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J. Biol. Chem., Vol. 275, Issue 30, 22905-22915, July 28, 2000

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Formation of the Catecholamine Release-inhibitory Peptide Catestatin from Chromogranin A

DETERMINATION OF PROTEOLYTIC CLEAVAGE SITES IN HORMONE STORAGE GRANULES^*

Carolyn V. Taylor, Laurent Taupenot, Sushil K. Mahata, Manjula Mahata, Hongjiang Wu, Sukkid Yasothornsrikul, Thomas Toneff, Carlo Caporale, Qijiao Jiang, Robert J. Parmer, Vivian Y. H. Hook, and Daniel T. O'Connor

From the Department of Medicine and Center for Molecular Genetics, University of California, and San Diego Veterans Affairs Healthcare System, San Diego, California 92161

Received for publication, February 14, 2000, and in revised form, April 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 Met³⁴⁶ 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 (IC₅₀ ~2.01 versus ~0.35 µM). 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chromogranin A, the major soluble protein in catecholamine storage vesicles, not only stabilizes the core of such vesicles (1) but also serves as a prohormone cleaved into several biologically active peptides, including the insulin release-inhibitory fragments pancreastatin (2) and -granin (3-5), the vasodilator vasostatin (6, 7), and the parathyroid hormone release-inhibitory parastatin (8). Recently we described an additional chromogranin A fragment, catestatin (bovine chromogranin A_344-364), which acts specifically as a potent (IC₅₀ ~200-400 nM) nicotinic cholinergic antagonist to inhibit catecholamine release (9, 10), suggesting a novel autocrine feedback mechanism controlling sympathochromaffin exocytosis. Initial studies of catestatin relied on synthetic peptides (10); hence, the existence of the peptide in vivo remains unexplored, as do its precise cleavage sites from chromogranin A.

In this study, we explored processing of the catestatin region of chromogranin A in secretory granules of chromaffin cells and sympathetic nerves, as well as in human pheochromocytomas. We documented catestatin cleavage from chromogranin A and determined the precise endogenous cleavage sites bounding catestatin in bovine and human chromaffin granules by chromatographic separations coupled with amino-terminal amino acid sequencing, immunoprecipitation, and matrix-assisted laser desorption ionization (MALDI)¹ mass spectrometry. We confirmed catestatin's regulated secretion by chromaffin cells. A major form of catestatin was processed at dibasic sites (bovine chromogranin A_332-364 or human chromogranin A_340-372), while a smaller form was also identified (bovine chromogranin A_343-362). Both larger and smaller size forms were synthesized; each displayed specific antagonism of nicotinic cholinergic-stimulated catecholamine release, while the smaller form had greater potency of inhibition.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Preparation of Tissue Fractions-- All preparative steps on tissues or protein fractions were conducted at 0-4 °C. Bovine adrenal medullary chromaffin granules were prepared by centrifugation on 0.3 M/1.6 M sucrose density step gradients, as described previously (10). After granule hypotonic lysis and centrifugal removal of granule membranes, the soluble proteins and peptides in the supernatant were size-fractionated on a 2.6 × 80-cm Sephacryl S-300 column (Amersham Pharmacia Biotech), eluting with the volatile buffer 0.3 M ammonium acetate, pH 6.5, as described previously (11). The buffer was removed by lyophilization before further studies. In some experiments, bovine chromaffin granule proteins/peptides (200 µl containing 8 mg of protein) were size-fractionated on a Superdex 75 HR 10/30 FPLC gel filtration column (10 × 300 mm, 24-ml bed volume; Amersham Pharmacia Biotech), eluting at 1 ml/min with 0.3 M ammonium acetate, 1 mM EDTA, pH 7.0, collecting fractions every 0.5 ml (0.5 min). Eluted fractions were analyzed for protein by on-line absorbance at 280 nm (A₂₈₀) and then lyophilized (to remove the volatile buffer) and resuspended in the same volume of radioimmunoassay (RIA) buffer (50 mM Tris-HCl, pH 8.3, 0.3% bovine serum albumin, 0.1% Triton X-100; see below). Chromaffin granules from human pheochromocytomas were also prepared by centrifugation on 0.3 M/1.6 M sucrose density step gradients, followed by hypotonic lysis and membrane removal by centrifugation, as described previously (10). Postganglionic sympathetic nerves were obtained by dissection of splenic nerve in samples from the local slaughterhouse. Nerves were placed in ice-cold 0.3 M sucrose at an ~10:1 ratio of tissue to buffer and then minced, homogenized, gauze-filtered, and centrifuged at 1000 × g for 10 min (to remove nuclei and debris). Supernatants were then centrifuged at 10,000 × g for 10 h to pellet a crude fraction of neuronal large dense core vesicles (12), which were lysed by resuspension in 10 mM Hepes, pH 7, and freezing/thawing.

Chromogranin A, Synthetic Peptides, and Antibodies-- Bovine or human chromogranin A was isolated from chromaffin granule soluble core proteins by affinity chromatography (to remove dopamine -hydroxylase) followed by gel filtration, as described previously (13). Recombinant human chromogranin A was expressed in Escherichia coli by a modification of previously described methods (14), except that a His₆ tag was used for affinity purification on a Ni²⁺-nitrilotriacetic acid column (15). Synthetic peptides (20-100-µmol scale) were prepared by the solid phase method, using Fmoc (N-(9-fluorenyl)methoxycarbonyl) protection chemistry. Purification was by C-18 reverse-phase HPLC (RP-HPLC). Authenticity of the resulting peptides was confirmed by mass spectrometry, using either MALDI or electrospray ionization. Polyclonal rabbit antisera recognizing the catestatin region of chromogranin A (Fig. 1), either bovine chromogranin A_344-364 (RSMRLSFRARGYGFRGPGLQL) or human chromogranin A_352-372 (SSMKLSFRARAYGFRGPGPQL), were developed by a modification of protocols previously described for other chromogranin peptides (11, 16). The polyclonal antibody recognizing the catestatin region of chromogranin A was further purified on an Amersham Pharmacia Biotech Hi Trap protein A column in 0.02 M sodium phosphate (pH 7.0), eluted with 0.1 M sodium citrate (pH 3), and the pH was then adjusted back to 7.0 with Tris-HCl (pH 8.8). A polyclonal rabbit antiserum recognizing bovine chromogranin A_316-329 was obtained from Dr. Marie-France Bader (INSERM U-338, Strasbourg, France), and a polyclonal rabbit antiserum recognizing human chromogranin A_367-391 was obtained from Dr. Reiner Fischer-Colbrie (University of Innsbruck, Austria).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Amino acid sequences in the catestatin (bovine chromogranin A344-364) region of the primary structures of bovine and human chromogranin A. The regions between dibasic cleavage sites ([KR] and [RR]) are shown. Previous studies (10) have established the catecholamine release-inhibitory activity of synthetic bovine chromogranin A344-364.

