title: Primary sequence characterization of catestatin intermediates and peptides defines proteolytic cleavage sites utilized for converting chromogranin a into active catestatin secreted from neuroendocrine chromaffin cells.
abstract: Catestatin is an active 21-residue peptide derived from the chromogranin A (CgA) precursor, and catestatin is secreted from neuroendocrine chromaffin cells as an autocrine regulator of nicotine-stimulated catecholamine release. The goal of this study was to characterize the primary sequences of high molecular mass catestatin intermediates and peptides to define the proteolytic cleavage sites within CgA that are utilized in the biosynthesis of catestatin. Catestatin-containing polypeptides, demonstrated by anti-catestatin western blots, of 54-56, 50, 32, and 17 kDa contained NH(2)-terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK downward arrow, KR downward arrow, R downward arrow, and KR downward arrow basic residue sites, respectively. The COOH termini of these catestatin intermediates were defined by the presence of the COOH-terminal tryptic peptide of the CgA precursor, corresponding to residues 421-430, which was identified by MALDI-TOF mass spectrometry. Results also demonstrated the presence of 54-56 and 50 kDa catestatin intermediates that contain the NH(2) terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA(344)(-)(364)) and variant peptide forms (CgA(343)(-)(368) and CgA(332)(-)(361)). These determined cleavage sites predicted that production of high molecular mass catestatin intermediates requires cleavage at the COOH-terminal sides of paired basic residues, which is compatible with the cleavage specificities of PC1 and PC2 prohormone convertases. However, it is notable that production of catestatin itself (CgA(344)(-)(364)) utilizes more unusual cleavage sites at the NH(2)-terminal sides of downward arrow R and downward arrow RR basic residue sites, consistent with the cleavage specificities of the chromaffin granule cysteine protease "PTP" that participates in proenkephalin processing. These findings demonstrate that production of catestatin involves cleavage of CgA at paired basic and monobasic residues, necessary steps for catestatin peptide regulation of nicotinic cholinergic-induced catecholamine release.
authors.affiliation: The Buck Institute for Age Research, Novato, California 94945, USA.
authors.forename: Jean C
authors.initials: JC
authors.lastname: Lee
authors.forename: Carolyn V
authors.initials: CV
authors.lastname: Taylor
authors.forename: Sara P
authors.initials: SP
authors.lastname: Gaucher
authors.forename: Thomas
authors.initials: T
authors.lastname: Toneff
authors.forename: Laurent
authors.initials: L
authors.lastname: Taupenot
authors.forename: Sukkid
authors.initials: S
authors.lastname: Yasothornsrikul
authors.forename: Sushil K
authors.initials: SK
authors.lastname: Mahata
authors.forename: Catherine
authors.initials: C
authors.lastname: Sei
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Parmer
authors.forename: John M
authors.initials: JM
authors.lastname: Neveu
authors.forename: William S
authors.initials: WS
authors.lastname: Lane
authors.forename: Bradford W
authors.initials: BW
authors.lastname: Gibson
authors.forename: Daniel T
authors.initials: DT
authors.lastname: O'Connor
authors.forename: Vivian Y H
authors.initials: VY
authors.lastname: Hook
mesh_terms: D000313:Adrenal Medulla / Q000737:chemistry / Q000166:cytology; D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism*; D002837:Chromaffin Granules / Q000201:enzymology; D053379:Chromogranin A; D002864:Chromogranins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004745:Enkephalins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism
chemical_list: D024361:Amino Acids, Basic; D053379:Chromogranin A; D002864:Chromogranins; D004745:Enkephalins; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; C109084:chromogranin A (344-364); C029992:proenkephalin; D014357:Trypsin