Sorting within the regulated secretory pathway occurs in the trans-Golgi network

Bioactive peptides cleaved from the egg-laying hormone precursor in the bag cell neurons of Aplysia are sorted into distinct dense core vesicle classes (DCVs). Bag cell prohormone processing can be divided into two stages, an initial cleavage occurring in a late Golgi compartment, which is not blocked by monensin, and later cleavages that occur within DCVs and are blocked by monensin. Prohormone intermediates are sorted in the trans-Golgi network. The large soma-specific DCVs turn over, while the small DCVs are transported to processes for regulated release. Thus, protein trafficking differentially regulates the levels and localization of multiple biologically active peptides derived from a common prohormone.     

Regional differences in processing of locally translated prohormone in peptidergic neurons of Aplysia californica

Earlier work showed that cell bodies and neurites of the peptidergic bag cell neurons of Aplysia californica contain mRNA for egg-laying hormone. The purpose of the present study was to determine if egg-laying hormone synthesis and prohormone processing is similar in the pleurovisceral connective nerves (containing neurites of bag cell neurons) and the bag cell neuron clusters (containing both cell bodies and neurites of bag cell neurons). Initial experiments confirmed by RT-PCR and sequencing that egg-laying hormone mRNA was present in the pleurovisceral connective nerves. To investigate possible regional differences in translation of mRNA and prohormone processing, clusters were separated from connective nerves and newly synthesized egg-laying hormone-immunoreactive proteins were analyzed. Results showed that synthesis and processing of prohormone occurred in both the clusters and isolated connective nerves; however, the relative abundance of prohormone, processing intermediates, and egg-laying hormone was different. Pulse-chase experiments showed that prohormone was processed more slowly in the connective nerves than in the clusters. These results show that mRNA in isolated neural processes of neuroendocrine cells can be translated, and that the cellular machinery for protein synthesis is present, but processing of the ELH prohormone is significantly compromised.     

Secretion of locally synthesized neurohormone from neurites of peptidergic neurons

Local protein synthesis in neuronal processes is a common phenomenon and may play an important role in synaptic plasticity and hormonal regulation. We have used neuroendocrine bag cells of Aplysia californica as a model system to study local protein synthesis. In our previous work we found that bag cell neurites are capable of synthesizing and processing the prohormone of egg-laying hormone (pro-ELH). In the present study, we found that bag cell neurites are also capable of releasing locally synthesized pro-ELH and ELH-related products via both constitutive and regulated pathways. However, an electrical afterdischarge did not enhance local pro-ELH synthesis, as it does in the bag cell soma. This is the first evidence that isolated neurites are capable of secreting locally synthesized proteins.     

Processing of the egg-laying hormone (ELH) precursor in the bag cell neurons of Aplysia

Egg laying in Aplysia is mediated by a battery of neuropeptides released from the bag cell neurons. Predominant intermediates in the proteolytic processing of the Aplysia egg-laying hormone neuropeptide precursor were characterized using biochemical and immunological techniques. Following removal of the signal peptide, a rapid cleavage at the tetrabasic sequence Arg-Arg-Lys-Arg separates the amino and carboxyl regions of the prohormone. Processing of the carboxyl-terminal portion of the precursor then proceeds rapidly via two further cleavages at dibasic residues, resulting in a well defined product mixture within 4 h of chase. By contrast, processing of the amino-terminal side of the molecule proceeds only partially to completion after 20 h of chase and a well defined set of intermediates is not observed. Molecular genetic, physiological, and behavioral studies in conjunction with the biochemical investigations presented here are defining the information flow which governs the egg-laying behavior of Aplysia.     

Processing of secretogranin II by prohormone convertases: Importance ofPC1 in generation of secretoneurin

Secretoneurin is a recently characterized neuropeptidepresent in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.     

