Pro-opiomelanocortin Converting Enzyme Research Articles

Cleavage of proglucagon by the prohormone converting enzymes PCSK1 and PCSK2 is not dictated by the amino acid sequence immediately adjacent to the cleavage site

The peptide hormones glucagon, glucagon‐like peptide‐1, and glucagon‐like peptide‐2 are all derived from proglucagon (PG), a 160 amino acid polypeptide containing multiple potential cleavage sites designated by either single or paired basic amino acids. Initial cleavage of PG takes place on the C‐terminal side of the K70‐R71 dibasic site, which liberates the intermediates glicentin and major proglucagon fragment (MPGF). My laboratory is interested in examining the specificity of the prohormone cleaving enzymes PCSK1 and PCSK2 using both PG and proinsulin (PI) as model substrates. PG is a useful model because it has one cleavage site (the glicentin/MPGF junction) recognized almost equally well by both enzymes and multiple potential cleavage sites that are not recognized by either enzyme. PI is a good model because it has one cleavage site (the B chain/C peptide junction) preferentially recognized by PCSK1 and a second cleavage site (the C peptide/A chain junction) preferentially recognized by PCSK2. My lab has previously described the expression and purification of these two model substrates. However, the purification of PG has presented a number of problems because of the presence of contaminants that obscure detection and participate in co‐precipitation during early stages of purification. I have now devised an improved, two‐step scheme using step elution from SP Sepharose as a capture step, followed by gradient elution from Q Sepharose as the major purification step. This method resolves the problems posed by the contaminants and generates PG samples suitable for analysis of the PCSK‐mediated cleavage reaction. An in vitro cleavage assay was then set up so that the PG substrate (~3 μM) incubated with either PCSK1 or PCSK2 (~100 nM at pH 6.0 or 5.4, respectively) results in ~30% cleavage at the glicentin/MPGF junction using either enzyme. As noted above, in contrast to the situation with PG, the normal maturation pathway for PI exhibits a marked preference for PCSK1‐mediated cleavage at the B chain/C peptide junction. To examine whether this cleavage selectivity is due to amino acids in the immediate vicinity of the cleavage site, the sequence EAED from the C‐terminal side of the B chain/C peptide cleavage site in PI was cloned into the C‐terminal side of the K70‐R71 cleavage site in PG. In contrast to expectations, this substitution resulted in a nearly 50% decrease in PG cleavage by PCSK1. Equally surprising was that this substitution had almost no effect on the efficiency of PCSK2‐mediated cleavage of the altered site, despite the fact that in the original context of the PI substrate, this sequence is recognized much less efficiently by PCSK2 than by PCSK1. Taken together, these results suggest that three‐dimensional features (secondary or tertiary structure) play a significant role in the cleavage site specificity exhibited by both PCSK1 and PCSK2.

Distinct proteomic profiles in post-mortem pituitary glands from bipolar disorder and major depressive disorder patients

Disturbances of the hypothalamic–pituitary–adrenal axis have been implicated in the pathophysiology of bipolar disorder (BD) and major depressive disorder (MDD). To examine this further, we carried out proteomic profiling of post-mortem pituitaries from 13 BD and 14 MDD patients, in comparison to 15 controls. Liquid chromatography–mass spectrometry (LC–MSE) analysis showed that BD patients had significantly increased levels of the major pituitary hormones pro-opiomelanocortin (POMC) and galanin. BD patients also showed changes in proteins associated with gene transcription, stress response, lipid metabolism and growth signalling. In contrast, LC–MSE profiling revealed that MDD patients had significantly decreased levels of the prohormone-converting enzyme carboxypeptidease E and follow-up enzymatic analysis showed decreased activity of prolyl-oligopeptidase convertase. This suggested that altered prohormone processing may occur in pituitaries of MDD patients. In addition, MDD patients had significant changes in proteins involved in intracellular transport and cytoskeletal signalling. Finally, we carried out selective reaction monitoring (SRM) mass spectrometry profiling for validation of protein changes in key biological pathways. This confirmed increased POMC levels in BD patients with no change in the levels of this prohormone in MDD. This study demonstrates that proteomic profiling analysis of the pituitary can lead to new insights into the pathophysiology of BD and MDD. Also, given that the pituitary directly releases a variety of bioactive molecules into the bloodstream, many of the proteins identified here could serve as focal points in the search for peripheral biomarkers in clinical or drug treatment studies of BD and MDD patients.

