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

• Published in: Biochemistry (Easton) Vol. 42; no. 23; pp. 6938 - 6946
• Main Authors: Lee, Jean C., Taylor, Carolyn V., Gaucher, Sara P., Toneff, Thomas, Taupenot, Laurent, Yasothornsrikul, Sukkid, Mahata, Sushil K., Sei, Catherine, Parmer, Robert J., Neveu, John M., Lane, William S., Gibson, Bradford W., O'Connor, Daniel T., Hook, Vivian Y. H.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.06.2003
• Subjects: Adrenal Medulla - chemistry; Adrenal Medulla - cytology; Amino Acid Sequence; Amino Acids, Basic - genetics; Amino Acids, Basic - metabolism; Animals; Binding Sites; Blotting, Western; Cattle; Chromaffin Cells - metabolism; Chromaffin Cells - secretion; Chromaffin Granules - enzymology; Chromogranin A; Chromogranins - biosynthesis; Chromogranins - chemistry; Chromogranins - genetics; Chromogranins - metabolism; Enkephalins - metabolism; Molecular Sequence Data; Molecular Weight; Peptide Fragments - biosynthesis; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide Mapping; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Protein Precursors - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Trypsin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0300433

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 NH2-terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK↓, KR↓, R↓, and KR↓ 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 NH2 terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA344 - 364) and variant peptide forms (CgA343 - 368 and CgA332 - 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 (CgA344 - 364) utilizes more unusual cleavage sites at the NH2-terminal sides of ↓R and ↓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.