars intermedia of the rat pituitary. At the electron microscope level, the secretory granules that contain ACTH were also stained for 16,000-dalton fragment. In the human pituitary, the 16,000-dalton fragment was also observed in all the secretory granules of lipocorticotrophs. These results suggest that, after enzymatic cleavage, fragment(s) of the common precursor and/or the whole common precursor remain packaged within the secretory granules with peptides of known activity.     

An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in AtT20 cells before its cleavage

We have raised a rabbit antiserum against a synthetic peptide corresponding to the cleavage site between beta-lipotropic hormone and the ACTH moieties of murine pro-opiomelanocortin (POMC). After affinity purification, the anti-cleavage site antibody immunoprecipitates POMC from extracts of AtT20 cells but it does not immunoprecipitate the ACTH in such extracts or any of the other products of cleavage of POMC. By contrast, an antiserum raised against pure swine ACTH immunoprecipitates both POMC and ACTH from AtT20 cell extracts. Using the anti-cleavage site antibody we have shown that all the POMC synthesized during a 15-min pulse-labeling with [35S]methionine is cleaved at this site within 1 h. By immunoelectron microscopy we show that approximately 25-30% of peripheral secretory granules in AtT20 cells can be labeled with the anti-cleavage site antibody while anti-ACTH antiserum labels all these granules. This establishes that at least some POMC is packaged into secretory granules before its proteolytic cleavage.     

Reactivity of basic amino acid pairs in prohormone processing: model of pro-ocytocin/neurophysin processing domain

Statistical analysis of several potential dibasic cleavage sites reveals differences in the distribution of basic doublets when the in vivo cleaved sites were compared to those which are not cleaved. Analysis of the substrate specificity of protease Kex2 towards the pro-ocytocin/neurophysin processing domain (pro-OT/Np(7-15) with altered basic pairs shows a cleavage efficiency order in accord with the statistical data. Structural analysis of these substrates indicates that each basic pair is associated with a local and specific conformational change. Thus, the in vivo cleavage hierarchy of dibasic sites is encoded by both the nature of basic pairs and the plasticity of proteolytic processing domains.     

An in vivo characterization of the cleavage site specificity of the insulin cell prohormone processing enzymes

Most peptide hormones and neurotransmitters are synthesized as larger precursor proteins, which are post-translationally processed to mature bioactive products. An early event in prohormone maturation is endoproteolytic cleavage, occurring usually at pairs of basic amino acids (e.g. Lys-Arg). Since many of the characteristics of a prohormone endoprotease are unknown, distinguishing these enzymes from other cellular proteases in vitro has been difficult. In this report, the substrate specificity of a model prohormone processing system, the insulinoma cell line Rin m5F, was characterized in vivo to establish a set of criteria by which putative proinsulin endoproteases may be assessed. To determine the role of composition of the paired basic amino acid site in directing cleavage, a series of mutant prohormones containing altered cleavage sites was constructed and expressed in Rin m5F cells. Proopiomelanocortin (POMC) was used as a substrate since this prohormone was previously shown to be processed by these cells. To control for positional effects, all four permutations of lysine and arginine (Lys-Arg, Arg-Arg, Arg-Lys, and Lys-Lys) were introduced at both the efficiently processed cleavage site separating the ACTH and beta-lipotropin (beta-LPH) domains of POMC and at the inefficiently processed site in the beta-endorphin sequence near the COOH-terminus of the precursor. His-Arg and Met-Arg sites were also introduced at the ACTH/beta-LPH junction to assess the requirement for paired lysines and arginines. Identification of POMC-derived peptides demonstrated efficient processing of Lys-Arg and inefficient processing of Lys-Lys and Arg-Lys sites at both positions in the prohormone. The Arg-Arg sequence, however, was processed in a position-dependent manner, being efficiently cleaved between ACTH and beta-LPH but only about 50% processed within beta-endorphin. His-Arg was not cleaved in Rin m5F cells, although surprisingly Met-Arg was partially processed. These results indicate a strict preference of the insulinoma prohormone endoprotease(s) for paired basic amino acids ending in arginine, but that processing efficiency of some sequences may be modulated by location within the precursor molecule.     