The role of small molecule GPR119 agonist, AS1535907, in glucose-stimulated insulin secretion and pancreatic β-cell function

AS1535907, a small molecule agonist of GPR119, was assessed for its glucose-stimulated insulin secretory activity and pancreatic β-cell function in type 2 diabetes. Both in vitro and in vivo tests were conducted using NIT-1 and HEK293 cell lines, male normal and db/db mice and isolated perfused rat pancreas preparations. AS1535907 had an EC₅₀ value of 1.5 µM for human GPR119 transfected in HEK293 cells. AS1535907 enhanced insulin secretion in NIT-1 cells and in the perfused rat pancreas. A transient increase in the human insulin promoter activity was also observed in NIT-1 cells. First-phase insulin secretion was particularly more evident in the AS1535907-treated perfused rat pancreas than that in the nateglinide or glibenclamide-treated group. Oral glucose tolerance improved following a single dose of AS1535907 in normal and db/db mice. Subsequently, 2 weeks of multiple dosing significantly increased plasma insulin levels and decreased blood glucose levels in db/db mice. After 3 weeks of treatment in db/db mice, the numbers of insulin and proliferation cell nuclear antigen-positive cells and the islet area were significantly higher than those in the vehicle-treated mice. As compared with the vehicle, gene expression analysis revealed that AS1535907 significantly upregulated transcription factors (Nkx 2.2, Nkx 6.1, NeuroD and activin A), responsible for β-cell regulation and prohormone-converting enzyme 1 responsible for insulin biosynthesis. These results suggest that AS1535907 can potentially regulate first-phase insulin secretion and exert a protective effect on pancreatic β-cell function via regulation of transcription factors.

The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects

The chromogranins A (CgA) and B (CgB) and secretogranin II (SgII) constitute the main members of a family of uniquely acidic secretory proteins in elements of the diffuse neuroendocrine system. These genetically distinct proteins, CgA, CgB, SgII and the less well known secretogranins III-VII are collectively referred to as 'granins' and characterised by numerous pairs of basic amino acids as potential cleavage sites for processing by the co-stored prohormone converting enzymes PC 1/3 and PC2. This review is directed towards comparative and functional aspects of the granins with emphasis on their phylogenetically conserved sequences. Recent developments provide ample evidence of widely different effects and targets for the intact granins and their derived peptides, intracellularly in the directed trafficking of storage components during granule maturation and extracellularly in autocrine, paracrine and endocrine interactions. Most of the effects assigned to the granin derived peptides fit into patterns of direct or indirect inhibitory modulations of major functions. So far, peptides derived from CgA (vasostatins, chromacin, pancreastatin, WE-14, catestatin and parastatin), CgB (secretolytin) and SgII (secretoneurin) are the most likely candidates for granin-derived regulatory peptides, of postulated relevance not only for homeostatic processes, but also for tissue assembly and repair, inflammatory responses and the first line of defence against invading microorganisms.

