The yellow fever and dengue fever carrying mosquito, Aedes aegypti, requires blood feeding for eg... more The yellow fever and dengue fever carrying mosquito, Aedes aegypti, requires blood feeding for egg production. The blood proteins are digested in the midgut to yield amino acids which are the nutritional source for oogenesis. Serine proteases are important enzymes that participate in the process of blood protein digestion. The identification of the corresponding genes may have significant implications in the control of mosquito-borne diseases. A gut-specific chymotrypsin-like cDNA was isolated and sequenced. The 938 bp clone encodes a preproenzyme with a putative 18 amino acid signal peptide sequence, a 7 amino acid activation peptide sequence rich in serine and charged residues, and a mature enzyme of 268 amino acids. The deduced amino acid sequence has a typical catalytic triad region for serine proteases (His 57, Asp 102 and Ser 195 in bovine chymotrypsin numbering system), and the hydrophobic substrate binding pocket with most features of chymotrypsins. Six cysteine residues are present in the sequence which are characteristically involved in disulfide bond formation in invertebrate serine proteases. Characterization of the gene expression and the protein synthesis, as well as the enzymatic activity in the midgut, clearly demonstrated that (1) the chymotrypsin gene is newly transcribed after eclosion and the mRNA is present almost steadily during the digestion of a meal; (2) the chymotrypsin synthesis and its corresponding activity are induced and increased significantly by the ingestion of a meal. In vitro studies of the recombinant protease derived from the cDNA clone indicated several unique properties of the mosquito chymotrypsin compared with its bovine analog
Robert J. Parmer, 1Sushil K. Mahata, 1Qijiao Jiang, 1Laurent Taupenot, 2Yun Gong, 1Manjula Mahata... more Robert J. Parmer, 1Sushil K. Mahata, 1Qijiao Jiang, 1Laurent Taupenot, 2Yun Gong, 1Manjula Mahata, 1Daniel T. O'Connor, and 2Lindsey A. Miles 1Department of Medicine and Center for Molecular Genetics, University of California, and Veterans Affairs Medical Center, San Diego Healthcare System, San Diego, California 921 6191II, 1Department of Vascular Biology, The Scripps Research Institute, La Jolla, Calfornia 92037 1
This protocol describes the primary culture of individual chromaffin cells derived by enzymatic d... more This protocol describes the primary culture of individual chromaffin cells derived by enzymatic digestion from the adrenal medulla of the bovine adrenal gland. Since the late 1970s, such cells have provided a useful model system to study neurotransmitter biosynthesis, storage and release in the catecholaminergic system. The protocol can be divided into three stages: isolation of cells (4-6 h), determination of viable cell numbers (approximately 30 min) and growth in culture (3-7 d). An alternative procedure is to perform studies in a continuous chromaffin (pheochromocytoma) cell line, such as PC12, although such transformed cells are typically less highly differentiated than primary cells. The bovine chromaffin cell procedure should yield approximately 10-20 million cells, suitable for several experiments over the subsequent 3-7 d. Typical experiments involve transmitter biosynthesis, vesicular storage, exocytotic release, stimulus coupling (signal transduction) toward secretion or transcription, or morphology, including ultrastructure. The total time, from adrenal gland harvest until functional experiments, is typically 4-8 d.
