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.     

Pro-opiomelanocortin-derived peptides in the pig pituitary: alpha- and gamma 1-melanocyte-stimulating hormones and their glycine-extended forms

Pro-opiomelanocortin (POMC)-related peptides in extracts of anterior and neurointermediate pituitary lobes from pigs were characterized by gel chromatography, reversed-phase chromatography and radioimmunoassays. The peptide content was ca. 3-fold greater in the anterior lobe compared to the neurointermediate lobe (19.8 nmol POMC/anterior lobe vs 7.0 nmol/neurointermediate lobe). In the neurointermediate lobe 93% of POMC was processed to alpha-melanocyte-stimulating hormone (alpha-MSH) and analogs exclusively of low molecular weight. Most of the remaining adrenocorticotropic hormone (ACTH)-related material consisted of the glycine-extended intermediate ACTH-(1-14) and analogs. In contrast only one fourth to one third of the N-terminal part of POMC (N-POMC) was processed to amidated gamma-MSH and its C-terminal glycine-extended precursor. The relative amount of amidated gamma-MSH was the same as alpha-MSH and analogs (94%). However, more than 95% of these peptides were of high molecular weight. In the anterior lobe 2.3% of N-POMC was processed and 94% was amidated gamma-MSH of only high molecular weight. These results show that gamma-MSH and alpha-MSH are amidated to the same extent and that gamma 1-MSH and gamma 2-MSH immunoreactivity are present in both the anterior lobe and the neurointermediate lobe. The results suggest that the production of amidated peptides is not regulated by the amidation process itself but at an earlier step (e.g. at the proteolytic cleavage).     

Subcellular Localization, Partial Purification, and Characterization of a Dynorphin Processing Endopeptidase from Bovine Pituitary

An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is subsantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7:1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.     

Effect of tunicamycin on biosynthesis, processing and release of proopiomelanocortin-derived peptides in the intermediate lobe of the frog Rana ridibunda

The intermediate lobe of the pituitary gland synthesizes a glycoprotein, proopiomelanocortin (POMC), which is cleaved by specific proteolytic enzymes to generate several hormonal peptides. The purpose of the present study was to examine the possible role of the carbohydrate moiety in the synthesis, intracellular processing and release of POMC-derived peptides in frog (Rana ridibunda) intermediate lobe cells. In vitro incorporation of [3H]-labelled glucosamine gave rise to three major radioactive products. Trypsin digestion of each of these glycopeptides gave a single glucosamine-labelled tryptic fragment with identical chromatographic characteristics. We conclude that Rana POMC is glycosylated in only one site (its gamma-MSH region) and that intracellular processing of this prohormone gives rise to smaller glycopeptides including glycosylated gamma-MSH. Treatment with the antibiotic tunicamycin (10 micrograms/ml, 6 hr) inhibited the glycosylation of POMC but did not significantly alter the neosynthesis of the peptide moiety of the precursor. Pulse-chase experiments combined with high-performance liquid chromatography analysis of the peptides derived from POMC revealed that inhibition of glycosylation by tunicamycin had no effect on the enzymatic cleavage of the precursor nor on the release of mature peptides. Thus, it is concluded that, in the frog, glycosylation of POMC has no influence on the biosynthesis, processing and release of intermediate lobe hormones.     

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.     

Biosynthesis and processing of pro-opiomelanocortin-derived peptides during fetal pituitary development

We have investigated the molecular weight forms of pro-opiomelanocortin (POMC)-derived peptides present in rat pituitaries during fetal and early postnatal development (embryonic Day 14 to 3 day neonate). At all early ages examined, the major immunoreactive form of corticotropin (ACTH) was POMC. Only during late fetal and early postnatal stages did progressively larger amounts of 4.5K ACTH, a major POMC processing end product, appear. This form was found almost exclusively in isolated anterior lobes. In contrast, 3.5K size endorphin(s), another POMC derivative, were present in whole glands even at early stages (Day 14), and were the major POMC derivative(s) found in isolated intermediate-posterior lobes of older fetuses. Despite the early appearance of 3.5K endorphin(s), α-MSH did not appear until Day 19 and was detected only in isolated intermediate-posterior lobes. We have also cultured dispersed fetal pituitary cells in the presence of radioactive amino acids. After immunoprecipitation using affinity-purified antisera, followed by fractionation of the radiolabeled products, we found that POMC biosynthesis does occur in cultures of Day 14 embryonic pituitary cells, and that the major POMC-derived end product produced is 3.5K size endorphin(s). These findings demonstrate that POMC is synthesized at least by Day 14 of rat pituitary development and that lobe-specific processing characteristic of the corresponding adult lobe is apparent at the earliest stages that the lobes can be separated. The presence of 3.5K-sized endorphins at early ages is consistent with the possibility that POMC synthesis first occurs in the intermediate lobe. The noncoordinate appearance of α-MSH, 1–39 ACTH, and endorphins implies that the activities of certain cleavage enzymes and acetylation enzymes responsible for lobe-specific post-translational POMC processing may be expressed at different times during development.     