Immunoprecipitations-- Tissue homogenates, granule soluble core lysates, or gel filtration size-separated fractions were applied to Sep-Pak C-18 cartridges (Waters/Millipore), eluted with 30-40% acetonitrile, lyophilized, resuspended in 500 µl of immunoprecipitation buffer containing protease inhibitors (0.1% Triton X-100, 140 mM NaCl, 0.025% sodium azide, 10 mM Tris-HCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 1 mM EDTA, 1 mM N-ethylmaleimide), and then immunoprecipitated by a modification of the protocol of Wang et al. (17). To minimize nonspecific binding results, samples were first incubated with 25 µl of normal (preimmune) rabbit serum with constant rotator mixing at 4 °C for 12-18 h. 60 µl of protein G Plus/protein A-agarose beads (33% slurry; Calbiochem) were added, and rotational incubation was continued for 3 h, followed by centrifugation for 2 min at ~13,000 × g in an Eppendorf 5417 microcentrifuge, after which the pellet was discarded. To the supernatant, 20 µl of rabbit anti-bovine catestatin (10) were added, and rotational incubation was continued for another 12-18 h. 60 µl of fresh protein G Plus/protein A-agarose beads were added, and rotational incubation continued for another 3 h, after which the beads were collected by centrifugation, washed three times with immunoprecipitation buffer, and then washed twice with 50 mM Tris-HCl, pH 8 (to remove Na⁺ and detergent).

Mass Spectrometric Analyses of Immunoprecipitated Catestatin-- Immunoprecipitated catestatin was eluted from the immune complexes with 20 µl of trifluoroacetic acid/water/acetonitrile, 1:20:20 (v/v/v) (17). To identify methionine-containing peptides (by oxidation of methionine to methionine sulfoxide, thereby adding the mass of a single oxygen at 16 daltons), 10 µl were oxidized by adding sufficient 3% H₂O₂ to achieve 10 µM final H₂O₂ concentration. 1-2 µl were characterized by MALDI mass spectrometry on a Voyager-Elite mass spectrometer with delayed extraction (PerSeptive Biosystems, Framingham, MA). Samples were embedded in an -cyano-4-hydroxycinnamic acid matrix (18) and then irradiated with a nitrogen laser at 337 nm, and the ions produced were accelerated with a deflection potential of 30,000 V. Ions were then differentiated according to their mass/charge ratio (m/z) using a time-of-flight mass analyzer. The mass error of this method is characteristically 0.1% (i.e. 1000 ppm; Ref. 18).

Immunoblots-- After suspension of bovine chromaffin granule protein samples in loading buffer (10 mM Tris-HCl, 1 mM EDTA, 3% SDS, 20 mM dithiothreitol, 10% glycerol, 0.1% bromphenol blue), 0.8-50 µg of protein were electrophoresed through SDS-PAGE 10-20% acrylamide gradient gels, in the presence of Tris-Tricine buffer (Novex, San Diego, CA). This gradient gel system resolves peptides as small as 2 kilodaltons (19). Human pheochromocytoma chromaffin granule samples were electrophoresed on 10% nongradient acrylamide gels. After SDS-PAGE, gels were stained for total protein with 0.1% Coomassie Brilliant Blue R250 in water/methanol/acetic acid (50:40:10%) (20), followed by destaining in water/methanol/acetic acid (82.5:10:7.5%). Parallel gels were electroeluted onto nitrocellulose paper (BA85; Schleicher and Schuell, Keene, NH), followed by immunoreactive catestatin staining by immunoblotting (21). The primary immunoblotting antibodies were rabbit anti-bovine catestatin (chromogranin A_344-364; RSMRLSFRARGYGFRGPGLQL; titer 1:1000 (v/v)), rabbit anti-bovine chromogranin A_316-329 (titer 1:500), rabbit anti-bovine chromogranin A_367-391 (titer 1:500 (v/v)), or rabbit anti-human catestatin (chromogranin A_352-372; SSMKLSFRARAYGFRGPGPQL; titer 1:2000 (v/v)) (10). Second antibody staining was visualized by either enhanced chemiluminescence (horseradish peroxidase-labeled goat anti-rabbit IgG, titer 1:4000 (v/v); Amersham Pharmacia Biotech ECL kit) or color development (goat anti-rabbit IgG-alkaline phosphatase conjugate, titer 1:2000 (v/v); Bio-Rad).

After RP-HPLC of bovine chromaffin granule peptides (see below), 50 µl of each 500-µl HPLC fraction were vacuum-dried, resuspended in 100 µl of water, adsorbed by vacuum filtration onto a nitrocellulose membrane using a slot-blotting device (Minifold II; Schleicher and Schuell), and immunoblotted using the anti-bovine catestatin primary antibody (at 1:500 (v/v)) and peroxidase-conjugated anti-rabbit secondary antibody (at 1:7000 (v/v)) with an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech).

Densitometry analysis was performed on a Macintosh computer using the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet.

RP-HPLC-- Gel filtration size-fractionated bovine chromaffin granule peptides were further separated by RP-HPLC using a 25 × 0.5-cm C-18 column, equilibrated in 0.1% trifluoroacetic acid, and eluted with a linear 0-60% gradient of acetonitrile in 0.1% trifluoroacetic acid, over 60 min, at 1 ml/min. The elution was monitored by A₂₁₄ (peptide bond absorbance), and fractions were collected at 0.5-min (0.5-ml) intervals. 50-µl aliquots from each fraction were vacuum-dried and subjected to anti-catestatin slot immunoblotting (see above), and slot-blot positive fractions were further subjected to amino-terminal microsequencing (100 µl; see below) and MALDI mass spectrometry (1-2 µl; see above).