The mechanisms and specific proteases involved in the processing of prohormone precursors in the ... more The mechanisms and specific proteases involved in the processing of prohormone precursors in the neuroendocrine system are not fully established. Chromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is released with catecholamines by exocytosis from the chromaffin cells of the adrenal medulla and from sympathetic axons. CgA is found in secretory vesicles throughout the neuroendocrine system and may exemplify a prototypical prohormone precursor (1). Recent evidence suggests that CgA is a precursor of several peptides that affect cellular secretory function. These peptides include pancreastatin (porcine CgA 240-288 , first isolated from porcine pancreas; ref. 2), which inhibits glucose-stimulated insulin release and parathyroid hormone release from islet beta-cells and chief cells, respectively; vasostatin (human CgA 1-76 ; refs. 3, 4) or β-granin (rat CgA 1-114 ; refs. 5-7), which inhibits parathyroid hormone release and relaxes vascular smooth muscle; and parastatin (porcine CgA 347-419 ; ref. 8), which inhibits parathyroid hormone release. In addition, recent studies have revealed that CgA may function as a prohormone from which peptides are released that modulate the function of catecholaminergic cells. Trypsin digestion of CgA produces peptides that inhibit release of catecholamines from adrenal medullary cells in culture (9). Previous studies have demonstrated that CgA processing occurs in a specific tissue-dependent fashion with the extent and pattern of processing varying widely from one neuroendocrine site to the next (10). CgA processing may take place intracellularly as well as extracellularly, that is, after secretion into the extracellular space (9, 11, 12). Indeed, previous studies have shown that extracellular processing appears to be particularly important for generation of catecholaminerelease inhibitory activity from the parent CgA molecule in the vicinity of the chromaffin cell (9, 11). Although a number of studies have examined the role of prohormone convertases (13-15) that process prohormone precursors intracellularly to generate regulatory peptides, few studies have focused on the role of other proteases and mechanisms of extracellular processing in the generation of bioactive peptides. Recent studies have demonstrated that catecholamine release-inhibitory activity is generated when CgA is co-incubated with chromaffin cells, suggesting that at least some component of the cleavage occurs
Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a pr... more Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.
The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occur... more The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A 332-364 ; identity of the peptide was confirmed by diagnostic Met 346 oxidation. Further analysis revealed two additional forms: bovine chromogranin A 333-364 and A 343-362. Synthetic longer (chromogranin A 332-364) and shorter (chromogranin A 344-364) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC 50 ϳ2.01 versus ϳ0.35 M). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A 340-372 , bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.
A cDNA for a midgut chymotrypsin, induced by a blood meal, has been cloned and sequenced from the... more A cDNA for a midgut chymotrypsin, induced by a blood meal, has been cloned and sequenced from the mosquito Aedes aegypti. The 938 base sequence codes for a 268 amino acid protein, which contains an 18-residue signal peptide and a seven-residue activation peptide. The deduced amino acid sequence contains several features typical of chymotrypsin proteases, including the catalytic triad of serine proteases and the residues that determine the chymotrypsin substrate specificity pocket. The chymotrypsin mRNA, absent in larvae, pupae, males and newly emerged females, reaches detectable levels within 24 h post-emergence and attains a maximum level 3-7 days after emergence. Translation of the chymotrypsin mRNA is induced by feeding a protein meal, and there is a dramatic increase in midgut chymotrypsin enzymatic activity after feeding. Chymotrypsin activity remained high during protein digestion, but chymotrypsin protein levels and enzymatic activity were almost undetectable once digestion was completed, 48 h after feeding.
Recent studies suggest a crucial role for plasminogen activator inhibitor-1 (PAI-1) in mediating ... more Recent studies suggest a crucial role for plasminogen activator inhibitor-1 (PAI-1) in mediating stress-induced hypercoagulability and thrombosis. However, the mechanisms by which PAI-1 is released by stress are not well-delineated. Here, we examined catecholaminergic neurosecretory cells for expression, trafficking, and release of PAI-1. PAI-1 was prominently expressed in PC12 pheochromocytoma cells and bovine adrenomedullary chromaffin cells as detected by Northern blotting, Western blotting, and specific PAI-1 ELISA. Sucrose gradient fractionation studies and immunoelectron microscopy demonstrated localization of PAI-1 to catecholamine storage vesicles. Secretogogue stimulation resulted in corelease of PAI-1 with catecholamines. Parallel increases in plasma PAI-1 and catecholamines were observed in response to acute sympathoadrenal activation by restraint stress in mice in vivo. Reverse fibrin zymography demonstrated free PAI-1 in cellular releasates. Detection of high molecular ...
