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

Abstract

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 A332–364; identity of the peptide was confirmed by diagnostic Met346oxidation. Further analysis revealed two additional forms: bovine chromogranin A333–364 and A343–362. Synthetic longer (chromogranin A332–364) and shorter (chromogranin A344–364) 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 A340–372, bounded by dibasic sites. We conclude that catestatin is cleaved extensivelyin 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. 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 A332–364; identity of the peptide was confirmed by diagnostic Met346oxidation. Further analysis revealed two additional forms: bovine chromogranin A333–364 and A343–362. Synthetic longer (chromogranin A332–364) and shorter (chromogranin A344–364) 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 A340–372, bounded by dibasic sites. We conclude that catestatin is cleaved extensivelyin 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. matrix-assisted laser desorption ionization high pressure liquid chromatography reverse-phase HPLC fast protein liquid chromatography radioimmune assay N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine polyacrylamide gel electrophoresis Chromogranin A, the major soluble protein in catecholamine storage vesicles, not only stabilizes the core of such vesicles (1Videen J.S. Mezger M.S. Chang Y.M. O'Connor D.T. J. Biol. Chem. 1992; 267: 3066-3073Abstract Full Text PDF PubMed Google Scholar) but also serves as a prohormone cleaved into several biologically active peptides, including the insulin release-inhibitory fragments pancreastatin (2Tatemoto K. Efendic S. Mutt V. Makk G. Feistner G.J. Barchas J.D. Nature. 1986; 324: 476-478Crossref PubMed Scopus (622) Google Scholar) and β-granin (3Hutton J.C. Davidson H.W. Grimaldi K.A. Peshavaria M. Biochem. J. 1987; 244: 449-456Crossref PubMed Scopus (65) Google Scholar, 4Drees B.M. Rouse J. Johnson J. Hamilton J.W. Endocrinology. 1991; 129: 3381-3387Crossref PubMed Scopus (98) Google Scholar, 5Drees B.M. Hamilton J.W. Endocrinology. 1994; 134: 2057-2063Crossref PubMed Scopus (23) Google Scholar), the vasodilator vasostatin (6Aardal S. Helle K.B. Regul. Pept. 1992; 41: 9-18Crossref PubMed Scopus (182) Google Scholar,7Helle K.B. Marley P.D. Angeletti R.H. Aunis D. Galindo E. Small D.H. Livett B.G. J. Neuroendocrinol. 1993; 5: 413-420Crossref PubMed Scopus (55) Google Scholar), and the parathyroid hormone release-inhibitory parastatin (8Fasciotto B.H. Trauss C.A. Greeley G.H. Cohn D.V. Endocrinology. 1993; 133: 461-466Crossref PubMed Scopus (0) Google Scholar). Recently we described an additional chromogranin A fragment, catestatin (bovine chromogranin A344–364), which acts specifically as a potent (IC50 ∼200–400 nm) nicotinic cholinergic antagonist to inhibit catecholamine release (9Simon J.P. Bader M.F. Aunis D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1712-1716Crossref PubMed Scopus (186) Google Scholar, 10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar), suggesting a novel autocrine feedback mechanism controlling sympathochromaffin exocytosis. Initial studies of catestatin relied on synthetic peptides (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar); 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 A332–364 or human chromogranin A340–372), while a smaller form was also identified (bovine chromogranin A343–362). 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. 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 (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar). 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 (11Barbosa J.A. Gill B.M. Takiyyuddin M.A. O'Connor D.T. Endocrinology. 1991; 128: 174-190Crossref PubMed Scopus (83) Google Scholar). 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 (A 280) 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 (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar). 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 (12O'Connor D.T. Klein R.L. Thureson-Klein A.K. Barbosa J.A. Brain Res. 1991; 567: 188-196Crossref PubMed Scopus (32) Google Scholar), which were lysed by resuspension in 10 mm Hepes, pH 7, and freezing/thawing. Bovine or human chromogranin A was isolated from chromaffin granule soluble core proteins by affinity chromatography (to remove dopamine β-hydroxylase) followed by gel filtration, as described previously (13Syversen U. Waldum H.L. O'Connor D.T. Neuropeptides. 