Chromogranin A (CgA) is a 48 kDa acidic protein in neuroendocrine secretory vesicles whose primary structure is now known. We used synthetic peptides, synthetic peptide antisera, intact molecule antisera, chymotryptic peptide mapping, microsequencing, immunoblotting, and immunoprecipitation to probe the location of immunodominant domains within the CgA molecule. Polyclonal anti mid-molecule, anti N-terminal and anti C-terminal antibodies specifically visualized CgA (both bovine and human) in one and two dimensional immunoblots of adrenal chromaffin vesicles, and the stain CgA fragments further suggested bidirectional (both N- and C-terminal) cleavage or processing of CgA. Anti intact CgA immunoblotting of HPLC-separated peptides from chymotrypsin-digested bovine CgA revealed several strongly immunoreactive internal peptides, two of which were positioned by N-terminal amino acid sequencing: CgA91ff. and CgA197ff.. A single synthetic peptide (CgA79-113) was recognized by three antibodies developed against the intact CgA molecule: two polyclonal rabbit antisera as well as a monoclonal mouse antibody. Not all antigenicity algorithm-predicted domains were immunogenic, suggesting that some of these predicted domains may not be accessible. Polyclonal anti mid-molecule, anti N- and anti C-terminal synthetic peptide antisera specifically immunoprecipitated 125I-labeled bovine CgA from aqueous solution; mid-molecule antisera precipitated substantially greater amounts than terminal antisera. The immunoprecipitation results suggested exposed terminal as well as interior hydrophilic epitopes in the molecule in its intact, native conformation. 125I-human CgA was best precipitated by anti N-terminal antisera, consistent with greatest interspecies sequence conservation at the N-terminus of CgA. The terminal antisera reacted immunohistochemically in a granular pattern with adrenal medullary chromaffin cells (but not adrenal cortical cells) and pancreatic islet cells (but not pancreatic exocrine acini). Thus, synthetic and chymotryptic peptides yielded novel and specific insights into the structure, conformation, vesicular processing and interior immunodominant domains of CgA.
ASJC Scopus subject areas
- Endocrine and Autonomic Systems
- Cellular and Molecular Neuroscience