ADRENAL GROWTH IS CONTROLLED BY EXPRESSION OF SPECIFIC PRO-OPIOMELANOCORTIN SERINE PROTEASE IN THE OUTER ADRENAL CORTEX

As previous work had shown that extreme N-terminal fragments of the ACTH precursor pro-opiomelanocortin (POMC) not containing γ-melanotropin (γ-MSH) were active adrenal mitogens but an antiserum raised against γ-MSH paradoxically also inhibited adrenal growth we proposed that the adrenal mitogen is processed from pro-γ-MSH by a neurally controlled protease at the growing adrenal. To this end we have characterised a novel serine protease (named adrenal secretory protease (AsP) as Psort predicted a leader motif) which is expressed at the glomerulosa/fasciculata boundary where mitosis takes place. The expression of AsP was also found to be essential for mitosis of the adrenal cortical tumor Y1 cell-line in POMC containing media and 3D homology modeling revealed the presence of a catalytic pocket flanked by the classical His/Asp/Ser motifs. An usual feature of the model was a cluster of arginine residues on the underside of the protease suggesting that this basically charged face would tend to retain it on the cell surface on secretion-immunocytochemistry using an antiserum raised against a synthetic peptide spanning residues 1–25 of AsP showed that this was the case for Y1 cells. Specificity of AsP (affinity purified from Y1 media) was demonstrated by its inability to cleave model substrates for either trypsin or pro-hormone converting enzymes but was able to cleave an internally quenched POMC (44–55) model peptide. Interestingly mass spectral analysis of products of the latter predicts that the protease cleaves between the bond between Val52 and Met53 suggesting the natural adrenal mitogen is POMC (1–52).

Up-regulation of splenic prohormone convertases PC1 and PC2 in diabetic rats

Organisms respond to infection in a complex manner involving bidirectional interactions between the neuroendocrine and immune systems. Many of the bioactive endocrine/immune factors are synthesized in a precursor form and are expected to be activated by prohormone convertases (PCs). Since patients with both type 1 and type 2 diabetes have an increased incidence and severity of infections, we hypothesized that in a condition of hyperglycemia, these processing enzymes would be activated in an immune tissue, the spleen. To test this hypothesis, we treated rats with intraperitoneal streptozotocin (STZ) (50 mg/kg/day) daily for 5 days and measured splenic PC1 and PC2 mRNA by ribonuclease protection assay. We found that PC1 mRNA was increased 6.0±0.02-fold ( P<0.05) and PC2 mRNA was increased 1.80±0.01-fold ( P<0.005) in the spleen of rats that received STZ compared to rats that received vehicle. Western blot indicated that the 75-kDa form of PC1 was the only form of PC1 present in the spleen and that this form increased with STZ treatment. Immunohistochemistry revealed that PC1 was found in both the white pulp (T-lymphocytes) and red pulp (monocytes and macrophages) and that its increase in immunoreactivity occurred primarily in the white pulp. PC2 and pro-opiomelanocortin (POMC, a possible splenic substrate for PC1/PC2) immunoreactivity was found predominantly in the red pulp. STZ induced an increase in splenic PC1 and POMC, but not PC2 protein levels. We conclude that in the STZ model of diabetes, splenic PCs are induced, which could lead to an increased activation of many immune-derived hormones. We speculate that this up-regulation of prohormone converting enzymes may be related to the increased infections seen in patients with both type 1 and type 2 diabetes.

Regulation of pancreatic PC1 and PC2 associated with increased glucagon-like peptide 1 in diabetic rats.

The pancreatic processing enzymes, PC1 and PC2, convert proinsulin to insulin and convert proglucagon to glucagon and glucagon-like peptide 1 (GLP-1). We examined the effect of streptozotocin (STZ) treatment on the regulation of these enzymes and the production of insulin, glucagon, and GLP-1 in the rat. Pancreatic PC1 and PC2 mRNA increased >2-fold and >4-fold, respectively, in rats receiving intraperitoneal STZ (50 mg/kg) daily for 5 days. Immunocytochemistry revealed that, although pancreatic islet cells in the STZ-treated rats were sparse and atrophic PC1, PC2, glucagon, and GLP-1 immunoreactivity increased dramatically in the remaining islet cells. Heightened PC1 and PC2 expression was seen in cells expressing glucagon but not in insulin-expressing cells. Furthermore, in STZ-treated rats, bioactive GLP-1(7-36 amide) accumulated in pancreatic extracts and serum 3- and 2.5-fold, respectively, over control animals. This treatment also caused a 2-fold increase in the ratio of amidated forms of GLP-1 immunoreactivity to total glucagon immunoreactivity in the pancreas but did not affect the ratio of proinsulin to insulin. We conclude that hyperglycemic rats have an increased expression of prohormone converting enzymes in islet alpha cells, leading to an increase in amidated GLP-1, which can then exert an insulinotropic effect on the remaining beta cells.

Post-translational processing of human procorticotrophin-releasing factor in transfected mouse neuroblastoma and Chinese hamster ovary cell lines.

