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PubMed1995

Differential processing of proglucagon by the subtilisin-like prohormone convertases PC2 and PC3 to generate either glucagon or glucagon-like peptide.

Proglucagon is processed differently in the islet alpha cells and the intestinal endocrine L cells to release either glucagon or glucagon-like peptide 1-(7-37) (GLP1-(7-37)), peptide hormones with opposing actions in vivo. In previous studies with a transformed alpha cell line (alpha TC1-6) we demonstrated that the kexin/subtilisin-like prohormone convertase, PC2 (SPC2), is responsible for generating the typical alpha cell pattern of proglucagon processing, giving rise to glucagon and leaving unprocessed the entire C-terminal half-molecule known as major proglucagon fragment or MPGF (Rouillé, Y., Westermark, G., Martin, S. K., Steiner. D. F. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3242-3246). Here we present evidence, using mouse pituitary AtT-20 cells infected with a vaccinia viral vector encoding proglucagon, that PC3 (SPC3), the major neuroendocrine prohormone convertase in these cells, reproduces the intestinal L cell processing phenotype, in which MPGF is processed to release two glucagon-related peptides, GLP1 and GLP2, while the glucagon-containing N-terminal half-molecule (glicentin) is only partially processed to oxyntomodulin and small amounts of glucagon. Moreover, in AtT-20 cells stably transfected with PC2 (AtT-20/PC2 cells), glicentin was efficiently processed to glucagon, providing further support for the conclusion that PC2 is the enzyme responsible for the alpha cell processing phenotype. In other cell lines expressing both PC2 and PC3 (STC-1 and beta TC-3), proglucagon was also processed extensively to both glucagon and GLP1-(7-37), although STC-1 cells express lower levels of PC2 and processed the N-terminal domain to glucagon less efficiently. In contrast, GH4C1 and COS 7 cells, which express very little or no PC2 or PC3, failed to process proglucagon, aside from a low level of interdomain cleavage which occurred only in the GH4C1 cells. In vitro PC3 did not cleave at the single Arg residue in GLP1 to generate GLP1-(7-37), its truncated biologically active form, indicating the likelihood that another convertase is required for this cleavage.

Amino Acid Sequence↗
PubMed1996

Secretogranin III is a sulfated protein undergoing proteolytic processing in the regulated secretory pathway.

Secretogranin III (SgIII) is an acidic protein of unknown function that is present in the storage vesicles of many neuroendocrine cells. It is coexpressed with the prohormone proopiomelanocortin in the intermediate pituitary of Xenopus laevis. We developed an antiserum to investigate the biosynthesis of SgIII in pulse-chase incubated Xenopus neurointermediate lobes. SgIII was synthesized as a 61- or 63-kDa (N-glycosylated) protein and processed to a 48-kDa form which, in turn, was partially cleaved to fragments of 28 and 20 kDa. The 48-, 28-, and 20-kDa cleavage products, but not their precursors, were secreted. This secretion is regulated and can be blocked in parallel with that of proopiomelanocortin-derived peptides by the hypothalamic factors dopamine, gamma-aminobutyric acid, and neuropeptide Y. Coexpression of Xenopus SgIII with prohormone convertase (PC)1 or PC2 in transfected fibroblasts was sufficient to reconstitute the processing events observed in the neurointermediate lobes. Site-directed mutagenesis revealed that Xenopus SgIII is cleaved at two dibasic sites, namely Lys68-Arg69 and Arg237-Arg238. Pulse-chase incubations of lobes with Na2[35S]SO4 showed that SgIII is sulfated in the trans-Golgi network before it is processed. Finally, SgIII processing was found in several neuroendocrine cell types from various species. We conclude that SgIII is a precursor protein and that the intact molecule can only have an intracellular function, whereas an extracellular role can only be attributed to its cleavage products.

Adaptation, Physiological↗
PubMed1998

Regulatory roles of the P domain of the subtilisin-like prohormone convertases.

