The post-translational processing of chromogranin A in the pancreatic islet: involvement of the eukaryote subtilisin PC2

Abstract

The post-translational processing of chromogranin A (CGA) and the nature of the enzyme(s) involved were investigated in rat pancreatic islet and insulinoma tissue. Pulse-chase radiolabelling experiments using sequence-specific antisera showed that the 98 kDa (determined by SDS/PAGE) precursor was processed to an N-terminal 21 kDa peptide, a C-terminal 14 kDa peptide and a 45 kDa centrally located peptide with a rapid time course (t1/2 approx. 30 min) after an initial delay of 30-60 min. The 45 kDa peptide was, in turn, converted partially into a 5 kDa peptide with pancreastatin immunoreactivity and a 3 kDa peptide with WE-14 immunoreactivity over a longer time period. Incubation of bovine CGA with rat insulinoma secretory-granule lysate produced peptides of 18, 16 and 40 kDa via intermediates of 65 and 55 kDa. N-terminal sequence analysis indicated that cleavage occurred at the conserved paired basic sites Lys114-Arg115 and Lys330-Arg331, suggesting that cleavage of the equivalent sites (Lys129-Arg130 and Lys357-Arg358) in the rat molecule produced the initial post-translational products observed in intact pancreatic beta-cells. The enzyme activity responsible for the cleavage of bovine CGA co-chromatographed on DEAE-cellulose with the type-2 proinsulin endopeptidase and with PC2 immunoreactivity. The type-1 enzyme (PC1/3) appeared inactive towards CGA. The requirement for Ca2+ ions and an acidic pH for conversion was consistent with the involvement of a member of the eukaryote subtilisin family, and the composition of the released peptides in pulse-chase and secretion studies suggested that conversion occurred in the secretory-granule compartment. The overall catalytic rate as well as the relative susceptibilities of the Lys114-Arg115 and Lys330-Arg331 sites to cleavage were affected by pH, suggesting that the ionic environment of the processing compartment may play a role in the differential processing of CGA which is evident in various neuroendocrine cells.

Chromogranin A: current status as a precursor for bioactive peptides and a granulogenic/sorting factor in the regulated secretory pathway

“…The concentration of unprocessed granin in mature granules appears to be inversely proportional to the amount of co-stored processed peptide(s), which are directly proportional to the concentration of the prohormone converting (PC) enzymes PC1 and PC2 within the granule [88,89]. Thus, the granins could well be present in relatively high levels in the trans-Golgi network, where they contribute to granulogenesis, and in relatively low concentrations in the mature secretory granules following processing [17,88,90,91]. The second is that it is unlikely that CGA, CGB and SglI are the only regulated granule constituents that contribute to granulogenesis.…”

“…The extent of chromogranin A processing is highly tissuedependent: processing appears to be least complete in the adrenal medulla and progressively greater in intestine, stomach, peripheral nerves, and pancreas [27,103,[110][111][112]. The endocrine pancreas appears to exhibit the highest level of processing based on the ratio of intact CGA to fully processed /3-granin as well as pancreastatin [21,27,88]. CGA intragranular proteolytic processing in the endocrine pancreas is apparently completely calcium-dependent, and CGA is a much better substrate for PC2 than for PC1/3 kex2-1ike prohormone converting enzymes [88].…”

“…The endocrine pancreas appears to exhibit the highest level of processing based on the ratio of intact CGA to fully processed /3-granin as well as pancreastatin [21,27,88]. CGA intragranular proteolytic processing in the endocrine pancreas is apparently completely calcium-dependent, and CGA is a much better substrate for PC2 than for PC1/3 kex2-1ike prohormone converting enzymes [88]. According to Arden and co-workers, the higher concentrations of PC2 in pancreas compared to adrenal medulla may explain why the former tissue contains CGA polypeptides in more processed form than the latter [88].…”

“…Each of the peptides above have been demonstrated to exist in endocrine tissues by extraction, isolation, purification and peptide sequencing. The single exception is parastatin, the existence of which is inferred from the presence of a 14 kDa C-terminal CGA fragment generated in rat insulinoma cells, and the biological activity of the parastatin fragment generated by endoproteinase Lys-C digestion of purified CGA [88,108] The processing of CGA seems to occur preferentially at some but not all pairs of basic amino acids, and at some single basic amino acids, at the C-and N-termini of the molecule [27,103,110,111]. The extent of chromogranin A processing is highly tissuedependent: processing appears to be least complete in the adrenal medulla and progressively greater in intestine, stomach, peripheral nerves, and pancreas [27,103,[110][111][112].…”

“…3). These are (i) the vasostatin/fl-granin peptides, comprising the N-terminal 113 amino acids (flgranin/vasostatin II) of CGA and in some tissues (except in rat) a shorter N-terminal 76-amino acid polypeptide (vasostatin I) [91,103,104], (ii) pancreastatin [21,105], (iii) WE-14 [86,106,107] and (iv) the parastatin/GE-25 peptides [88,108,109]. Each of the peptides above have been demonstrated to exist in endocrine tissues by extraction, isolation, purification and peptide sequencing.…”