The yellow fever and dengue fever carrying mosquito, Aedes aegypti, requires blood feeding for eg... more The yellow fever and dengue fever carrying mosquito, Aedes aegypti, requires blood feeding for egg production. The blood proteins are digested in the midgut to yield amino acids which are the nutritional source for oogenesis. Serine proteases are important enzymes that participate in the process of blood protein digestion. The identification of the corresponding genes may have significant implications in the control of mosquito-borne diseases. A gut-specific chymotrypsin-like cDNA was isolated and sequenced. The 938 bp clone encodes a preproenzyme with a putative 18 amino acid signal peptide sequence, a 7 amino acid activation peptide sequence rich in serine and charged residues, and a mature enzyme of 268 amino acids. The deduced amino acid sequence has a typical catalytic triad region for serine proteases (His 57, Asp 102 and Ser 195 in bovine chymotrypsin numbering system), and the hydrophobic substrate binding pocket with most features of chymotrypsins. Six cysteine residues are present in the sequence which are characteristically involved in disulfide bond formation in invertebrate serine proteases. Characterization of the gene expression and the protein synthesis, as well as the enzymatic activity in the midgut, clearly demonstrated that (1) the chymotrypsin gene is newly transcribed after eclosion and the mRNA is present almost steadily during the digestion of a meal; (2) the chymotrypsin synthesis and its corresponding activity are induced and increased significantly by the ingestion of a meal. In vitro studies of the recombinant protease derived from the cDNA clone indicated several unique properties of the mosquito chymotrypsin compared with its bovine analog
Robert J. Parmer, 1Sushil K. Mahata, 1Qijiao Jiang, 1Laurent Taupenot, 2Yun Gong, 1Manjula Mahata... more Robert J. Parmer, 1Sushil K. Mahata, 1Qijiao Jiang, 1Laurent Taupenot, 2Yun Gong, 1Manjula Mahata, 1Daniel T. O'Connor, and 2Lindsey A. Miles 1Department of Medicine and Center for Molecular Genetics, University of California, and Veterans Affairs Medical Center, San Diego Healthcare System, San Diego, California 921 6191II, 1Department of Vascular Biology, The Scripps Research Institute, La Jolla, Calfornia 92037 1
This protocol describes the primary culture of individual chromaffin cells derived by enzymatic d... more This protocol describes the primary culture of individual chromaffin cells derived by enzymatic digestion from the adrenal medulla of the bovine adrenal gland. Since the late 1970s, such cells have provided a useful model system to study neurotransmitter biosynthesis, storage and release in the catecholaminergic system. The protocol can be divided into three stages: isolation of cells (4-6 h), determination of viable cell numbers (approximately 30 min) and growth in culture (3-7 d). An alternative procedure is to perform studies in a continuous chromaffin (pheochromocytoma) cell line, such as PC12, although such transformed cells are typically less highly differentiated than primary cells. The bovine chromaffin cell procedure should yield approximately 10-20 million cells, suitable for several experiments over the subsequent 3-7 d. Typical experiments involve transmitter biosynthesis, vesicular storage, exocytotic release, stimulus coupling (signal transduction) toward secretion or transcription, or morphology, including ultrastructure. The total time, from adrenal gland harvest until functional experiments, is typically 4-8 d.