Amino-terminal Amino Acid Microsequencing-- Eluted HPLC fractions (100-µl aliquots from 500-µl fractions) in 0.1% trifluoroacetic acid/acetonitrile/H₂O column buffer were analyzed for amino-terminal sequence by automated Edman microsequencing (10-100 pmol; ABI 494 Procise^® sequencing system with ABI 610 data analysis system; Applied Biosystems/Perkin Elmer).

Mass Spectrometric and Sequencing Analyses-- Molecular weights from MALDI mass spectra were interpreted, and peptide fragments within the chromogranin A primary structure were assigned by the program PAWS (Protein Analysis WorkSheet, version 8.1.1, for Macintosh; ProteoMetrics; freeware available on the Internet), assigning average isotopic MH⁺ values for chromogranin A peptides (18). Sequencing results were analyzed at each Edman cycle by the algorithm "Hydrosites" (for Macintosh) to deconvolute multiple amino-terminal sequences derived from the same parent molecule at a given cycle (22).

Activity of Synthetic Catestatins-- Peptides were subjected to a test of activity by inhibition of secretagogue-stimulated norepinephrine release from [³H]norepinephrine-prelabeled PC12 pheochromocytoma cells, over a 30-min secretion period, as described previously (10, 23). The stimuli to catecholamine release were either nicotinic cholinergic (60 µM nicotine) or membrane depolarization (by 55 mM KCl). In some control experiments, the peptide was immunoadsorbed overnight (4 °C, 1:100 antibody titer in secretion buffer) prior to test of secretion.

Bovine Chromaffin Cell Isolation, Culture, and Stimulation of Secretion-- Primary cultures of bovine chromaffin cells were prepared as described previously (24). Secretion was stimulated over a 15-min period. The stimuli were either nicotinic cholinergic (100 µM nicotine), membrane depolarization (by 55 mM KCl, in the presence or absence of 1 mM Ca²⁺), or vehicle (mock). Secretion media were collected, concentrated on a Millipore Ultrafree 10K filter, and analyzed by radioimmunoassay.

Catestatin RIA-- Synthetic catestatin (bovine chromogranin A_344-358; RSMRLSFRARGYGFR) was radioiodinated with ¹²⁵I by the IODO-GEN method (Pierce) on its endogenous tyrosine (Tyr³⁵⁵) to a specific activity of 3457 Ci/mmol, by Phoenix Pharmaceuticals (Mountain View, CA). The RIA incubation mixture contained 100 µl of rabbit anti-bovine chromogranin A_344-364 in RIA buffer (50 mM Tris-HCl, pH 8.3, 0.3% bovine serum albumin, 0.1% Triton X-100) at a titer of 1:1000 (v/v), and 100 µl of unlabeled catestatin (bovine chromogranin A_344-358; RSMRLSFRARGYGFR) calibration standards (2.5-5000 pg) or sample (unknown). After 24 h at 4 °C, 10,000 cpm of ¹²⁵I-labeled chromogranin A_344-358 in 100 µl of RIA buffer was added. After a second 24-h 4 °C incubation, the second antibody (goat anti-rabbit IgG (Calbiochem), titer 1:20 (v/v)) and carrier antiserum (2% bovine serum albumin) were added. After 2 h at ambient temperature, 500 µl of RIA buffer was added, samples were centrifuged at 3000 rpm for 25 min at 4 °C, the supernatant was discarded, and the samples were counted for 1 min in the -counter (ICN Biomedicals Inc.). Fetal bovine serum and adult equine serum were obtained from Life Technologies, Inc.

Statistics-- Results are expressed as the mean value ± one S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Catecholamine Release-inhibitory Activity of Catestatin Is Found in Bovine Adrenal Chromaffin Granule Low Molecular Weight Size Fractions-- Chromaffin granule soluble core proteins (chromogranins) were size-fractionated by gel filtration (Fig. 2); fractions were analyzed by SDS-PAGE (10), and low molecular weight peptide-containing fractions 42-52 were pooled for further study. After peptide adsorption and batch elution from a C-18 hydrophobic affinity matrix (SepPak), this fraction inhibited nicotinic cholinergic-stimulated catecholamine release from [³H]norepinephrine-prelabeled PC12 pheochromocytoma cells; 60 µM nicotine stimulated 19.1 ± 0.39% release of cellular norepinephrine, but inclusion of as little as 1.25 µg/ml of the peptide fraction diminished nicotinic-stimulated release to 1.7 ± 0.19% (10).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Size separation of bovine adrenal medullary chromaffin granule soluble core proteins and peptides. After chromaffin granule isolation and lysis, soluble proteins and peptides were fractionated by gel filtration on a 2.6 × 80-cm column of Sephacryl S-300 (Amersham Pharmacia Biotech), equilibrated and eluted with the volatile buffer 0.3 M ammonium acetate, pH 6.5. Low molecular weight (LMW) peptide fractions 42-52, devoid of chromogranin A by SDS-PAGE analysis, were used for further studies. By Coomassie Blue stain of SDS-PAGE, the low molecular weight fraction molecular mass values ranged from <55 kDa to the dye front.

