title: Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing.
abstract: Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC-peptide preferentially inhibited insulin secretory granule lysate processing of des-31,32-proinsulin (ID50 = microM) compared to proinsulin (ID50 = 330 microM), and not des-64,65-proinsulin. We conclude that the type I endopeptidase recognized des-64,65-proinsulin and proinsulin as similar substrates, whereas the type II endopeptidase has a stronger preference for des-31,32-proinsulin compared to intact proinsulin. Furthermore, we suggest that in intact proinsulin there exists a constraint to efficient processing that is relieved following type I processing. Structural flexibility, in addition to the presence of Lys64-Arg65, therefore appears to be important for type II endopeptidase specificity and may provide a molecular basis for a preferential route of proinsulin conversion via des-31,32-proinsulin.
authors.affiliation: E.P. Joslin Research Laboratory, Joslin Diabetes Center, Boston, Massachusetts 02215.
authors.forename: C J
authors.initials: CJ
authors.lastname: Rhodes
authors.forename: B
authors.initials: B
authors.lastname: Lincoln
authors.forename: S E
authors.initials: SE
authors.lastname: Shoelson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000201:enzymology*; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007340:Insulinoma / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000201:enzymology*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D011498:Protein Precursors; C070703:proinsulin, des(31,32)-; D011384:Proinsulin; D010450:Endopeptidases; C061496:proinsulin endopeptidase II