The mouse neuroblastoma cell line (Neuro 2 A) has been shown to contain the mRNA of a prohormone converting enzyme, PC2. The Chinese hamster ovary cell line (CHO) does not express PC2 mRNA, but is thought to contain the ubiquitous protease, furin. The enzyme(s) responsible for releasing corticotrophin-releasing hormone (CRH) from its precursor (proCRH) have not been identified, therefore to investigate the possible function(s) of PC2 or furin in the processing of proCRH, stable Neuro 2 A and CHO cell lines that express the 21 kDa human (h)proCRH were established. A specific two-site IRMA for CRH demonstrated that the hpreproCRH-expressing Neuro 2 A cell line cleaved the CRH precursor to the CRH peptide, and was able to release the mature peptide into cell medium at levels that were 4-fold higher than produced by the hproCRH-expressing CHO cells. RIA showed that the CHO cells secreted levels of CRH-containing peptides that were 10-fold higher than produced by the Neuro 2 A cells. Medium from the transfected CHO and Neuro 2 A cells was analysed by HPLC; this showed that CHO cells released a single protein corresponding to the unprocessed CRH precursor, whereas Neuro 2 A cells secreted two peptides, which could be identified as the 5 kDa CRH(1-41) and residual 16 kDa CRH peptides. These results suggest that Neuro 2 A cells, which contain PC2, can process proCRH to the mature peptide.

Pro-Opiomelanocortin and adrenal function

Pro-opiomelanocortin (POMC) is a polyhormone precursor produced predominantly in the pars distalis and pars intermedia of the pituitary gland where it undergoes tissue specific processing to produce a whole array of peptides. We have shown previously that peptides derived from the N-terminal region of POMC are involved in adrenal growth in rats. Using specific two site immunoradiometric assays we have found that the plasma of 17 week old fetal sheep contain a 50 fold excess of pro-gamma-MSH over ACTH. As term approached, the levels of pro-gamma-MSH fell and ACTH rose with evidence of fragmentation of pro-gamma-MSH, suggesting that these peptides act in concert in the development of the fetal adrenal cortex and also provide the necessary drive to bring about parturition. In an attempt to explore the pathophysiology of adrenal function we have cloned human POMC cDNA which led to the discovery of a 9bp addition/deletion mutation in the C-terminus of gamma 3-MSH between positions 67-73. Chinese hamster ovary cells (CHO) cells stably transfected with constructs containing the variant POMC cDNAs have shown a degree of partial processing. Work is currently underway to further investigate the effects of these mutations on the processing by the prohormone converting enzymes PC1 and PC2.

Super-CHO?A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6) cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6) cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Identification and expression of prohormone-converting enzymes in the rat stomach.

The conversion of regulatory peptide precursors to their active forms usually involves limited proteolysis that may be mediated by subtilisin-like prohormone convertases (PC). We have examined the representation of this enzyme family in rat gastric mucosa. With the use of polymerase chain reaction, employing primers to conserved sequences, we identified from rat antrum clones corresponding to PC1/3, PC2, PC5, and furin. Northern blots indicated that the mRNAs for PC1/3 and PC2 were substantially more abundant in mucosa compared with muscle, and that there were differences in expression in antrum and corpus. In the antrum a PC1/3 probe identified hands of 3 and 4.5 kb that were of equal intensity and were both increased in fasted rats; in corpus, the latter mRNA species predominated and did not change with fasting. In rats treated with omeprazole, there was a preferential increase in the antral 3-kb band. In both antrum and corpus, a PC2 probe hybridized with a band of 2.8 kb that increased in omeprazole-treated rats. The data suggest that 1) PC1/3 and PC2 are expressed in antral mucosa and so are candidates for gastric regulatory peptide processing, 2) there is selective processing of the mRNAs encoding prohormone convertases in different gastric cell populations, and 3) the expression of these enzymes is physiologically regulated.