Determination of Catestatin Cleavage Sites in Bovine Chromaffin Granules by Mass Spectrometry-- To determine whether the catestatin region of chromogranin A is discretely excised at particular amino acid residues, we turned to mass spectrometry on chromogranin A fragments formed endogenously in chromaffin granules. The low molecular weight peptide fraction (Fig. 2, fractions 42-52) was immunoprecipitated by a bovine catestatin (chromogranin A_344-364) antibody and subjected to MALDI mass spectrometry (Fig. 3). The initial spectrum revealed a major peak at m/z = 3829 (Fig. 3B), which, within the 431-amino acid bovine chromogranin A primary structure (25, 26), corresponds uniquely to chromogranin A_332-364 (LEGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL; calculated m/z = 3827.2); the 0.047% difference between experimental and calculated m/z values is within the range of acceptable error of the MALDI method (0.1% m/z; Ref. 18). Preimmune serum did not recognize a peak of this mass (Fig. 3A). Since mass spectra of peptides are susceptible to shifting by formation of peptide-cation (such as Na⁺) adducts, we repeated the spectrum on granule peptides that had been desalted by adsorption to a C-18 matrix (Fig. 3C); an m/z = 3827 peak remained the principal component, with some diminution in surrounding minor peaks. To confirm that this peak represents catestatin, the granule peptide sample was gently oxidized with H₂O₂ to convert any methionine residues into methionine sulfoxide, as described by Wang et al. in studies of -amyloid processing by mass spectrometry (17); since bovine catestatin contains a single methionine residue (bovine chromogranin A Met³⁴⁶; Fig. 1), this procedure provides a further specific diagnostic for the identity of the catestatin region. After oxidation, the major peak was now found at m/z = 3844 (Fig. 3D), a mass shift corresponding to the added 16-dalton oxygen atom in Met³⁴⁶-sulfoxide.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   MALDI mass spectrometry identification of catestatin in immunoprecipitated bovine adrenal medullary chromaffin granules. Low molecular weight chromaffin granule peptides, devoid of chromogranin A (fractions 42-52 from gel filtration; Fig. 2), were examined. Aliquots (200 µl) of the size fraction were immunoprecipitated (20 µl of antiserum) and then subjected to MALDI mass spectrometry (1-2 µl). A, immunoprecipitation by preimmune serum. B, immunoprecipitation by rabbit anti-bovine chromogranin A344-364 (RSMRLSFRARGYGFRGPGLQL). C, immunoprecipitation by rabbit anti-bovine chromogranin A344-364, followed by adsorption and elution from a C-18 (Sep-Pak) cartridge. D, immunoprecipitation by rabbit anti-bovine chromogranin A344-364 after Met346 oxidation by 10 µM H2O2.

Isolation and Characterization of Catestatin Fragments: RP-HPLC, Mass Spectrometry, and Amino-terminal Amino Acid Sequencing-- In a second approach to determine the boundaries of endogenous catestatin cleavage, the chromaffin granule low molecular weight peptide fraction (gel filtration fractions 42-52; Fig. 2) was first separated by RP-HPLC (Fig. 4, bottom), and fractions were immunoblotted with anti-bovine catestatin. HPLC fraction 27, containing intense catestatin immunoreactivity (Fig. 4, middle), was then analyzed (Fig. 4, top) by both MALDI mass spectrometry and amino-terminal amino acid sequencing. MALDI revealed two peaks, at m/z = 3718 and 2300 (Fig. 4, top). In the catestatin region of bovine chromogranin A (Fig. 1), m/z = 3718 is compatible with chromogranin A_333-364 (EGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL), while m/z = 2300 is compatible with chromogranin A_343-362 (DRSMRLSFRARGYGFRGPGL). Fraction 27 was amino-terminally sequenced over 10 residue cycles, with the result suggesting two peptides (22): (a) EGEEEEEEDP ... , corresponding to bovine chromogranin A_333-342, the first 10 amino acids of the 3718 m/z peptide EGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL (chromogranin A_333-364), and (b) DRS ... , corresponding to bovine chromogranin A_343-345, the first three amino acids of the 2300 m/z peptide DRSMRLSFRARGYGFRGPGL (chromogranin A_343-362).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Isolation and further characterization of catestatin size forms in bovine chromaffin granules by RP-HPLC, microsequencing, and mass spectrometry. Low molecular weight bovine chromaffin granule peptides, devoid of chromogranin A (fractions 42-52 from gel filtration; Fig. 2), were separated on a 0.5 × 25-cm C18 RP-HPLC column (bottom panel), and eluted fractions were tested for catestatin immunoreactivity by slot-blotting (middle panel) with rabbit anti-bovine chromogranin A344-364 (titer 1:2000). The fraction with catestatin immunoreactivity (fraction 27; 1-2 µl) was then subjected two analyses (top panel): amino-terminal amino acid sequencing and MALDI mass spectrometry.

Cleavage of the Catestatin Region of Chromogranin A in Neurons: Mass Spectrometry-- Bovine splenic nerve was homogenized, desalted by adsorption to/elution from a C-18 matrix (SepPak), immunoprecipitated by an anti-catestatin (bovine chromogranin A_344-364) antibody, and subjected to MALDI mass spectrometry. MALDI revealed a peak at m/z = 3832 (Fig. 5, top), which, in the catestatin region of bovine chromogranin A (Fig. 1), is within 0.125% of 3827.2, the calculated MH⁺ of chromogranin A_332-364 (LEGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL). After oxidation by H₂O₂, the m/z = 3832 peak diminished, while an m/z = 3843 peak became more prominent (Fig. 5, bottom), a mass shift corresponding to the added 16-dalton oxygen atom in Met³⁴⁶-sulfoxide in chromogranin A_332-364 (predicted MH⁺ = 3843.2).

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Cleavage of the catestatin region from chromogranin in bovine sympathetic neurons: splenic sympathetic nerve. Bovine splenic nerve large dense core granule fractions were desalted by adsorption to/elution from a C-18 matrix (SepPak), immunoprecipitated by an anti-catestatin (bovine chromogranin A344-364) antibody, and subjected to MALDI mass spectrometry. MALDI revealed a peak at m/z = 3832 (top), which, in the catestatin region of bovine chromogranin A (Fig. 1), is within 0.125% of 3827.2, the calculated MH+ of chromogranin A332-364 (LEGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL). After oxidation by H2O2, the m/z = 3832 peak diminished, while an m/z = 3843 peak became more prominent (bottom), a mass shift corresponding to the added 16-dalton oxygen atom in Met346-sulfoxide in chromogranin A332-364 (predicted MH+ = 3843.2).