A unique feature of the eukaryotic subtilisin-like proprotein convertases (SPCs) is the presence of an additional highly conserved sequence of approximately 150 residues (P domain) located immediately downstream of the catalytic domain. To study the function of this region, which is required for the production of enzymatically active convertases, we have expressed and characterized various P domain-related mutants and chimeras in HEK293 cells and alpha-TC1-6 cells. In a series of C-terminal truncations of PC3 (also known as PC1 or SPC3), PC3-Thr594 was identified as the shortest active form, thereby defining the functional C-terminal boundary of the P domain. Substitutions at Thr594 and nearby sites indicated that residues 592-594 are crucial for activity. Chimeric SPC proteins with interchanged P domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp616), both PC3/PC2Pd and PC3/FurPd had elevated activity on several synthetic substrates as well as reduced calcium ion dependence, whereas Fur/PC2Pd was only slightly decreased in activity as compared with truncated furin (Fur-Glu583). Of the three active SPC chimeras tested, all had more alkaline pH optima. When PC3/PC2Pd was expressed in alpha-TC1-6 cells, it accelerated the processing of proglucagon into glicentin and major proglucagon fragment and cleaved major proglucagon fragment to release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain exchanges generated fully active chimeric proteases in several instances but not in all (e.g. PC2/PC3Pd was inactive). The observed property changes indicate a role for the P domain in regulating the stability, calcium dependence, and pH dependence of the convertases.

Animals↗
PubMed2001

Impaired prohormone convertases in Cpe(fat)/Cpe(fat) mice.

A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene results in a loss of CPE activity that correlates with the development of late onset obesity (Nagert, J. K., Fricker, L. D., Varlamov, O., Nishina, P. M., Rouille, Y., Steiner, D. F., Carroll, R. J., Paigen, B. J., and Leiter, E. H. (1995) Nat. Genet. 10, 135-142). Examination of the level of neuropeptides in these mice showed a decrease in mature bioactive peptides as a result of a decrease in both carboxypeptidase and prohormone convertase activities. A defect in CPE is not expected to affect endoproteolytic processing. In this report we have addressed the mechanism of this unexpected finding by directly examining the expression of the major precursor processing endoproteases, prohormone convertases PC1 and PC2 in Cpe(fat) mice. We found that the levels of PC1 and PC2 are differentially altered in a number of brain regions and in the pituitary. Since these enzymes have been implicated in the generation of neuroendocrine peptides (dynorphin A-17, beta-endorphin, and alpha- melanocyte-stimulating hormone) involved in the control of feeding behavior and body weight, we compared the levels of these peptides in Cpe(fat) and wild type animals. We found a marked increase in the level of dynorphin A-17, a decrease in the level of alpha-melanocyte-stimulating hormone, and an alteration in the level of C-terminally processed beta-endorphin. These results suggest that the impairment in the level of these and other peptides involved in body weight regulation is mainly due to an alteration in carboxypeptidase and prohormone convertase activities and that this may lead to the development of obesity in these animals.

Amino Acid Sequence↗
PubMed2003

Biological processing of the cocaine and amphetamine-regulated transcript precursors by prohormone convertases, PC2 and PC1/3.

