title: 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.
authors.affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
authors.forename: S D
authors.initials: SD
authors.lastname: Arden
authors.forename: N G
authors.initials: NG
authors.lastname: Rutherford
authors.forename: P C
authors.initials: PC
authors.lastname: Guest
authors.forename: W J
authors.initials: WJ
authors.lastname: Curry
authors.forename: E M
authors.initials: EM
authors.lastname: Bailyes
authors.forename: C F
authors.initials: CF
authors.lastname: Johnston
authors.forename: J C
authors.initials: JC
authors.lastname: Hutton
mesh_terms: D000311:Adrenal Glands / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000737:chemistry; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism; D007163:Immunosorbent Techniques; D007340:Insulinoma; D007515:Islets of Langerhans / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010190:Pancreatic Neoplasms; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D045706:Proprotein Convertase 2; D011499:Protein Processing, Post-Translational; D051381:Rats; D013381:Subtilisins / Q000378:metabolism*; D013462:Sulfur Radioisotopes
chemical_list: D053379:Chromogranin A; D002864:Chromogranins; D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; C506153:chromogranin A, rat; D008715:Methionine; D013381:Subtilisins; D045706:Proprotein Convertase 2