Determination of Catestatin Cleavage Sites in Human Chromogranin A: Mass Spectrometry-- After anti-catestatin immunoprecipitation of human pheochromocytoma chromaffin granules, m/z values of 3770-3771 were noted (Fig. 6), corresponding uniquely within the chromogranin A primary structure to chromogranin A_340-372 (KRLEGQEEEEDNRDSSMKLSFRARAYGFRGPGPQLRR; calculated m/z = 3771.1), which is bounded on either side by dibasic recognition sites for prohormone cleavage (underlined) (Fig. 1). Upon H₂O₂ oxidation, in each case these m/z 3770-3771 peaks shifted to m/z = 3787, consistent with the addition of an oxygen (16 daltons) to form Met³⁵⁴-sulfoxide. Other peaks in this region did not shift upon oxidation. Experimental and calculated m/z values are well within the 0.1% expected experimental error of MALDI mass determination (18).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   MALDI mass spectrometry identification of catestatin in immunoprecipitated human pheochromocytoma chromaffin granules. Aliquots (200 µl) of pheochromocytoma chromaffin granule soluble core proteins and peptides were immunoprecipitated (20 µl) and then subjected to MALDI mass spectrometry (1-2 µl). Top panels, immunoprecipitation by rabbit anti-human chromogranin A352-372 (SSMKLSFRARAYGFRGPGPQL). Bottom panels, immunoprecipitation after oxidation of Met354 with 10 µM H2O2. A, pheochromocytoma 272-17; B, pheochromocytoma 1062-8.

Reproduction of Catestatin Activity in Synthetic Peptides Corresponding to Cleavage Sites: Potency and Specificity of Nicotinic Cholinergic Antagonism-- To test the activity and specificity of the bovine catestatin peptides predicted by MALDI mass spectrometry and amino-terminal sequencing (Figs. 3 and 4), we synthesized both longer (chromogranin A_332-364; LEGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL) and shorter (chromogranin A_344-364; RSMRLSFRARGYGFRGPGLQL) versions of catestatin as well as whole chromogranin A and evaluated their effects on catecholamine release from PC12 pheochromocytoma cells (Fig. 7).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Catecholamine release-inhibitory activity of synthetic catestatins: specificity of mechanism and potency of effect. Longer (bovine chromogranin A332-364; LEGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL) versus shorter (bovine chromogranin A344-364; RSMRLSFRARGYGFRGPGLQL) regions of bovine chromogranin A were synthesized, representing longer and shorter cleavage site versions of bovine catestatin identified in these studies (Figs. 3 and 4). Secretion was tested in PC12 pheochromocytoma cells prelabeled with L-[3H]norepinephrine. bCgA, bovine chromogranin A; hCgA, human chromogranin A. A, specificity of mechanism. Inhibition of nicotinic cholinergic stimulated (60 µM nicotine) versus membrane depolarization-stimulated (55 mM KCl) secretion by a 10 µM concentration of a longer version of bovine catestatin (bovine chromogranin A332-364; LEGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL) is shown. B, potency. Dose-response (0.1-10 µM) curves are shown for the effect of different size forms of bovine catestatin (longer bovine chromogranin A332-364 versus shorter bovine chromogranin A344-364) or full-length chromogranin A (recombinant human chromogranin A1-439) on secretion stimulated by 60 µM nicotine. C, specificity of effect. For synthetic human catestatin (human chromogranin A352-372; SSMKLSFRARAYGFRGPGPQL), the specificity of its action to block norepinephrine secretion is shown. Secretion was triggered by 60 µM nicotine from L-[3H]norepinephrine-prelabeled PC12 pheochromocytoma cells. The peptide was used either alone or after preincubation (1 h) with either rabbit anti-human catestatin (titer 1:100 (v/v)) or preimmune serum.

As previously reported for the shorter version of catestatin (10), the longer version also selectively inhibited catecholamine release evoked by nicotinic cholinergic stimulation but not that evoked by membrane depolarization (Fig. 7A). Both peptides and chromogranin A showed concentration-dependent inhibition of nicotine-induced secretion (Fig. 7B): intact chromogranin A had an IC₅₀ of ~4.2 µM, chromogranin A_344-364 had an IC₅₀ of ~0.35 µM, and the chromogranin A_332-364 IC₅₀ was ~2.01 µM. At high dose (10 µM) each peptide completely blocked nicotinic-stimulated secretion, while whole chromogranin A only partially blocked secretion.

A peptide corresponding to the catestatin region (chromogranin A_352-372; SSMKLSFRARAYGFRGPGPQL) of human chromogranin A (see Fig. 1) also inhibited nicotinic cholinergic-stimulated catecholamine secretion (by 74% at 10 µM peptide), and the inhibition was specifically reversed by a rabbit antiserum directed toward the catestatin region of human chromogranin A (chromogranin A_352-372; SSMKLSFRARAYGFRGPGPQL), although not by preimmune serum (Fig. 7C).

Catestatin Immunoreactivity Has a Variety of Size Forms in Bovine Chromaffin Granule Radioimmunoassay-- Chromaffin granule soluble core proteins were size-fractionated on a standard calibrated gel filtration column (Fig. 8), and fractions were analyzed by radioimmunoassay. Four major peaks of catestatin immunoreactivity occurred in fractions 16-22, 25-27, 28-34, and 35-40 (Fig. 8). Peak 1 contained 71.7 nmol of catestatin immunoreactivity and 54% of the total catestatin immunoreactivity, while peak 2 contained 19.1 nmol and 13.2% of total, peak 3 contained 28.2 nmol and 21.4% of total, and peak 4 contained 7.5 nmol of catestatin immunoreactivity and 8% of total catestatin immunoreactivity. Of note, peak 4 elutes in the size position (fractions 37 and 38) of chromogranin A_344-364, the small, potent form of catestatin (Fig. 7B). Synthetic peptides corresponding to chromogranin A_332-364 and chromogranin A_344-364 were size-fractionated on the same column, eluting in fractions 35 and 36 and fractions 37 and 38, respectively. A standard curve of the gel filtration elution was created using bovine serum albumin (67 kDa), ovalbumin (43 kDa), ribonuclease (13.7 kDa), a larger form of catestatin (chromogranin A_332-364; 3827 kDa), and a smaller form of catestatin (chromogranin A_344-364; 2541.3 kDa).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Determination of size distribution of catestatin immunoreactivity in bovine chromaffin granules: gel filtration and radioimmunoassay. Bovine chromaffin granule soluble core proteins (200 µl containing 8 mg of protein) were size-fractionated on a Superdex 75 HR 10/30 FPLC gel filtration column (10 × 300 mm, 24-ml bed volume; Amersham Pharmacia Biotech), eluting at 1 ml/min with 0.3 M ammonium acetate, 1 mM EDTA, pH 7.0, collecting fractions every 0.5 ml (0.5 min). Eluted fractions were analyzed for protein by on-line absorbance at 280 nm (A280) and then lyophilized (to remove the volatile buffer) and resuspended in the same volume of RIA buffer (50 mM Tris-HCl, pH 8.3, 0.3% bovine serum albumin, 0.1% Triton X-100). 100 µl of each fraction were then analyzed by the bovine catestatin RIA, using 125I-labeled chromogranin A344-358 and rabbit anti-bovine chromogranin A344-364 (see "Materials and Methods"). RIA results are expressed as nmol/ml. A (top), RIA values. Column size standards are shown at the top. V0, column void volume (determined by elution of blue dextran 2000). Vt, column total internal volume. Peaks 1-4 are catestatin immunoreactivity forms of descending size. B (bottom), A280 values.