Proteolytic dissection of rat brain hexokinase: determination of the cleavage pattern during limited digestion with trypsin

Limited treatment of rat brain hexokinase (ATP: D-hexose-6-phosphotransferase; EC 2.7.1.1) with trypsin causes cleavage of the Mr 98K enzyme into three major fragments having molecular weights of 10K, 40K, and 50K, with intermediates of Mr 60K and 90K being detected. This information, in conjunction with N- and C-terminal analysis of the intact enzyme and tryptic cleavage products, has established the tryptic cleavage pattern as where T1 and T2 indicate tryptic cleavage sites; cleavage at only T1 or T2 gives rise to the 90K or 60K intermediate, respectively. Confirmation of this cleavage pattern has been provided by two-dimensional peptide mapping using Staphylococcus aureus V8 protease, and epitope mapping with two monoclonal antibodies directed against rat brain hexokinase. The epitopes recognized by one of the monoclonal antibodies is located within the 40K C-terminal fragment while the epitope for the other monoclonal antibody lies within the 50K fragment. A two-dimensional peptide mapping-immunoblotting technique has permitted a more defined localization of these epitopes to specific regions within these major tryptic cleavage fragments. Complete tryptic cleavage of the enzyme occurs with only modest (approximately 20%) loss of catalytic activity, and the cleaved enzyme retains many of the properties of intact hexokinase. Specifically, there was no effect of cleavage on the Km for Glc or the Ki for Glc-6-P, though a slight decrease in Km for ATP was consistently noted to result from cleavage. Furthermore, like the intact enzyme, cleaved hexokinase retained the ability to bind to outer mitochondrial membranes in a Glc-6-P-sensitive manner. Under nondenaturing conditions, the cleaved fragments remain associated by noncovalent forces. Thus, the cleaved enzyme sedimented at a rate comparable to intact enzyme during centrifugation on sucrose density gradients, and migrated only slightly faster when electrophoresed on gradient acrylamide gels under nondenaturing conditions.     

Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates.

In addition to kallikrein hK3, a serine protease generally reported as PSA (prostate-specific antigen), at least two other enzymes in human seminal plasma also cleave synthetic peptidyl substrates derived from the sequence of human semenogelins. We have identified one of these as prostatic acid phosphatase (PAP), a major component of prostatic fluid whose physiological function is unclear. The other is a high Mr basic protein present at low concentrations in seminal plasma and that remains to be characterized. PAP was purified to homogeneity from freshly ejaculated seminal plasma. Its N-terminal sequence and its phosphatase properties (hydrolysis of para-nitrophenylphosphate at low pH) were determined, and its inhibition by sodium fluoride measured. Both purified and commercial PAP also had amidolytic activity on peptide substrates derived from the semenogelin sequence at neutral and slightly basic pH. The k(cat)/K(m) values were in the 10(2)-10(3) m(-1) x s(-1) range using fluorogenic semenogelin-derived substrates whose peptidyl moiety included cleavage sites that had been identified ex vivo. PAP cleavage sites differed from those of hK3 and were mainly at P1 = Gln residues or between residues bearing hydroxyl groups. PAP amidolytic activity was poorly inhibited by all currently used wide spectrum proteinase inhibitors. Only 3-4 dichloroisocoumarin and benzamidine inhibited purified PAP. Purified human semenogelin was cleaved by purified and commercial PAP at neutral pH; the two main cleavage sites were at Tyr292 and Ser170 (semenogelin I sequence), only the former has been identified ex vivo by analysis of seminal plasma.     

Generation of Lys-gamma 3-melanotropin from pro-opiomelanocortin 1-77 by a bovine intermediate lobe secretory vesicle membrane-associated aspartic protease and purified pro-opiomelanocortin converting enzyme

The ability of bovine intermediate lobe secretory vesicle membrane-associated enzyme(s) and purified, soluble paired basic residue-specific, pro-opiomelanocortin converting enzyme (Loh, Y.P., Parish, D. C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) to cleave bovine NH2-terminal pro-opiomelanocortin1-77 (N-POMC 1-77) was investigated. Purified pro-opiomelanocortin converting enzyme and an enzyme activity associated with the secretory vesicle membrane were shown to cleave bovine N-POMC1-77 to two major products: N-POMC1-49 and Lys-gamma 3-melanotropin (MSH), and one minor product, gamma 3-MSH. These products were identified by their retention times on high performance liquid chromatography, immunological characteristics, and for Lys-gamma 3-MSH, amino acid composition. The products generated indicate cleavage preferentially between Arg 49-Lys 50 of bN-POMC1-77 (where b indicates bovine), which is identical to the processing pattern found in the bovine intermediate lobe in situ. The membrane converting activity was shown to be stimulated by 5 mM Ca2+ and has a pH optimum of 4-5 and an inhibitor profile characteristic of an aspartic protease. This suggests that the membrane-associated enzyme involved is very similar or identical to the purified, soluble pro-opiomelanocortin converting enzyme, which has previously been reported to be an acidic, aspartic protease responsible for the initial steps of POMC processing. The results of this study lead to the proposal that the lack of processing of the Arg49-Lys50 site in POMC in the anterior lobe versus the intermediate lobe of the pituitary in vivo may be due to other regulatory mechanisms rather than invoking the existence in the intermediate lobe of another enzyme specific for this site, different from pro-opiomelanocortin converting enzyme.     