Subcellular Fractionation of Prohormone Processing Products in the Bag Cell Neurons

Abstract: Multiple biologically active peptides arising from a common prohormone are sorted into distinct classes of dense core vesicles within the bag cell neurons of Aplysia californica . In this study, pulse-chase analysis, combined with subcellular fractionation on Percoll gradients, are used to define the location of the prohormone processing events within the secretory pathway. Initial cleavage of the prohormone occurs in a light cellular compartment associated with the Golgi apparatus. The amino-terminal processing intermediate then accumulates in a denser compartment containing small dense cores enclosed in membranous sacs, as well as larger immature vesicles. After 4 h, amino-terminai products are found primarily in a much denser compartment which consists of large and small dense core vesicles. These large and small vesicles can be separated from each other using Percoll gradient centrifugation and are found to be enriched in amino- and carboxy-terminal products, respectively. Lastly, membrane association experiments suggest differential binding to membranes, or integral membrane proteins, as a possible mechanism for sorting of amino- and carboxy-terminal products.     

Phylogenetic analysis of Amphioxus genes of the proprotein convertase family, including aPC6C, a marker of epithelial fusions during embryology

The proprotein convertases (PCs) comprise a family of subtilisin-like endoproteases that activate precursor proteins (including, prohormones, growth factors, and adhesion molecules) during their transit through secretory pathways or at the cell surface. To explore the evolution of the PC gene family in chordates, we made a phylogenetic analysis of PC genes found in databases, with special attention to three PC genes of the cephalochordate amphioxus, the closest living invertebrate relative to the vertebrates. Since some vertebrate PC genes are essential for early development, we investigated the expression pattern of the C isoform of the amphioxus PC6 gene (aPC6C). In amphioxus embryos and larvae, aPC6C is expressed at places where epithelia fuse. Several kinds of fusions occur: ectoderm-to-ectoderm during neurulation; mesoderm-to-ectoderm during formation of the preoral ciliated pit; and endoderm-to-ectoderm during formation of the mouth, pharyngeal slits, anus, and external opening of the club-shaped gland. Presumably, at all these sites, aPC6C is activating proteins favoring association between previously disjunct cell populations.     

Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting

Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.     

Multiple neuropeptides derived from a common precursor are differentially packaged and transported

The ELH prohormone is proteolytically processed into at least nine peptides which govern egg-laying behavior in Aplysia. Quantitative immunocytochemistry demonstrates that peptides derived from the prohormone are packaged into distinct vesicle classes. Further experiments suggest the segregation occurs via a rapid initial proteolytic cleavage of the prohormone followed by sorting at the trans Golgi. Egg-laying hormone (ELH) immunoreactivity is localized to the cell body and processes, while bag cell peptide (BCP) immunoreactivity is greater in the cell body. Steady state levels of the amino-terminal set of peptides including the BCPs are 3- to 8-fold lower than the carboxy-terminal cleavage products, such as ELH. Thus, intracellular packaging and routing of the peptides cleaved from a single prohormone regulate their localization and levels in these neurons.     

The bag cell neurons ofAplysia

Egg laying in Aplysia involves a well-characterized series of behaviors that can last for several hours. The behaviors are controlled by two bilateral clusters of peptidergic neurons in the abdominal ganglion. Following brief stimulation, these neurons, which have been termed the bag cell neurons, undergo a sequence of changes in their excitability lasting many hours. The bag cell neurons have served as a model system for studying the molecular mechanisms involved in the synthesis, processing, and release of neuroactive peptides and in the regulation of prolonged changes in neuronal excitability.     

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2

The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.     

ProSAAS-Derived Peptides Are Differentially Processed and Sorted in Mouse Brain and AtT-20 Cells

ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes) and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides.     

The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.     

