Processing of chromogranin A in the parathyroid: generation of parastatin-related peptides

Chromogranin A (CgA) is a glycoprotein present in secretory granules of end ocrine cells. In the parathyroid, it is costored and cosecreted with parath ormone (PTH) in response to hypocalcemia. CgA is the precursor of several b ioactive peptides including pancreastatin and betagranin. Parastatin (PARA, pCgA(347-419)) is a novel peptide that we generated in vitro by enzymatic digestion of pCgA. In vitro, it inhibits low Ca2+-stimulated parathyroid se cretion. Full activity resides in its first 19 residues. In order to determ ine if PARA or PARA-derived peptides are natural products of the parathyroi d, we generated an antiserum directed against pCgA,,,,,, corresponding to t he bioactive N-terminal sequence of pPARA (pPARA(1-13) antiserum), and deve loped a specific radioimmunoassay that we used in conjunction with various chromatographic separations. We identified small peptides carrying the pPAR A(1-13) immunoactivity in extracts and secretion medium of porcine parathyr oid glands. Continuous and pulse-chase radiolabeling studies, along with im munoprecipitation using PARA(1-13) antiserum demonstrate that a newly-synth esized PARA-related peptide fraction with a Mr of 11 kDa is secreted by the parathyroid cells and accumulates in the secretion medium. Edman degradati on of the 11 kDa PARA-related peptide band by Edman degradation yielded thr ee major N-terminal sequences: S-K-M-D-R-L-A-K-E-L-(residues 313-322), D-R- L-A-K-E-L-T-A-E-(residues 316-325), and A-K-E-L-T-A-E-K-R-L-(residues 319-3 29), in a molar ratio of approximately 1:2:1. The peptide bonds required to be cleaved to yield these peptides, Trp-Ser, Met-Asp and Leu-Ala, suggest that a chymotrypsin-like endopeptidase participated in their formation. The molecular size and the results of amino acid compositional analysis, indic ate that the C-termini of these peptides extended variably to residues 384- 401 of pCgA. These results demonstrate that processing of CgA by the parath yroid gland generates bioactive PARA-related peptides that could affect the gland's secretory activity. (C) 2000 Elsevier Science Inc. All rights rese rved.