Cleavage within the Catestatin Region of Bovine Chromogranin A: Immunoblots of Chromaffin Granules with Antisera Directed to Flanking Peptides-- Chromaffin granule soluble proteins were separated by SDS-PAGE and then immunoblotted with not only an antibody directed against the catestatin region (bovine chromogranin A_344-364) but also antibodies directed against peptide regions that are bounded by dibasic cleavage sites and lie either directly amino-terminal (bovine chromogranin A_316-329) or directly carboxyl-terminal (bovine chromogranin A_367-391) to catestatin (Fig. 9). Each antibody recognized not only intact chromogranin A (at a molecular mass of ~60-70 kDa) but also several lower molecular mass chromogranin A fragments, ranging from ~10 to 50 kDa. All of the lower molecular mass chromogranin A fragments are prominently recognized by all three antisera, with the exception of a ~19-kDa fragment (Fig. 9, arrow), which is visualized by anti-chromogranin A_316-329 and anti-chromogranin A_344-364, although not by anti-chromogranin A_367-391. Thus, the ~19-kDa fragment probably represents the peptide just amino-terminal to a dibasic cleavage at Arg³⁶⁵-Arg³⁶⁶ in bovine chromogranin A. 

View larger version (36K): [in this window] [in a new window]   Fig. 9.   Determination of size distribution of catestatin immunoreactivity in bovine chromaffin granules: SDS-PAGE with chromogranin A region-specific immunoblots. Bovine chromaffin granule soluble core proteins were subjected to SDS-PAGE (10-20% Tris-Tricine gradient gels; Novex) and then transferred electrophoretically to nitrocellulose (Hybond; Amersham Pharmacia Biotech), blocked with 10% fetal calf serum in TBST (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.5% Tween 20), and stained with 1:500 to 1:1000 (v/v) primary antibodies in TBST containing 1% (w/v) powdered nonfat milk (Carnation). The color was developed by a secondary antibody (1:4000 (v/v) horseradish peroxidase-labeled goat anti-rabbit IgG (Amersham Pharmacia Biotech) in TBST, followed by enhanced chemiluminescence detection with Kodak X-Omat AR5 film. Arrow, ~19-kDa chromogranin A immunoreactive fragment bearing epitopes for chromogranin A316-329 (WE14) and chromogranin A344-364 (catestatin), although not chromogranin A367-392 (GE25).

When the low molecular weight chromaffin granule peptides (Fig. 2, fractions 42-52) were subjected to catestatin immunoblotting, followed by densitometry of the immunoreactive bands, more than half of the catestatin immunoreactivity was found in fractions of lower molecular mass than chromogranin A: 23.7% in a ~34-kDa band and 27.1% in a ~15 kDa band.

Chromogranin A Processing to Catestatin in Human Pheochromocytoma Chromaffin Granules: Immunoblot-- The anti-human catestatin antibody (rabbit anti-human chromogranin A_352-372) recognized intact human chromogranin A, at M_r ~70 kDa (Fig. 10, lane 1) in pheochromocytoma chromaffin granule immunoblots. All pheochromocytomas studied exhibited at least some processing of the catestatin region. Low molecular weight peptides, migrating near the tracking dye and bearing the catestatin epitope, are readily apparent in two pheochromocytomas (Fig. 10, lanes 4 and 5), and the overall pattern of catestatin processing from human chromogranin A was strikingly similar in two of five pheochromocytomas (Fig. 10, lanes 4 and 5). In three of the five tumors, more than 50% of the catestatin immunoreactivity still resided in the intact chromogranin A parent molecule (Fig. 10, lanes 4-6). Similar processing was noted on catestatin immunoblots of chromaffin granules from eight additional pheochromocytomas (data not shown).

View larger version (63K): [in this window] [in a new window]   Fig. 10.   Catestatin immunoblots of human pheochromocytoma chromaffin granule soluble proteins and peptides. Chromaffin granules were prepared from fresh human pheochromocytomas, and the soluble proteins and peptides were separated from granule membranes after granule lysis and centrifugation. SDS-PAGE was conducted on 10% acrylamide (nongradient) gels (50 µg of protein/lane). Left, Coomassie Brilliant Blue stain of the gel for total protein. Right, catestatin immunoblot with rabbit anti-human chromogranin A352-372 (titer 1:2000). Second antibody color development was by the alkaline phosphatase method. Lane 1, purified human chromogranin A; lanes 2-6, chromaffin granule soluble lysates from five separate human pheochromocytomas.

Catestatin Is Released by Chromaffin Cells, in Secretagogue-regulated Fashion-- Bovine chromaffin cells in primary culture were stimulated by either the nicotinic cholinergic agonist nicotine (100 µM) or membrane depolarization (by 55 mM KCl), each in the presence of extracellular calcium (1 mM) (Fig. 11A). Under basal (unstimulated) circumstances, cells released 90.7 ± 9.7 nmol/ml of catestatin, rising to 20,700 ± 7850 nmol/ml after nicotine (~228-fold stimulation) or 4570 ± 1130 nmol/ml after membrane depolarization (~50-fold stimulation).

