Sulfation of porcine parathyroid secretory protein I. Detection of tyrosine sulfate

Secretory Protein I (SP-I) is an acidic glycoprotein that is stored and co-secreted with parathormone by parathyroid glands. It has been found to be chemically similar, if not identical, to chromogranin A of the adrenal medulla and to be present in most endocrine cells. In the present study, 35SO4 was shown to be incorporated into SP-I and several other proteins of porcine parathyroid tissue incubated in vitro. The predominant sulfated species secreted to the medium was SP-I. Up to 20% of the tyrosine residues in secreted SP-I were labeled with 35SO4. Both the cellular and secreted forms migrated on sodium dodecyl sulfate gels as a pair of proteins with apparent molecular weights of 82,000 and 78,000. The 82-kDa protein could be converted to the 78-kDa species by treatment with neuraminidase. Sulfate exists in SP-I as tyrosine sulfate based on the identification of this amino acid by thin layer electrophoresis following alkaline hydrolysis. Extracellular Ca2+ (3 mM) greatly suppressed the secretion of 35SO4-labeled SP-I without affecting the intracellular sulfation of the molecule or the secretion of a minor sulfated protein unrelated to SP-I. The ratio of incorporated 35SO4 to 3H-amino-acid was greater in secreted SP-I than in tissue SP-I, suggesting that much sulfation of this protein occurred during or just before secretion.     

Secretion of sulfated and nonsulfated forms of parathyroid chromogranin A (secretory protein-I)

Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and [3H]Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues.     

Basic Fibroblast Growth Factor (bFGF) Immunoreactivity Is Present in Chromaffin Granules

Basic fibroblast growth factor (bFGF) has recently been isolated from bovine adrenal glands. Immunohistological data revealed its presence in both adrenal cortex and adrenal medulla. Using immuno-electronmicroscopy, we found that in medullary chromaffin cells bFGF-immunoreactivity is localized in the secretory granules. Immunoreactivity also was observed by electronmicroscopy in isolated granules. Western blot analysis revealed the presence of the typical 18-kDa bFGF and additional immunoreactive materials with molecular masses of approximately 24, 30, and 46 kDa in whole bovine adrenal, and in cortex and medulla. Similar results were obtained with proteins from bovine chromaffin granules, with the following two exceptions: the 46-kDa immunoreactivity was found to be highly enriched when compared with medulla or cortex, and the 18-kDa band could be detected with only an antiserum against a synthetic peptide comprising the 24 NH2-terminal amino acids of bFGF, and not with an antiserum against purified bovine pituitary bFGF. All fractions enriched for bFGF-immunoreactivity showed neurotrophic activity for chick ciliary ganglion neurons, which could be blocked by antibodies. These results demonstrate for the first time the localization and occurrence of bFGF in a cellular secretory organelle, and present further evidence for the existence of higher molecular weight immunoreactive forms of bFGF.     

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.     

Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Signal-mediated sorting of neuropeptides and prohormones: secretory granule biogenesis revisited

Neuropeptides and hormones, in contrast to constitutive secretory proteins, are sorted to and stored in secretory granules and released upon a stimulus. During the last two decades, signals and mechanisms involved in their sorting to the regulated pathway of protein secretion have been addressed in numerous studies. Taken together these studies revealed three important features of regulated secretory proteins: aggregation, sorting signal motifs and membrane binding. Here we try to dissect the sorting process with regard to these features and discuss their relevance in the context of current sorting models. We especially address the question where in the secretory pathway sorting takes place and discuss a possible role of sorting receptors.     

α1-Antichymotrypsin-Like Proteins I and II Purified from Bovine Adrenal Medulla Are Enriched in Chromaffin Granules and Inhibit the Proenkephalin Processing Enzyme "Prohormone Thiol Protease"

Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K(i,app) of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

An antibody against secretogranin I (chromogranin B) is packaged into secretory granules

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.     

The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br- cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.     

Tissue distribution of smg p25A, a ras p21-like GTP-binding protein, studied by use of a specific monoclonal antibody

We made a monoclonal antibody specifically recognizing smg p25A among many ras p21-like GTP-binding proteins and investigated the tissue distribution of smg p25A by use of this antibody. By immunoblot analysis, smg p25A was detected in rat brain and bovine adrenal medulla but not in bovine adrenal cortex or other rat tissues including thymus, spleen, lung, heart, liver and kidney. However, by immunocytochemical studies, smg p25A was detected not only in the synaptic areas of rat brain and the chromaffin cells of bovine adrenal medulla but also in the endocrine cells of rat pancreatic islets, the acinar cells of rat exocrine pancreas and the exocrine cells of rat submaxillary gland. These results suggest that smg p25A is involved in the regulation of secretory processes not only in synapses but also in other endocrine and exocrine secretory cells.     

Sulfated secreted forms of bovine and porcine parathyroid chromogranin A (secretory protein-I)

Chromogranin A (secretory protein-I) is an acidic sulfated glycoprotein found in secretory granules of most endocrine and neuroendocrine cells. In the parathyroid it is co-stored and secreted with parathormone in response to hypocalcemia. Differences in post-translational modifications have been reported between chromogranin A from the bovine adrenal and porcine parathyroid glands. The former has been reported to be sulfated mainly on oligosaccharide residues and apparently includes a proteoglycan form, whereas the latter was previously reported to be tyrosine sulfated with little of the proteoglycan form present. Here we have directly compared 35SO4-labeled parathyroid chromogranin A from the pig and the cow to determine if these reported differences were tissue or species specific. We find that the chromogranin A secreted by the bovine gland contains a proteoglycan form, whereas that from the porcine gland does not. Moreover, chromogranin A of both species is primarily sulfated on oligosaccharide residues with little if any tyrosine sulfate detected. Differences were detected in the structure of sulfated O-linked oligosaccharides in bovine and porcine parathyroid chromogranin A.     