1992; 22: 235-240Crossref PubMed Scopus (22) Google Scholar). Recombinant human chromogranin A was expressed in Escherichia coli by a modification of previously described methods (14Taupenot L. Remade J.E. Helle K.B. Aunis D. Bader M.F. Regul. Pept. 1995; 56: 71-88Crossref PubMed Scopus (30) Google Scholar), except that a His6 tag was used for affinity purification on a Ni2+-nitrilotriacetic acid column (15Sheibani N. Prep. Biochem. Bio/Technol. 1999; 29: 77-90Crossref PubMed Scopus (45) Google Scholar). 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 A344–364 (RSMRLSFRARGYGFRGPGLQL) or human chromogranin A352–372 (SSMKLSFRARAYGFRGPGPQL), were developed by a modification of protocols previously described for other chromogranin peptides (11Barbosa J.A. Gill B.M. Takiyyuddin M.A. O'Connor D.T. Endocrinology. 1991; 128: 174-190Crossref PubMed Scopus (83) Google Scholar, 16Gill B.M. Barbosa J.A. Hogue-Angeletti R. Varki N. O'Connor D.T. Neuropeptides. 1992; 21: 105-108Crossref PubMed Scopus (16) Google Scholar). The polyclonal antibody recognizing the catestatin region of chromogranin A was further purified on an Amersham Pharmacia Biotech Hi protein A column in m with m and the pH was then to with A polyclonal rabbit recognizing bovine chromogranin obtained from Bader and a polyclonal rabbit recognizing human chromogranin was obtained from of granule soluble core or gel filtration fractions were to C-18 with resuspended in μl of buffer containing Triton mm 10 pH 1 mm 1 1 mm EDTA, 1 mm and then by a modification of the of R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). samples were with μl of rabbit serum with at for μl of protein were and was for followed by centrifugation for min at × g in an which the pellet was the μl of rabbit catestatin (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar) were and was for μl of protein were and for which the were by centrifugation, with and then with mm Tris-HCl, pH 8 (to remove and catestatin was from the with μl of R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). peptides of to the mass of a at 10 μl were by to 10 μl were by MALDI mass on a mass with were in an acid G. for Scholar) and then with a laser at and the were with a of V. were then to ratio using a mass The mass of this is G. for Scholar). After of bovine chromaffin granule protein samples in buffer (10 mm Tris-HCl, 1 mm EDTA, mm 0.1% of protein were in the of buffer gel peptides as as H. G. Biochem. 1987; PubMed Scopus Google Scholar). Human pheochromocytoma chromaffin granule samples were on After were for protein with 0.1% in acid in Scholar), followed by in acid were and followed by catestatin by H. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The were rabbit catestatin (chromogranin rabbit chromogranin rabbit chromogranin or rabbit catestatin (chromogranin (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar). antibody was by either Amersham Pharmacia Biotech or After of bovine chromaffin granule peptides μl of each HPLC fraction were resuspended in μl of by filtration a membrane using a and and using the catestatin antibody and antibody with an (Amersham Pharmacia analysis was on a using the at the of and on the filtration size-fractionated bovine chromaffin granule peptides were further by using a × C-18 in 0.1% and with a of in 0.1% at 1 The was by A and fractions were at from each fraction were and to and fractions were further to amino-terminal see and MALDI mass see Eluted HPLC fractions from in 0.1% column buffer were analyzed for amino-terminal by with analysis from MALDI mass were and peptide fragments the chromogranin A structure were by the version for on the for chromogranin A peptides G. for Scholar). were analyzed at each by the to amino-terminal from the same at a V. Google Scholar). were to a of by of release from pheochromocytoma cells, a secretion as described previously (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar, M. Mahata S.K. Parmer R.J. O'Connor D.T. PubMed Scopus Google Scholar). The to catecholamine release were either nicotinic cholinergic or membrane mm In some experiments, the peptide was antibody in secretion buffer) to of of bovine chromaffin cells were prepared as described previously S. C.A. S. Endocrinology. 