Tissue distribution and processing of proSAAS by proprotein convertases

The conversion of inactive precursor proteins into bioactive neuropeptides and peptide hormones involves regulated secretory proteins such as prohormone convertases PC1 and PC2. The neuroendocrine protein 7B2 represents a specific binding protein for PC2, and the protein proSAAS, which interacts with PC1, exhibits certain structural and functional homologies with 7B2. With the intention of better understanding the physiological role of proSAAS and its derived peptides, we investigated its tissue localization using a new radioimmunoassay (RIA) to a C-terminal proSAAS-derived peptide. Immunoreactivity corresponding to this SAAS-derived peptide is mostly localized to the brain and gut. Analysis of the brain distribution of the proSAAS-derived peptides indicates that the hypothalamus and pituitary are the two richest areas, consistent with the previously described high expression of PC1 in these two areas. In order to investigate the cleavage of proSAAS by prohormone convertases, we incubated recombinant His-tagged proSAAS with recombinant mouse proPC2 or furin, separated the cleavage products using high-pressure gel permeation chromatography and analyzed the products by RIA. Our results indicate that either PC2 or furin can accomplish in vitro rapid removal and efficient internal processing of the C-terminal peptide, exposing the inhibitory hexapeptide to possible further digestion by carboxypeptidases. Finally, we also studied proSAAS processing in the brains of wild-type and PC2 null mice and found that proSAAS is efficiently processed in vivo. Whereas the C-terminal peptide is mostly internally cleaved in wild-type mouse brain, it is not processed as efficiently in the brain of PC2 null mice, suggesting that PC2 is partially responsible for this cleavage in vivo.     

Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells

The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.     

Mammalian neural and endocrine pro-protein and pro-hormone convertases belonging to the subtilisin family of serine proteinases.

Conversion of pro-hormones and precursor proteins into biologically active peptides and proteins involves the concerted action of a number of convertases and post-translation modification enzymes. The identification of the yeast convertase kexin as a prototype processing enzyme led to the discovery of the mammalian convertase designated furin, PC1 and PC2. Whereas furin is ubiquitously expressed, PC1 and PC2 are found only in endocrine and neural tissues and cell lines. In man and mouse, the genes coding for furin, PC1 and PC2 reside on three different chromosomes. The analysis of the intracellular processing of PC1 and PC2 and the removal of their pro-segment is presented, together with a summary of the cleavage specificity of these enzymes for precursors such as pro-opiomelanocortin (POMC) and human pro-renin. The distinct tissue distribution of PC1 and PC2 and their coregulation with POMC in the pituitary neurointermediate lobe adds credence to their physiological role as convertases involved in the tissue-specific processing of precursor proteins.     

Influence of Ca2+ and pH on the folding of the prourotensin II precursor

Proper folding is a crucial step for the trafficking of proteins through the secretory pathway. We hypothesized that the secretory granules of endocrine cells provide optimal folding conditions of prohormone precursors for cleavage. Here, using circular dichroism and in vitro processing on purified prourotensin II (ProUII), we show that the precursor undergoes pH- and Ca(2+)-dependent conformational and stability changes. ProUII has a stable tertiary structure at pH 5.5 in presence of Ca(2+) and is correctly cleaved in these conditions by prohormone convertases. Taken together, our results support the notion that precursors may need to be optimally folded in the lumen of secretory granules for their processing.     

Neuropeptide-processing carboxypeptidases

Neuropeptides are generally produced from precursor proteins by selective cleavage at specific sites, usually involving basic amino acids. Enzymes such as the prohormone convertases and carboxypeptidase E are highly specific for these basic amino acid-containing sites. In addition to this "traditional" pathway, several neuropeptides are known to be cleaved at non-basic sites, and the enzymes responsible for these cleavages have not been conclusively identified. In a recent search for novel members of the metallocarboxypeptidase family, we found three human genes. One of these, named "CPA-5," has a specificity for C-terminal hydrophobic amino acids and mRNA expression in brain, pituitary, and testis. To test whether CPA-5 protein has a distribution pattern in pituitary that is consistent with a role for this enzyme in the non-basic processing of proopiomelanocortin-derived peptides such as beta-endorphin and adrenocorticotropin, we examined the distribution of CPA-5 using immunocytochemistry. In the pituitary, CPA-5 is detected in the neurointermediate lobe and in scattered cells in the anterior lobe. In the AtT-20 corticotroph cell line, CPA-5 has a perinuclear distribution. Taken together, these results are consistent with a role for CPA-5 in the intracellular processing of proopiomelanocortin-derived peptides at non-basic sites.