title: The post-translational processing and intracellular sorting of carboxypeptidase H in the islets of Langerhans.
abstract: The post-translational processing and intracellular sorting of the proinsulin-converting enzyme carboxypeptidase H (CPH) was studied in isolated rat islets of Langerhans. Pulse-chase-radiolabelling experiments using sequence-specific antisera showed that CPH was synthesized initially as a 57-kDa glycoprotein which was processed to a 54-kDa mature form by proteolytic processing at the N-terminus. Processing of the CPH precursor occurred rapidly (t(1/2) = 30) after an initial delay of 15-30 min and the enzyme was secreted in parallel with the insulin-related peptides in response to glucose-stimulation within 1 h after radiolabelling. This indicated that the proteins were packaged into nascent secretory granules at approximately the same rate following synthesis. Conversion of proinsulin and the 57-kDa form was inhibited markedly by chase incubation of islets at 20 degrees C, indicating that maturation of both proteins occurs in a post-Golgi compartment. Affinity purification of the enzyme from insulinoma subcellular fractions showed that the 57-kDa form was associated with endoplasmic reticulum or Golgi elements, and the 54-kDa form was present in secretory granules. Structural analysis showed that the granule form of the enzyme had an N-terminal amino acid sequence beginning at residue 42 of rat CPH, thereby implicating cleavage of the precursor after the fourth Arg in a site containing five consecutive Arg residues. These findings indicate that post-translational processing of CPH is mediated by an endoprotease which cleaves at sites containing multiple basic amino acid residues upon segregation of the enzyme to the secretory granules.
authors.affiliation: University of Cambridge, Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK.
authors.forename: P C
authors.initials: PC
authors.lastname: Guest
authors.forename: S D
authors.initials: SD
authors.lastname: Arden
authors.forename: N G
authors.initials: NG
authors.lastname: Rutherford
authors.forename: J C
authors.initials: JC
authors.lastname: Hutton
mesh_terms: D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000378:metabolism*; D003080:Cold Temperature; D003594:Cytoplasmic Granules / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000378:metabolism; D007163:Immunosorbent Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma / Q000737:chemistry / Q000648:ultrastructure; D007515:Islets of Langerhans / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011384:Proinsulin / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D013347:Subcellular Fractions / Q000737:chemistry; D013462:Sulfur Radioisotopes
chemical_list: D007328:Insulin; D010455:Peptides; D011498:Protein Precursors; D013462:Sulfur Radioisotopes; D011384:Proinsulin; D006026:Glycoside Hydrolases; D002268:Carboxypeptidases; D043423:Carboxypeptidase H