Cocaine and amphetamine-regulated transcript (CART), a neuroendocrine peptide influencing reward, feeding/appetite, and stress responses is derived from two peptide precursors of 129 and 116 amino acid (aa) residues that arise via alternative splicing from a single Cart gene in rats and mice. The signal peptide constitutes the first 27 aa resulting in pro-CART molecules of either 102 or 89 aa. In the present study, we have shown that pro-CART is a substrate for the neuroendocrine subtilisin/kexin-like prohormone convertases, PC2 (SPC2) and PC1/3 (SPC3). By using different neuroendocrine cell lines, with or without endogenous expression of either PC2 or PC1/3 or both enzymes, we have demonstrated through transient transfection studies that long pro-CART gives rise to an intermediate peptide, residues 33-102, and the two major bioactive CART forms, residues 55-102 (I) and 62-102 (II), respectively. Likewise, short pro-CART also generates three peptides, an intermediate, residues 10-89, and the two identical bioactive CART forms. We have confirmed the identities of the bioactive and intermediate CART molecules by microsequencing and/or high performance liquid chromatography and mass spectrometry. We have shown that PC2 is more efficient in generating bioactive CART I compared with PC1/3, whereas the production of the smaller bioactive CART II is exclusively carried out by PC2. PC1/3 is predominantly responsible for generating the intermediate CART fragments, 33-102 and 10-89, from long and short pro-CART, respectively. To compare in vitro and in vivo processing of pro-CART, we have examined its processing in PC2, 7B2, and PC1/3 knock-out mouse hypothalamic extracts and demonstrated that, as in vitro, PC2 is more potent than PC1/3 in generating bioactive CART I whereas bioactive CART II is solely generated by PC2. Also, in vivo, we have shown that PC1/3 is predominantly active in liberating the two intermediate CART fragments, 33-102 and 10-89. These findings confirm the key roles of PC2 and PC1/3 acting together or separately to carry out CART processing in selected sites in vivo.

Animals↗
PubMedDate not supplied

cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases.

Based on the concept of sequence conservation around the active sites of serine proteinases, polymerase chain reaction applied to mRNA amplification allowed us to obtain a 260-bp probe which was used to screen a mouse pituitary cDNA library. The primers used derived from the cDNA sequence of active sites Ser* and Asn* of human furin. Two cDNA sequences were obtained from a number of positive clones. These code for two similar but distinct structures (mPC1 and mPC2), each being homologous to yeast Kex2 and human furin. In situ hybridization (mPC1) and Northern blots (mPC1 = 3.0 kb and mPC2 = 2.8 and 4.8 kb) demonstrated tissue and cellular specificity of expression, only within endocrine and neuroendocrine cells. These data suggest that mPC1 and mPC2 represent prime candidates for tissue-specific pro-hormone converting proteinases.

Amino Acid Sequence↗
PubMed1995

Maintained PC1 and PC2 expression in the AtT-20 variant cell line 6T3 lacking regulated secretion and POMC: restored POMC expression and regulated secretion after cAMP treatment.

Two variant cell lines were recently established from parent AtT-20 cells. Whereas HYA.15.10.T.2 have a reduced level of secretory granules, HYA.15.6.T.3 are completely devoid of both the regulated pathway of secretion and of dense-core secretory granules. AtT-20 cells normally express the processing enzymes PC1, PC2, furin, carboxypeptidase E, and peptidylglycine alpha-amidating monooxygenase, as well as proopiomelanocortin, chromogranin B, and 7B2. We measured the expression of these mRNAs in both variant cell lines. Although some differences in mRNA level were noted, HYA.15.10.T.2 and HYA.15.6.T.3 cell lines maintained their expression of the processing enzymes and of 7B2. Furthermore, PC1 and PC2 were shown to be functionally active in the HYA.15.6.T.3 cells. In contrast, proopiomelanocortin and chromogranin B mRNA levels were no longer detectable in HYA.15.6.T.3 cells. Interestingly, stimulation of the HYA.15.6.T.3 cells with cAMP restored proopiomelanocortin mRNA, beta-endorphin immunoreactivity, and dense-core granules. Furthermore, at the ultrastructural level, beta-lipotropin immunoreactivity was detected in granules of cAMP-induced HYA.15.6.T.3 cells. Finally, depolarization of cAMP-induced HYA.15.6.T.3 cells with 56 mM potassium chloride resulted in a marked increase in the release of beta-endorphin immunoreactivity. These observations demonstrate that cAMP restores the regulated pathway of secretion in HYA.15.6.T.3 cells, which under untreated conditions do not demonstrate regulated release. These variant cell lines are unique models to understand better the relationship of the regulated pathway and the expression of the processing enzymes.

Animals↗
PubMed1996

Molecular cloning of Aplysia neuronal cDNAs that encode carboxypeptidases related to mammalian prohormone processing enzymes.