Homologous IRCM-Serine Protease 1 from pituitary,heart atrium and ventricle: A common pro-hormone maturation enzyme?

IRCM-Serine Protease 1 (IRCM-SP1) has recently been isolated and characterized from porcine pituitary anterior and neurointermediate lobes (Cromlishet al., 1986a,J. Biol. Chem. 261:10850–10858; Cromlishet al., 1986b,J. Biol. Chem. 261:10859–10870). This pituitary serine protease was shown to selectively cleave human proopiomelanocortin (POMC)-derived peptides at both pairs of basic residues and C-terminal to specific Arg residues, all known to be cleavedin vivo. Here, a similar enzyme was isolated from rat heart atria and ventricles. Rat IRCM-SP1 was shown to be highly specific for the same cleavage sites in POMC, as the porcine pituitary homologue. Furthermore, the rat and the porcine enzymes cleave rat pro-Atrial Natriuretic Factor (pro-ANF 1–126) to yield ANF 103–126, 102–126 and 99–126 in that order of preference. This suggests thatin vitro the cleavage sites preferred in pro-ANF resemble those found in brain and hypothalamus. The enzyme is nine times more abundant in atria versus ventricles/mg protein. It is concluded that IRCM-SP1, could well represent a common pro-hormone maturation enzyme for POMC and Pro-ANF and possibly many other pro-hormones.     

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.     

Corticotrophs and peptides

Corticotrophs were long thought to be a static, homogeneous population of cells that respond positively to hypothalamic stimulation, are inhibited by glucocorticoid feedback and secrete a single biologically active peptide, ACTH(1-39). Our current understanding is that this is an oversimplification and corticotrophs are a dynamic and more complex group of cells. The biosynthetic precursors of ACTH and other cleavage products of proopiomelanocortin (POMC) have been found to be secreted by anterior pituitary cells, to circulate and to have biological activity. POMC and the biosynthetic intermediate, pro-ACTH, exert activity antagonistic to ACTH(1-39) on glucocorticoid secretion by adrenal cells, and other derivatives of POMC are mitogenic to adrenocortical cells. In terms of responses to hypothalamic and peripheral factors, corticotrophs are functionally heterogeneous. This is reflected in the sensitivity of individual subtypes of corticotrophs to CRH, vasopressin and glucocorticoids. There is a functional plasticity amongst the various types of corticotrophs. During gestation, in fetal sheep, changes occur in the overall ACTH-secretory responses to CRH relative to vasopressin, the proportions of total corticotrophs that respond to the respective peptides and the average secretory response of individual cells. Corticotrophs also respond to locally produced pituitary factors. Local actions of leukaemia inhibitory factor are demonstrated by the effects of immunoneutralization of the peptide in pituitary cells. Urocortin and preproTRH(178-199) are locally produced peptides with potent stimulatory and inhibitory actions on corticotrophs, respectively. The specific roles of these peptides are under investigation.     

"Joining peptide" of pro-opiomelanocortin. II. Interspecies heterogeneity of the joining peptide fragment

The use of an antiserum raised against the joining peptide sequence -23 to -14 of bovine pro-opiomelanocortin (POMC) enabled the detection of related immunoreactive sequences of peptides in bovine, porcine, mouse and guinea-pig pituitaries, as well as in mouse brain and cerebral cortex, guinea-pig cerebral cortex, and bovine hypothalamus. Gel chromatography of pituitary extracts (Sephadex G-75 and Bio-Gel P-4) indicated the presence of several immunoreactive joining peptide fragments ranging in the molecular weight range (Mr) of 1,500 to 2,300. Furthermore, high molecular weight (Mr greater than 22,500) immunoreactive-precursor from bovine anterior pituitary was readily digested with trypsin into an immunoreactive fragment of approximately Mr 1,500. Analyses of these immunoreactive peptides by reverse-phase high-performance liquid chromatography (HPLC) led to their resolution into six distinct peptides. The only apparent correspondence in the elution profiles of immunoreactive peptide profiles between different mammalian species was the identification of a similar fragment (Mr 2,000) from bovine and guinea-pig pituitaries. Thus, we conclude that immunoreactivity to the joining peptide region of POMC from various mammalian species exhibits a degree of heterogeneity in its composition. The relatively low levels of immunoreactivity in comparison to that of ACTH also suggest that the joining peptide domain may be further processed. The hormonal status of the joining peptide region remains to be determined.     

Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate

Cleavage of specific peptide bonds occurs with aging in the alpha A subunit of bovine alpha-crystallin. One of the breaks occurs at residue Asn-101. This same residue undergoes in vivo deamidation, isomerization, and racemization. Deamidation and isomerization are known to occur via succinimide ring formation of labile asparagine residues. Model studies on peptides have shown that imide formation can also lead to peptide bond cleavage (Geiger, T., and Clarke, S. (1987) J. Biol. Chem. 262, 785-794). In that case, both asparagine and aspartic acid amide would be expected as C termini of the truncated polypeptide, and this is indeed the case in the alpha A-(1-101)-chain. This thus represents a first example of nonenzymatic in vivo peptide bond cleavage in an aging protein through the formation of a succinimide intermediate. In addition, we found that in bovine lens no detectable conversion (through the action of protein-carboxyl methyltransferase) of isoaspartyl to normal aspartyl residues occurs in vivo after deamidation of Asn-101.     

"Joining peptide" of pro-opiomelanocortin. I. Radioimmunoassay and extraction of related peptides from pituitary glands

In order to immunoassay the specific region of bovine pituitary pro-opiomelanocortin (POMC) between ACTH and gamma-MSH, referred to as "joining peptide," antisera were prepared against the synthetic amidated decapeptide Val-Ala-Val-Gly-Glu-Gly-Pro-Gly-Pro-Arg-NH2. The non-amidated peptide represents residues -23 to -14 of bovine POMC. An NH2-terminal tyrosine analog of the decapeptide was used as the radioligand. Under optimal conditions, immunoassay with selected antisera exhibited a sensitivity (50% displacement of the radioligand) toward the decapeptide in the range of 31-55 pg. Immunoreactivity found in extracts of fresh or lyophilized bovine pituitary glands displaced the iodinated Tyr-decapeptide in the RIA in a parallel manner. The amount of immunoreactive (ir)-material was dependent upon the state of preservation of the tissue, the method of extraction, and the particular antiserum used. Extractable immunoreactivity was separated into low (Mr 1,500) and high (Mr 17,000) molecular weight peptides using gel chromatography (G-75). Additional ir-material appeared in the void volume (Mr greater than 22,500). Thus, these antisera have the capacity to interact not only with a region of the joining peptide but also with its larger, and apparent precursor forms. The immunoassay developed should be valuable in understanding the disposition and processing in this specific region of POMC.     

Differential glycosylation of N-POMC1-77 regulates the production of gamma 3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing.

The amino terminus of bovine pro-opiomelanocortin (N-POMC^1–77) is partially processed in the intermediate lobe of the pituitary to N-POMC^1–49 and lys-γ₃ -melanotropin. Two pools of N-POMC^1–77 were isolated which were differentially glycosylated at threonine⁴⁵, while N-POMC^1–49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC^1–77 may regulate cleavage at the Arg⁴⁹-Lys⁵⁰ processing site. We tested this hypothesis by incubating N-POMC^1–77 glycoforms with purified pro-opiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC^1–77 and O-glycosylated N-POMC^1–77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-γ₃ -melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.     

A novel prohormone processing site in Aplysia californica: the Leu-Leu rule

Neuropeptides are a complex set of signaling molecules produced through enzymatic cleavages from longer prohormone sequences. The most common cleavage sites in prohormones are basic amino acid residues; however, processing is observed at non-basic sites. Cleavage at Leu-Leu sequences has been observed in three Aplysia californica prohormones. To further investigate this unusual event, native and non-native synthetic peptides containing Leu-Leu residues are incubated with homogenates of Aplysia californica ganglia and the resulting products monitored with MALDI MS. Cleavage near and between Leu-Leu residues is observed in the abdominal and buccal ganglia homogenates, confirming the presence of an unidentified peptidase. In addition, fractions from an HPLC separation of buccal ganglia homogenates also produce cleavages at Leu-Leu residues. Products resulting from cleavage at Leu-Leu sites are observed and are produced in larger amounts in acidic and neutral pH ranges, and cleavage is inhibited by the addition of EDTA, suggesting a metal is required for activity.