1999; Scopus Google Scholar). was a The were either nicotinic cholinergic membrane mm in the or of 1 or were on a and analyzed by Synthetic catestatin (bovine chromogranin was by the on its endogenous to a specific of by The μl of rabbit chromogranin A344–364 in buffer (50 mm Tris-HCl, pH 8.3, 0.3% bovine serum albumin, 0.1% Triton at a of and μl of catestatin (bovine chromogranin or After h at 10,000 of chromogranin in μl of buffer was After a the antibody and bovine serum were After h at μl of buffer was samples were centrifuged at for min at the supernatant was and the samples were for 1 min in the bovine serum and serum were obtained from expressed as the Chromaffin granule soluble core proteins were size-fractionated by gel filtration fractions were analyzed by (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar), and fractions were for further After peptide and from a C-18 affinity this fraction inhibited nicotinic cholinergic-stimulated catecholamine release from pheochromocytoma release of but of as as of the peptide fraction release to (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar). the catestatin region of chromogranin A is at amino acid we to mass on chromogranin A fragments in chromaffin granules. The peptide fraction (Fig. fractions was by a bovine catestatin (chromogranin A344–364) antibody and to MALDI mass The revealed a major at (Fig. the acid bovine chromogranin A structure A. E. M. Nature. 1986; PubMed Scopus Google Scholar, R. J. J. J. 1986; 5: PubMed Scopus Google Scholar), to chromogranin A332–364 the between and is the of of the MALDI G. for Scholar). serum not a of this mass (Fig. mass of peptides to by of as we the on granule peptides that had been by to a C-18 an the with some in that this the granule peptide was with to into as described by in studies of processing by mass R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar); bovine catestatin a (bovine chromogranin A 1), this a further specific diagnostic for the identity of the catestatin After the major was found (Fig. a mass to the in In a to the of endogenous catestatin the chromaffin granule peptide fraction filtration fractions was by (Fig. and fractions were with HPLC fraction containing catestatin (Fig. was then analyzed (Fig. by MALDI mass and amino-terminal amino acid MALDI revealed two at and (Fig. In the catestatin region of bovine chromogranin A (Fig. 1), is with chromogranin is with chromogranin was 10 with the suggesting two peptides V. Google to bovine chromogranin the 10 amino of the peptide (chromogranin and to bovine chromogranin the amino of the peptide (chromogranin A343–362). Bovine splenic nerve was homogenized, by from a C-18 by an (bovine chromogranin A344–364) and to MALDI mass spectrometry. MALDI revealed a at (Fig. in the catestatin region of bovine chromogranin A (Fig. 1), is of the of chromogranin A332–364 After by the while a mass to the in in chromogranin A332–364 After of human pheochromocytoma chromaffin granules, of were (Fig. the chromogranin A structure to chromogranin which is bounded on either by dibasic sites for prohormone cleavage (Fig. in each to with the of an to form in this region not and well the 0.1% of MALDI mass G. for Scholar). the and of the bovine catestatin peptides by MALDI mass and amino-terminal and we longer (chromogranin A332–364; and shorter (chromogranin versions of catestatin as well as chromogranin A and on catecholamine release from pheochromocytoma cells (Fig. previously for the shorter version of catestatin (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar), the longer version also inhibited catecholamine release by nicotinic cholinergic but not that by membrane (Fig. Both peptides and chromogranin A of secretion (Fig. chromogranin A had an of chromogranin A344–364 had an of and the chromogranin was ∼2.01 high (10 each peptide while chromogranin A only A peptide to the catestatin region (chromogranin of human chromogranin A also inhibited nicotinic cholinergic-stimulated catecholamine secretion at 10 and the was specifically by a rabbit the catestatin region of human chromogranin A (chromogranin not by serum (Fig. Chromaffin granule soluble core proteins were size-fractionated on a gel filtration column (Fig. and fractions were analyzed by major of catestatin in fractions and (Fig. 1 of catestatin and of the catestatin