View larger version (27K): [in this window] [in a new window]   Fig. 11.   Catestatin is released in secretagogue-regulated fashion from chromaffin cells. Bovine chromaffin cells were isolated and maintained in primary culture (2,000,000 cells/well, six-well plates) prior to stimulation of secretion. Secretion of catestatin was monitored over 15 min by bovine catestatin radioimmunoassay using 125I-labeled chromogranin A344-358 and rabbit anti-bovine chromogranin A344-364 (see "Materials and Methods"). Results are expressed as nmol/ml. A, secretion by nicotinic cholinergic stimulation or by membrane depolarization. Secretion was stimulated by either the nicotinic cholinergic agonist nicotine (100 µM) or by membrane depolarization (55 mM KCl). In each case, calcium (CaCl2, 1 mM) was present in the extracellular medium. B, secretion dependence on extracellular calcium. Membrane depolarization (with 55 mM KCl) was triggered in either the absence or the presence of extracellular calcium (CaCl2, 1 mM). In this experiment, secretion of methionine-enkephalin was also monitored by RIA (24).

We also tested the calcium-dependence of catestatin secretion (Fig. 11B); in the presence of extracellular calcium (1 mM), membrane depolarization (by 100 mM KCl) stimulated catestatin release by ~40-fold (from 31.5 ± 5.6 to 1260 ± 19.4 nmol/ml), while in the absence of extracellular calcium this stimulation was abolished (back to 18.7 ± 1.0 nmol/ml). Qualitatively similar results were observed for enkephalin secretion (Fig. 11B); in the presence of extracellular calcium, membrane depolarization (by 100 mM KCl) stimulated enkephalin release by ~27-fold (from 546 ± 104 to 15,000 ± 3710 nmol/ml), while in the absence of extracellular calcium this stimulation was virtually abolished (back to 626 ± 430 nmol/ml).

Using this radioimmunoassay, we detected catestatin immunoreactivity in fetal bovine serum (at 2.71 nM) and adult equine serum (at 22.2 nM).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The catestatin region of chromogranin A (bovine chromogranin A_344-364) is a potent and specific inhibitor of chromaffin cell catecholamine release when triggered by nicotinic cholinergic stimulation, the physiologic pathway for such cells (10); catestatin also antagonizes nicotinic desensitization of the process (27). In these studies, we documented cleavage of the catestatin region in normal chromaffin granules (Figs. 3, 4, 8, and 9) and sympathetic nerve catecholamine storage vesicles (Fig. 5) as well as chromaffin granules from human pheochromocytoma (Figs. 6 and 10). Indeed, cleavage of the catestatin region occurs at high frequency in chromogranin A: ~46% of catestatin immunoreactivity in chromaffin granules was of a lower molecular size form than intact chromogranin A (Fig. 8A, peaks 2-4), and ~8% of catestatin immunoreactivity co-eluted with the small peptide bovine chromogranin A_344-364 (Fig. 8A, peak 4). Furthermore, catestatin is subject to calcium-dependent, secretagogue-regulated release by chromaffin cells (Fig. 11).

Size separation of chromaffin granule peptides (Fig. 2), followed by immunoprecipitation and MALDI mass spectrometry (Fig. 3), revealed a major catestatin form (bovine chromogranin A_332-364) cleaved, as expected, at dibasic sites: KRLEGEEEEEEDPDRSMRLSFRARGYGFRGPGLQLRR. Recognition of dibasic recognition sites by prohormone convertases is well described in chromogranin A; we (28) and others (29) have shown that chromogranin A is a substrate in vivo for prohormone convertases 1 and 2 as well as furin. Chromogranin B and secretogranin II are also cleaved by prohormone convertases (30). Other proteases recognizing dibasic sites may also be active in chromaffin granules (31). Since proteases recognizing dibasic sites cleave to the carboxyl-terminal side of such sites (32), the lack of the carboxyl-terminal residues Arg³⁶⁵-Arg³⁶⁶ suggests, in addition, carboxypeptidase B (33, 34) removal of the remaining Arg³⁶⁵-Arg³⁶⁶ residues after initial cleavage by the prohormone convertase(s). Further resolution of chromaffin granule peptides on reverse-phase chromatography (Fig. 4), followed by MALDI mass spectrometry and amino-terminal sequencing, revealed two further forms: a long form (EGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL; chromogranin A_333-364) lacking the amino-terminal Leu³³² of the form previously identified (Fig. 3) and a shorter form (DRSMRLSFRARGYGFRGPGL; chromogranin A_343-362). Radioimmunoassay confirmed the secretion and physiological relevance of catestatin.

Both longer (chromogranin A_332-364; LEGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL) and shorter (chromogranin A_344-364; RSMRLSFRARGYGFRGPGLQL) synthetic forms of bovine catestatin showed specific antagonism of nicotinic cholinergic-stimulated catecholamine release (Fig. 7A), although the shorter form had superior potency (IC₅₀ ~0.35 versus ~2.01 µM; Fig. 7B), suggesting that cleavage of the longer to the shorter version may remove an inhibitory domain (LEGEEEEEEDP; bovine chromogranin A_332-342) from catestatin, although the enzymology of such further internal cleavage is uncertain. Whatever the cleavage mechanism, removal of the acidic amino terminus (LEGEEEEEEDP) seems to delete an inhibitory domain, thus potentiating the biological activity (IC₅₀) of the peptide. Finally, catestatin cleavage from intact chromogranin A magnifies its potency by ~12-fold (IC₅₀ ratio, 4.2/0.35 µM; Fig. 7B).

Pheochromocytoma provides an accessible source of human chromaffin granules (35, 36). Synthetic human catestatin (chromogranin A_352-372) specifically inhibited chromaffin cell catecholamine release (Fig. 7C), and liberation of catestatin from human chromogranin A between dibasic sites at residues of chromogranin A_340-372 (KRLEGQEEEEDNRDSSMKLSFRARAYGFRGPGPQLRR) was demonstrated by mass spectrometry (Fig. 6).