Identification and expression of prohormone converting enzymes in the rat stomach : J.A.Macro, R.Dimaline and G.J.Dockray. Physiological Laboratory, University of Liverpool, Liverpool, UK

Identification and expression of prohormone converting enzymes in the rat stomach : J.A.Macro, R.Dimaline and G.J.Dockray. Physiological Laboratory, University of Liverpool, Liverpool, UK

A C-terminal domain, which prevents secretion of the neuroendocrine protein 7B2 in Saccharomyces cerevisiae, inhibits Kex2 yet is processed by the Yap3 protease

Recent reports reveal that the C-terminal half of the neuroendocrine polypeptide 7B2 selectively inhibits and binds PC2, a mammalian prohormone converting enzyme that is homologous to the yeast pro-α-factor processing protease Kex2. During attempted secretion of the 185 amino-acid human 7B2 in Saccharomyces cerevisiae, we observe that the protein is mostly retained inside the cell. However a mutant polypeptide (7B2Δ1), where the C-terminal 48 amino acids of 7B2 are deleted, is efficiently secreted. Two shorter C-terminal truncations either permit poor secretion or no secretion at all. Surprisingly, full-length 7B2 but not 7B2Δ1 abolishes the catalytic activity of Kex2, indicating that C-terminal residues of 7B2 might also be important for inhibition of the yeast protease. When the KEX2 gene is disrupted, yeast cells unexpectedly secrete a 7B2 variant similar in size to 7B2Δ1, suggesting involvement of the alternate yeast prohormone convertase Yap3 in processing. Secretion is enhanced by overexpression of Yap3 and by the presence of a Lys-Arg residue at the processing site of precursor 7B2. These results purport that, in neuroendocrine cells too, secretion of 7B2 could be mediated by a homologue of Yap3.

Proteases and the emerging role of protease inhibitors in prohormone processing.

Peptide hormones and neurotransmitters constitute a large class of neurohumoral agents that mediate cell-cell communication in neuroendocrine systems. Their biosynthesis requires proteolytic processing of inactive protein precursors into active neuropeptides. Elucidation of the proteolytic components required for prohormone processing is important for identifying key proteases that may control the production of neuropeptides. This article compares the subtilisin-like PC1/3 and PC2 processing enzymes identified through molecular biological approaches, and several candidate processing enzymes identified biochemically, including the 'proopiomelanocortin converting enzyme' (PCE) and the 'prohormone thiol protease' (PTP), as well as others of different classes (aspartyl, cysteine, metallo, and serine proteases). A role for PTP in cellular proenkephalin processing is suggested by blockade of forskolin-stimulated (Met)enkephalin production by Ep453 that is converted intracellularly to E-64c, a selective cysteine protease inhibitor that potently inhibits PTP. A possible role for endogenous protease inhibitors in prohormone processing represents a new aspect of cellular mechanisms that may regulate neuropeptide biosynthesis. Future studies of the enzymology and molecular biology of processing enzymes and endogenous protease inhibitors will be necessary to elucidate mechanisms of prohormone processing.

Unique cleavage specificity of ‘prohormone thiol protease’ related to proenkephalin processing

‘Prohormone thiol protease’ (PTP) represents the major enkephalin precursor processing activity in chromaffin granules. In this study, cleavage specificity of PTP for paired basic and monobasic residues was examined with a series of model peptide-MCA (-methylcoumarinamide) substrates. Monobasic peptides were cleaved at the COOH- and NH 2-terminal sides of the single basic residue. Dibasic peptides, however, were preferentially cleaved at the NH 2-terminal side of the pair, or between the two basic residues, with low cleavage at the COOH-terminal side of the pair. Inhibition by the peptide inhibitor ( d-Tyr)-Glu-Phe-Lys-Arg-CH 2Cl provided further evidence for ptp's specificity for the dibasic Lys-Arg site. Inhibition by Z-Leu-Val-Gly-CHN 2; and Z-Arg-Leu-Val-Gly-CHN 2; suggests involvement of Val-Gly in substrate binding to PTP; these two cystatin C-related inhibitors also indicate PTP as a cysteine protease. These results demonstrate PTP's unique cleavage specificity that differs from other processing endopeptidases, including the subtilisin-related proprotein convertases, PC1/PC3, and PC2, as well as the pituitary proopiomelanocortin-converting enzyme, PCE. This study provides further evidence for PTP as a novel prohormone processing enzyme that belongs to the class of cysteine proteases.