The mechanisms and specific proteases involved in the processing of prohormone precursors in the ... more The mechanisms and specific proteases involved in the processing of prohormone precursors in the neuroendocrine system are not fully established. Chromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is released with catecholamines by exocytosis from the chromaffin cells of the adrenal medulla and from sympathetic axons. CgA is found in secretory vesicles throughout the neuroendocrine system and may exemplify a prototypical prohormone precursor (1). Recent evidence suggests that CgA is a precursor of several peptides that affect cellular secretory function. These peptides include pancreastatin (porcine CgA 240-288 , first isolated from porcine pancreas; ref. 2), which inhibits glucose-stimulated insulin release and parathyroid hormone release from islet beta-cells and chief cells, respectively; vasostatin (human CgA 1-76 ; refs. 3, 4) or β-granin (rat CgA 1-114 ; refs. 5-7), which inhibits parathyroid hormone release and relaxes vascular smooth muscle; and parastatin (porcine CgA 347-419 ; ref. 8), which inhibits parathyroid hormone release. In addition, recent studies have revealed that CgA may function as a prohormone from which peptides are released that modulate the function of catecholaminergic cells. Trypsin digestion of CgA produces peptides that inhibit release of catecholamines from adrenal medullary cells in culture (9). Previous studies have demonstrated that CgA processing occurs in a specific tissue-dependent fashion with the extent and pattern of processing varying widely from one neuroendocrine site to the next (10). CgA processing may take place intracellularly as well as extracellularly, that is, after secretion into the extracellular space (9, 11, 12). Indeed, previous studies have shown that extracellular processing appears to be particularly important for generation of catecholaminerelease inhibitory activity from the parent CgA molecule in the vicinity of the chromaffin cell (9, 11). Although a number of studies have examined the role of prohormone convertases (13-15) that process prohormone precursors intracellularly to generate regulatory peptides, few studies have focused on the role of other proteases and mechanisms of extracellular processing in the generation of bioactive peptides. Recent studies have demonstrated that catecholamine release-inhibitory activity is generated when CgA is co-incubated with chromaffin cells, suggesting that at least some component of the cleavage occurs
Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a pr... more Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.
The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occur... more The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A 332-364 ; identity of the peptide was confirmed by diagnostic Met 346 oxidation. Further analysis revealed two additional forms: bovine chromogranin A 333-364 and A 343-362. Synthetic longer (chromogranin A 332-364) and shorter (chromogranin A 344-364) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC 50 ϳ2.01 versus ϳ0.35 M). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A 340-372 , bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.
A cDNA for a midgut chymotrypsin, induced by a blood meal, has been cloned and sequenced from the... more A cDNA for a midgut chymotrypsin, induced by a blood meal, has been cloned and sequenced from the mosquito Aedes aegypti. The 938 base sequence codes for a 268 amino acid protein, which contains an 18-residue signal peptide and a seven-residue activation peptide. The deduced amino acid sequence contains several features typical of chymotrypsin proteases, including the catalytic triad of serine proteases and the residues that determine the chymotrypsin substrate specificity pocket. The chymotrypsin mRNA, absent in larvae, pupae, males and newly emerged females, reaches detectable levels within 24 h post-emergence and attains a maximum level 3-7 days after emergence. Translation of the chymotrypsin mRNA is induced by feeding a protein meal, and there is a dramatic increase in midgut chymotrypsin enzymatic activity after feeding. Chymotrypsin activity remained high during protein digestion, but chymotrypsin protein levels and enzymatic activity were almost undetectable once digestion was completed, 48 h after feeding.
Recent studies suggest a crucial role for plasminogen activator inhibitor-1 (PAI-1) in mediating ... more Recent studies suggest a crucial role for plasminogen activator inhibitor-1 (PAI-1) in mediating stress-induced hypercoagulability and thrombosis. However, the mechanisms by which PAI-1 is released by stress are not well-delineated. Here, we examined catecholaminergic neurosecretory cells for expression, trafficking, and release of PAI-1. PAI-1 was prominently expressed in PC12 pheochromocytoma cells and bovine adrenomedullary chromaffin cells as detected by Northern blotting, Western blotting, and specific PAI-1 ELISA. Sucrose gradient fractionation studies and immunoelectron microscopy demonstrated localization of PAI-1 to catecholamine storage vesicles. Secretogogue stimulation resulted in corelease of PAI-1 with catecholamines. Parallel increases in plasma PAI-1 and catecholamines were observed in response to acute sympathoadrenal activation by restraint stress in mice in vivo. Reverse fibrin zymography demonstrated free PAI-1 in cellular releasates. Detection of high molecular ...
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