The bag cell neurons of Aplysia synthesize an egg-laying hormone (ELH) precursor that initially is cleaved into two fragments in the Golgi apparatus, and the fragments are differentially packaged in separate granule populations and further processed. Aplysia Afurin, Afurin2, prohormone convertase 1 (PC1), and PC2 are thought to be involved in the posttranslational processing of the ELH prohormone. In the present study, we have cloned Aplysia neuronal cDNAs that encode an enzyme most closely related to mammalian carboxypeptidase E (CPE), a peptide hormone processing enzyme that removes basic residues during prohormone processing. Northern blot analysis identified a single Aplysia CPE mRNA (approximately 5.2 kb) in central nervous system tissue. The C-terminal region of Aplysia CPE contains amphiphilic alpha-helices that may serve as a hydrophobic membrane anchor. A novel neuronal Aplysia enzyme was also identified by the polymerase chain reaction that was most closely related to the carboxypeptidase D (CPD)-related duck protein gp180 and the Drosophila silver gene carboxypeptidases. Aplysia CPE and the CPD-related enzyme are candidate processing enzymes that may play a role in the processing of the ELH prohormone and other Aplysia prohormones.

Amino Acid Sequence↗
PubMed1997

Convertase PC2 and the neuroendocrine polypeptide 7B2 are co-induced and processed during neuronal differentiation of P19 embryonal carcinoma cells.

Convertases of the subtilisin/kexin family are responsible for the biological activation of a variety of pro-proteins, pro-hormones, and pro-trophic factors, and thus can modulate various aspects of embryonic development. We investigated the expression of each convertase by Northern hybridization during cell differentiation in vitro, using the mouse embryonal carcinoma cell line P19 as a model. The neuroendocrine convertase PC2 and 7B2, its specific binding protein, are co-induced during neuronal differentiation of P19 cells with retinoic acid, whereas the other convertases are not or follow different patterns of temporal expression. The mature forms of PC2 and 7B2 proteins are detected together by immunoblotting following induction of mRNA expression, indicating that these proteins are processed early during brain development. These results demonstrate that PC2 and 7B2 gene expression and protein processing are in a close temporal association during neuronal differentiation and point to the value of the P19 cell model to study the significance and the regulation of this relationship in mammalian brain development.

Animals↗
PubMed1994

Expression patterns of messenger ribonucleic acids encoding prohormone convertases (PC2 and PC3) in human pituitary adenomas.

OBJECTIVE: Prohormone convertases (PC2 and PC3) have been reported to play an important role for prohormone processing in rodent pituitaries. However, expression of mRNAs encoding these enzymes has not been characterized in human pituitaries. In addition, altered or insufficient prohormone processing has been reported in some human pituitary adenomas. Thus, the expression patterns of these mRNAs in non-tumorous and tumorous human pituitaries should be examined. DESIGN: Total RNAs were extracted from non-tumorous or tumorous human pituitaries to analyse PC2 and PC3 mRNA expression. SAMPLES: Five ACTH producing adenomas, 11 GH producing adenomas, one PRL producing adenoma and five non-functioning adenomas were obtained at surgery. Two non-tumorous pituitaries were also included in this study. MEASUREMENTS: The contents were quantitatively measured by Northern blot analysis using rat PC3 cDNA or human PC2 cDNA as a probe. The method was also developed for the detection of PC2 mRNA by Southern blot analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products. RESULTS: PC2 and PC3 mRNAs in non-tumorous samples were detected by Northern blot analysis whereas their contents in tumorous samples varied from high levels to undetectable. Marked variation of PC3 mRNA levels was observed among GH producing adenomas. ACTH producing adenomas were observed to express PC3 mRNA. Northern blot analysis also revealed that PC2 mRNA levels in ACTH producing adenomas were low except for one sample. PC2 mRNA expression in GH producing adenomas was confirmed by Southern blot analysis of RT-PCR products. This procedure also confirmed the various levels of PC2 mRNA expression among ACTH producing adenomas. CONCLUSION: The expression of PC2 and PC3 mRNA in human pituitaries has been confirmed. However, their expression has been observed to vary quantitatively and not to be restricted to certain types of pituitary cells.