while and of and of and of catestatin and of catestatin in the size and of chromogranin the potent form of catestatin (Fig. Synthetic peptides to chromogranin A332–364 and chromogranin A344–364 were size-fractionated on the same eluting in fractions and and fractions and A of the gel filtration was using bovine serum a larger form of catestatin (chromogranin A332–364; and a smaller form of catestatin (chromogranin Chromaffin granule soluble proteins were by and then with not only an antibody the catestatin region (bovine chromogranin A344–364) but also peptide that bounded by dibasic cleavage sites and either amino-terminal (bovine chromogranin or carboxyl-terminal (bovine chromogranin to catestatin (Fig. antibody not only chromogranin A a mass of but also several mass chromogranin A from to All of the mass chromogranin A fragments by with the of a fragment (Fig. which is by and not by the fragment the peptide amino-terminal to a dibasic cleavage at in bovine chromogranin A. the chromaffin granule peptides (Fig. fractions were to catestatin followed by of the of the catestatin was found in fractions of mass chromogranin in a and in a The catestatin antibody chromogranin human chromogranin A, at (Fig. in pheochromocytoma chromaffin granule All pheochromocytomas at some processing of the catestatin peptides, the and the catestatin in two pheochromocytomas (Fig. and and the of catestatin processing from human chromogranin A was in two of pheochromocytomas (Fig. and In of the of the catestatin in the chromogranin A (Fig. processing was on catestatin of chromaffin granules from additional pheochromocytomas not Bovine chromaffin cells in were by either the nicotinic cholinergic or membrane mm each in the of (Fig. cells released of to or membrane We also the of catestatin secretion in the of membrane mm catestatin release by to while in the of this was to were for secretion (Fig. in the of membrane mm release by to while in the of this was to this we catestatin in bovine serum nm) and serum The catestatin region of chromogranin A (bovine chromogranin A344–364) is a potent and specific inhibitor of chromaffin catecholamine release by nicotinic cholinergic the pathway for such cells (10Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar); catestatin also nicotinic of the S.K. Mahata M. Parmer R.J. O'Connor D.T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In we documented cleavage of the catestatin region in chromaffin granules and and sympathetic nerve catecholamine storage vesicles (Fig. as well as chromaffin granules from human pheochromocytoma and cleavage of the catestatin region at high in chromogranin of catestatin in chromaffin granules was of a size form chromogranin A (Fig. 8 and of catestatin with the peptide bovine chromogranin A344–364 (Fig. 8 A, catestatin is to release by chromaffin cells (Fig. of chromaffin granule peptides (Fig. followed by and MALDI mass (Fig. revealed a major catestatin form (bovine chromogranin A332–364) as at dibasic of dibasic sites by prohormone is well described in chromogranin we A. Wu H. 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Radioimmunoassay confirmed the secretion and of Both longer (chromogranin A332–364; and shorter (chromogranin synthetic forms of bovine catestatin specific antagonism of nicotinic cholinergic-stimulated catecholamine release (Fig. the shorter form had superior potency ∼2.01 suggesting that cleavage of the longer to the shorter version may remove an bovine chromogranin from the of such further cleavage is the cleavage removal of the amino to an the of the peptide. catestatin cleavage from chromogranin A its potency by (IC50 an of human chromaffin granules D.T. R.L. Clin. A PubMed Scopus Google Scholar, D.T. R.L. PubMed Scopus Google Scholar). Synthetic human catestatin (chromogranin specifically inhibited chromaffin catecholamine release (Fig. and of catestatin from human chromogranin A between dibasic sites at of chromogranin was demonstrated by mass (Fig. studies several cleavage sites in the catestatin region of bovine chromogranin A. bovine chromaffin granules, Hogue-Angeletti R. 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Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (338) Google Scholar). We protein by in the of of of and mass by and Wu in the of Chromogranin A were by chromogranin or of of and Bader chromogranin or