Previous studies established several cleavage sites in the catestatin region of bovine chromogranin A. Within bovine chromaffin granules, Metz-Boutigue et al. (37) found peptides with amino termini at chromogranin A₃₃₂ (Leu³³²; i.e. after dibasic site Lys³³⁰-Arg³³¹), chromogranin A₃₅₁ (Arg³⁵¹), and chromogranin A₃₅₄ (Gly³⁵⁴); upon secretion into the extracellular space, cleavage was also detected before Gly³⁵⁹. Sigafoos et al. (38) also detected a fragment with the amino terminus chromogranin A₃₄₂ (Pro³⁴²). Evidence for cleavage at the dibasic site Arg³⁶⁵-Arg³⁶⁶ in chromogranin A has not heretofore been obtained. Such previous studies used amino-terminal amino acid sequencing but occurred before the widespread availability of protein and peptide mass spectrometry; thus, carboxyl-terminal boundaries of the detected peptides could only be deduced imprecisely.

Studies on the formation of peptides flanking catestatin, i.e. bovine chromogranin A_316-329 (referred to as WE14 (39)) and bovine chromogranin A_367-392 (referred to as GE25 (40)), also provide evidence of cleavage in the catestatin (bovine chromogranin A_344-364) region. Our region-specific immunoblots of the catestatin region (Fig. 9) indicate that chromogranin A fragments bearing the catestatin epitope are at least as prominent as fragments bearing the WE14 or GE25 epitopes and document dibasic cleavage at Arg³⁶⁵-Arg³⁶⁶ in bovine chromogranin A.

What prompted cleavage of amino-terminal Leu³³² from bovine chromogranin A_332-364 (Fig. 3), to yield chromogranin A_333-364 (EGEEEEEEDPDRSMRLSFRARGYGFRGPGLQL; Fig. 4, top)? Chromaffin granules contain lysine- and arginine-aminopeptidase activities (41), although an aminopeptidase recognizing aliphatic hydrophobic residues (such as Leu³³²) has not been described in chromaffin cells; if present, the exopeptidases leucine aminopeptidase or aminopeptidase M could accomplish this cleavage (32).

Likewise, the enzymology giving rise to endoproteolytic cleavage between bovine chromogranin A residues Pro³⁴²Asp³⁴³, to yield chromogranin A_343-362 (DRSMRLSFRARGYGFRGPGL) (Fig. 4), is not certain, although a post-proline-cleaving enzymatic activity (at ProXaa) would suffice (42, 43). Asp-Pro peptide bonds are unstable in acidic solution (42), but acidic cleavage at Asp³⁴¹Pro³⁴² would still yield a peptide (chromogranin A_342-364) with amino-terminal Pro³⁴² (PDRSMRLSFRARGYGFRGPGLQL), unlike chromogranin A_343-362 (DRSMRLSFRARGYGFRGPGL).

What prompted cleavage of carboxyl-terminal Gln³⁶³-Leu³⁶⁴ from bovine chromogranin A_343-364 (DRSMRLSFRARGYGFRGPGLQL), to yield chromogranin A_343-362 (DRSMRLSFRARGYGFRGPGL) (Fig. 4)? Chromaffin granules do contain carboxypeptidase B (34), but carboxypeptidase B cleaves preferentially at basic amino acids (Arg, Lys); carboxypeptidase types P or Y (32) could catalyze sequential removal of Gln³⁶³-Leu³⁶⁴, but such carboxypeptidases have not been described in chromaffin granules. Cleavage at chromogranin A residues Leu³⁶²Gln³⁶³ may represent a chymotrypsin-like cleavage, at the hydrophobic residue Leu³⁶²; chymotrypsin-like enzymatic cleavages are known to occur in neuroendocrine peptides (44), and a chymotrypsin inhibitor has been isolated from chromaffin granules (45).

Since peptide fractions characterized in these experiments came from sucrose density gradient-purified chromaffin granules, artifactual proteolysis is unlikely to be problematic here.

We detected catestatin immunoreactivity in bovine and equine serum (see "Results"). Using chromogranin A radioimmunoassays directed to regions overlapping the catestatin portion of human (chromogranin A_344-374; Ref. 46) or rat (chromogranin A_359-389; Ref. 47) chromogranin A, Yanaihara and co-workers (46-49) also found catestatin region immunoreactivity in the circulation (46, 47) as well as in saliva, where its release was triggered by autonomic stimulation (48, 49). Since catestatin administration into the bloodstream exerts profound effects upon blood pressure (50), detection of catestatin in serum has implications for control of the circulation.

Thus, the catestatin fragment of chromogranin A is formed by endogenous proteolytic cleavage in vivo. Such authentic cleavage sites provide a useful starting point in analysis of the relationship between structure and function for this potent and specific catecholamine release-inhibitory peptide (10).

	ACKNOWLEDGEMENTS

We appreciate protein sequencing assistance by Matthew Williamson in the laboratory of Dr. Paul Price (Department of Biology, University of California San Diego, La Jolla, CA) and mass spectrometry assistance by Ken Harris and Dr. Jane Wu in the laboratory of Dr. Gary Siuzdak (The Scripps Research Institute, La Jolla, CA). Chromogranin A region-specific antibodies were kindly provided by Drs. Reiner Fischer-Colbrie (anti-bovine chromogranin A_367-391, or "GE-25"; Department of Pharmacology, University of Innsbruck, Austria) and Marie-France Bader (anti-bovine chromogranin A_316-329, or "WE-14"; INSERM U-338, Strasbourg, France).

	FOOTNOTES

^* This work was supported by grants from the Department of Veterans Affairs, the National Institutes of Health, and the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Medicine and Center for Molecular Genetics (9111H), University of California, San Diego, 3350 La Jolla Village Dr., San Diego, CA 92161. Tel.: 858-552-8585 (ext. 7373); Fax: 858-642-6331; E-mail: doconnor@ucsd.edu.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M001232200

	ABBREVIATIONS

The abbreviations used are: MALDI, matrix-assisted laser desorption ionization; HPLC, high pressure liquid chromatography; RP-HPLC, reverse-phase HPLC; FPLC, fast protein liquid chromatography; RIA, radioimmune assay; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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