Adenoma↗
PubMed1996

Dissociation of the complex between the neuroendocrine chaperone 7B2 and prohormone convertase PC2 is not associated with proPC2 maturation.

7B2 is a highly conserved neuroendocrine protein that is associated with the proform of the prohormone convertase PC2 in the early stages of the secretory pathway in intermediate pituitary cells of Xenopus laevis. Subsequent processing of 7B2 and dissociation of the 7B2/proPC2 complex is thought to be associated with the conversion of proPC2 to the mature enzyme. Here, we report that, in both Xenopus and mouse intermediate cells, proPC2 maturation does not take place when the proenzyme is associated with the 7B2 precursor and that, in contrast to the previous notion, dissociation of the complex between proPC2 and the N-terminal 7B2 fragment precedes, and is thus not directly linked to, proPC2 maturation. In vitro, conversion of newly synthesized proPC2 was efficiently blocked by recombinant 7B2 and studies with truncation mutants indicated that a short segment in the C-terminal region of 7B2 is necessary and sufficient for this inhibitory effect. Our results indicate that, after 7B2 precursor processing and dissociation of the N-terminal fragment, the C-terminal fragment of 7B2 may remain associated with proPC2, thereby preventing autocatalytic conversion of the proenzyme until the appropriate site for activation in the secretory pathway is reached.

Animals↗
PubMed1996

Prohormone convertases (PC1/3 and PC2) in rat and human pancreas and islet cell tumors: subcellular immunohistochemical analysis.

Prohormone convertase 1/3 (PC1/3; also termed PC1 or PC3) and PC2 are enzymes that activate prohormones by cleaving the pairs of basic amino acids. This mechanism was initially inferred from the series of several endocrine and neuroendocrine precursor proteins, including proinsulin and proglucagon. To determine the cellular and subcellular distribution of PC1/3 and PC2 in the rat and human pancreas, immunohistochemistry was performed using polyclonal antisera against mouse PC1/3 (ST-28) and mouse PC2 (ST-29). These studies showed light and electron microscopic co-localization of insulin, PC1/3 and PC2, and the coexistence of glucagon and PC2 in the pancreatic islets. This tendency of colocalization was also depicted in one case of human insulinoma and three cases of human glucagonomas, as well as in rat insulinomas. In two cases of human insulinomas, incomplete processing of proinsulin was suggested by the absence of PC2. At the subcellular level in the rat pancreatic islet, the colocalization of PC1/3 and insulin, and that of PC2 and glucagon, were observed in the same secretory granules by immunoelectron microscopy and image analysis. These studies suggest that PC1/3 and PC2 can function with the specificities in the processing of proinsulin and proglucagon into their active forms, respectively, in the normal and neoplastic pancreatic islets.

Adenoma, Islet Cell↗
PubMed1993

Peptidylglycine alpha-amidating monooxygenase and other processing enzymes in the neurointermediate pituitary.

Studies on the mRNAs encoding PAM and on the various PAM proteins have begun to reveal some of the intricate mechanisms used to optimize the ability of this enzyme to carry out the alpha-amidation of peptides. Comparison of the regulatory elements governing expression of the various enzymes involved in peptide processing should reveal common elements. Knowledge of the processing enzymes themselves should help us to understand how these enzymes function in the secretory granule environment. In addition to their catalytic domains, other processing enzymes, like PAM, may well have processing domains and routing domains designed to optimize their ability to function in secretory granules.

Amino Acid Sequence↗
PubMed2002

Proteolytic processing of chromogranins is modified in brains of transgenic mice.

In normal brain, secretogranin II and chromogranins A and S are extensively converted by endopeptidases to the peptides secretoneurin, GE-19, and PE-11, respectively. After genetic knockout of PC2 or its helper protein 7B2, but not after mutation of carboxypeptidase E, endoproteolytic processing decreased, as indicated by appearance of intermediate-sized processing products.

Animals↗