Secretory granule formation

A deeper understanding of the regulated exocytic pathway, and for that matter the constitutive exocytic pathway, will depend on our ability to characterize the proteins in the vesicle membranes. Characterizing the protein composition of secretory granule membrane has proven to be a formidable task, and as far as I know, the work done to date has not told us a great deal about the mechanisms involved in sorting the contents of regulated secretory granules, or bringing about constitutive or regulated fusion with the plasma membrane. Without knowing a great deal more about the membranes, there seems to be little prospect of real further progress in understanding the key properties of the regulated exocytic pathway.     

Sorting of growth hormone-erythropoietin fusion proteins in rat salivary glands

Neuroendocrine and exocrine cells secrete proteins in either a constitutive manner or via the regulated secretory pathway (RSP), but the specific sorting mechanisms involved are not fully understood. After gene transfer to rat salivary glands, the transgenic model proteins human growth hormone (hGH) and erythropoietin (hEpo) are secreted primarily into saliva (RSP; exocrine) and serum (constitutive; endocrine), respectively. We hypothesized that fusion of hGH at either the C-terminus or the N-terminus of hEpo would re-direct hEpo from the bloodstream into saliva. We constructed and expressed two fusion proteins, hEpo-hGH and hGH-hEpo, using serotype 5-adenoviral vectors, and delivered them to rat submandibular glands in vivo via retroductal cannulation. Both the hEpo-hGH and hGH-hEpo fusion proteins, but not hEpo alone, were secreted primarily into saliva (p < 0.0001 and p = 0.0083, respectively). These in vivo studies demonstrate for the first time that hGH, in an N- as well as C-terminal position, influences the secretion of a constitutive pathway protein.     

Neurotrophin-4, alone or heterodimerized with brain-derived neurotrophic factor,is sorted to the constitutive secretory pathway

Nerve growth factor and neurotrophin-3 (NT-3) are processed within the constitutive secretory pathway of neurons and neuroendocrine cells and are released continuously in an activity-independent fashion. In contrast, brain-derived neurotrophic factor (BDNF) is processed in the regulated secretory pathway, stored in vesicles, and released in response to neuronal activity, consistent with its role in modulating synaptic plasticity. In this study, we used vaccinia virus infection and transfection methods to monitor the processing and sorting of neurotrophin-4 (NT-4) in AtT-20 cells, which have been used as a model for the sorting of secretory proteins in neurons. Our data show that NT-4 is processed in the constitutive secretory pathway. The molecule is diffusely distributed within the cells and released, soon after being synthesized, in a manner that is not affected by cell depolarization. We further show that NT-4 and BDNF, when co-expressed, can form heterodimers that are constitutively released. In contrast, heterodimers of NT-3 and BDNF have been shown to be released through the regulated secretory pathway. Thus, NT-4, alone or when co-expressed with BDNF, is processed within and secreted by the constitutive secretory pathway.     

Distinct linkage between post-translational processing and differential secretion of progastrin derivatives in endocrine cells

Prohormones often undergo extensive cellular processing prior to secretion. These post-translational processing events occur in organelles of the constitutive or regulated secretory pathway. The aim of this study was to examine the relationship between post-translational modifications and the secretory pathways taken by peptides derived from progastrin, the prohormone of gastrin, which in vivo is secreted by cells of the pyloric glands and stimulates the release of gastric acid. Targeting progastrin to compartments of the early secretory pathway shows that endoproteolytic processing is initiated in a pre-trans-Golgi network compartment of endocrine but not non-endocrine cells. The resulting N-terminal fragments of progastrin are secreted via the constitutive pathway, whereas endoproteolytically processed C-terminal fragments are secreted via the regulated or constitutive-like pathways. C-terminal fragments derived from progastrin differ in characteristic manners in levels and patterns of carboxyamidation and tyrosine sulfation in accordance with the secretory pathway taken. Point mutations introduced into a sorting motif disrupt these patterns, suggesting that differences in post-translational modifications are attributable to differential intracellular sorting of precursors. The results suggest a two-step sorting mechanism for progastrin leading to differential secretion of processed fragments via different secretory pathways.     

cAMP-dependent protein phosphorylation of membrane proteins in the parotid gland, platelets and liver. Comparison of a 22-kDa phosphoprotein from rat parotid microsomes (protein III) with phosphoproteins of similar molecular size from platelet and liver membranes

Stimulation of secretion in exocrine secretory glands leads to the phosphorylation of a 22-kDa membrane protein (protein III) whose function is still unknown [Jahn et al. (1980) Eur. J. Biochem. 112, 345-352; Jahn & S?ling (1980) Proc. Natl Acad. Sci. USA 78, 6903-6906]. This report describes the comparison of this protein with phosphorylated membrane proteins of similar molecular mass in platelets and liver. Incubation of platelets with agents which raise the intracellular cAMP concentration results in the phosphorylation of a 22-kDa protein which is also phosphorylated in membrane preparations by endogenous kinases or by exogenous cAMP-dependent protein kinase. It is shown that this protein is distinct from protein III although both proteins have the same molecular mass and are substrates of cAMP-dependent protein kinase. In contrast to platelets, protein III could be demonstrated in liver microsomes. This indicates that the function of protein III is not exclusively linked to the stimulus-secretion coupling in exocrine cells.