TRAF1 is a substrate of caspases activated during tumor necrosis factor receptor-alpha-induced apoptosis

TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-alpha- and Fas-induced apoptosis in vivo. (160)LEVD(163) was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)- and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1- and TRAF2-mediated nuclear factor-kappaB activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF- and Fas-induced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.     

Expression of variant forms of proopiomelanocortin, the common precursor to corticotropin and beta-lipotropin in the rat pars intermedia

Proopiomelanocortin, the common glycoprotein precursor to adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), is the most abundant protein synthesized in rat neurointermediate lobes. It represents 30% of the total amount of radioactive proteins obtained after a 1-h pulse incubation with [3H]phenylalanine. Several forms of this protein can be separated by a high-resolution two-dimensional gel electrophoresis technique. The three most abundant species which can be reproducibly characterized by their apparent molecular weights (Mr) and isoelectric points (pI) were called form I (Mr 34 000; pI 8.2), form II (Mr 36 000; pI 8.2), and form III (Mr 35 000; pI 7.3). Additional minor forms, representing together approximately 30% of the total forms I, II, and III combined, are also observed. They have very close molecular weights but differ by their isoelectric points. When glycosylation is prevented by tunicamycin, forms I and II are replaced by a new molecule with the same pI of 8.2 but a slightly lower Mr (32 000). This form is referred to as form T1. Similarly, form III is replaced by form T2 (Mr 33 000; pI 7.3). Forms T1 and T2 are supposed to be nonglycoslyated peptides. They were further characterized by microsequencing and peptide mapping. They both have the same N-terminal amino acid sequence with leucine residues in positions 3 and 11, and they both contain identical [3H]phenylalanine-labeled tryptic fragments, two of them corresponding to the sequences 1-8 of ACTH and 61-69 of beta-LPH. However, a limited digestion with the Staphylococcus aureus (V8 strain) protease generates a collection of peptides different for each form. These results suggest the presence of at least two different gene products corresponding to the major forms of proopiomelanocortin in the rat pars intermedia.     

Limited proteolysis of filamin is catalyzed by caspase-3 in U937 and Jurkat cells

Members of the caspase family have been implicated as key mediators of apoptosis in mammalian cells. However, few of their substrates are known to have physiological significance in the apoptotic process. We focused our screening for caspase substrates on cytoskeletal proteins. We found that an actin binding protein, filamin, was cleaved from 280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135, 120, and 110 kDa major C-terminal fragments when apoptosis was induced by etoposide in both the human monoblastic leukemia cell line U937, and the human T lymphoblastic cell line Jurkat. The cleavage of filamin was blocked by a cell permeable inhibitor of caspase-3-like protease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ac-YVAD-cho. These results suggest that filamin is cleaved by a caspase-3-like protease. To examine whether caspase-3 cleaves filamin in vitro, we prepared a recombinant active form of caspase-3 directly using a Pichia pastoris overexpression system. When we applied recombinant active caspase-3 to the cell lysate of U937 and Jurkat cells, filamin was cleaved into the same fragments seen in apoptosis-induced cells in vivo. Platelet filamin was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-terminal fragments, and the cleavage pattern was the same as observed in apoptotic human cells in vivo. These results suggest that filamin is an in vivo substrate of caspase-3.     

Purification of an endopeptidase from bovine adrenal medulla granules which cleaves in vitro at paired but not at single basic residues

An endopeptidase was isolated from bovine adrenomedullary granules by fast protein liquid chromatography, including two ion exchange, one hydrophobic interaction, and one gel filtration step. The enzyme assay was based on the HPLC detection of the degradation of the dodecapeptide BAM 12P from the sequence of proenkephalin. After a 1200-fold purification, the enzyme fraction was homogeneous on polyacrylamide gel electrophoresis. The apparent molecular weight of the monomeric protein was 68,000 and its pH optimum was 5.6, in agreement with the internal pH of the granules. The specificity of the protease was determined initially by analysis of the degradation fragments of BAM 12P which showed that cleavage occurred at the double but not at the single Arg site of BAM 12P. The cleavage pattern of other substrates showed that the enzyme also recognized other pairs of basic amino acids. The behavior of the enzyme toward specific inhibitors showed that it was an acid thiol protease different from serine proteases and from lysosomal cathepsin B. The endopeptidase may act as a maturation enzyme in vivo.     

Cytoimmunological and histochemical study of cat adenohypophyseal cells, made fluorescent by the Falck and Hillarp technic]

In the Cat, after Falck and Hillarp method, all the fluorescent cells of the PI and the anterior lobe of adenohypophysis can be revealed with specific anti-sera to ACTH(1-24), ACTH(17-39), bovine beta-MSH and porcine beta-LPH. With the lead hematoxyline staining method, two types of cells are recognizable in the anterior lobe, in which the non hormonal constituents of the granules must be different.