Release of the prohormone convertase PC1 from AtT-20 cells

AtT-20 cells are known to synthesize two molecular weight forms of the prohormone converting enzyme PC1 with molecular masses of 87 and 66 kDa. In this study we have analyzed basal and stimulated secretion of these proteins. Western blot results show that basal secretion medium of cultured AtT-20 cells contained low concentrations of both the 87 and 66 kDa forms of PC1 with the former protein predominant. During the stimulation period with CRF, cAMP and cAMP + BaCl 2, increased release of both proteins was observed, but the 66 kDa protein predominated. Secretion medium obtained from stimulated and unstimulated cells was enzymatically active against the Cbz-Arg-Ser-Lys-Arg-AMC fluorogenic substrate as well as against 35S-proenkephalin. This activity was Ca +2 dependent and was inhibited by the chelating agent EDTA. The activity was insensitive to acid and thiol proteinase inhibitors as well as to N-alpha-p-tosyl-L-Lys-chloromethyl ketone; it was slightly sensitive to phenylmethyl sulfonyl fluoride and was strongly inhibited by D-Tyr-Ala-Lys-Arg-chloromethyl ketone. This inhibitor profile exhibits strong similarities to furin and kexin. After partial purification of medium by gel filtration chromatography, a portion of the enzymatic activity and immuno-reactivity for both 87 kDa and 66 kDa proteins eluted with an apparent molecular weight of 400 kDa (suggesting aggregation); however the highest activity appeared in the elution position of the 66 kDa monomer. When the 87 kDa protein was removed from the medium by means of an affinity column containing an antibody against the carboxyl terminal portion of PC1, the column flow-through, which included the 66 kDa protein, still remained enzymatically active. These data support the notion that the 66 kDa protein, which is the most concentrated PC1 product stored in AtT-20 cells and is released during stimulation, is enzymatically active.

Prohormone-Converting Enzymes: Regulation and Evaluation of Function Using Antisense RNA

Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, the question is still open as to which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2. The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases. PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues. In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H. In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids. When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing. These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s).

Differential glycosylation of N-POMC 1–77 regulates the production of γ 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-γ 3 -melanotropin. Two pools of N-POMC 1–77 were isolated which were differentially glycosylated at threonine 45, 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 Arg 49-Lys 50 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-γ 3 -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.

Heterologous expression of peptide hormone precursors in the yeast Saccharomyces cerevisiae. Evidence for a novel prohormone endoprotease with specificity for monobasic amino acids

The peptide somatostatin (SRIF) exists as two different molecular species. In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid amino-terminally extended form of the tetradecapeptide, SRIF-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which, upon cleavage, generate either SRIF-14 or -28, respectively. In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone whereas in some species of fish separate genes encode two distinct but homologous precursors prepro-SRIF-I and -II that give rise to SRIF-14 and -28, respectively. To investigate the molecular basis for differential processing of the prohormones we introduce their cDNAs into yeast cells (Saccharomyces cerevisiae). The signal peptides of both precursors were poorly recognized by the yeast endoplasmic reticulum translocation apparatus, consequently only low levels of SRIF peptides were synthesized. To circumvent this problem a chimeric precursor consisting of the alpha-factor signal peptide plus 30 residues of the proregion was fused to pro-SRIF-II. This fusion protein was efficiently transported through the yeast secretory pathway and processed to SRIF-28 exclusively, which is identical to the processing of the native precursor in pancreatic islet D-cells. Most significantly, cleavage of the precursor to SRIF-28 was independent of the Kex 2 endoprotease since processing occurred efficiently in a kex 2 mutant strain. We conclude that in addition to the Kex 2 protease, yeast possess a distinct prohormone converting enzyme with specificity toward monobasic processing sites.

Generation of Lys-γ3-melanotropin from pro-opiomelanocortin1–77 by a bovine intermediate lobe secretory vesicle membrane-associated aspartic protease and purified pro-opiomelanocortin converting enzyme

Abstract 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.