Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro

C E Higham, R L Hull, L Lawrie, K I J Shennan, J F Morris, N P Birch, K Docherty, A Clark

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.

Original languageEnglish
Pages (from-to)4998-5004
Number of pages7
JournalEuropean Journal of Biochemistry
Volume267
Publication statusPublished - 2000

Keywords

  • PC2
  • PC3
  • processing
  • proinsulin
  • pro-islet amyloid polypeptide
  • PANCREATIC-ISLETS
  • BETA-CELLS
  • B-CELLS
  • 7B2
  • LOCALIZATION
  • PURIFICATION
  • PROINSULIN
  • EXPRESSION
  • MATURATION
  • CLEAVAGE

Cite this

Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro. / Higham, C E ; Hull, R L ; Lawrie, L ; Shennan, K I J ; Morris, J F ; Birch, N P ; Docherty, K ; Clark, A .

In: European Journal of Biochemistry, Vol. 267, 2000, p. 4998-5004.

Research output: Contribution to journalArticle

Higham, C E ; Hull, R L ; Lawrie, L ; Shennan, K I J ; Morris, J F ; Birch, N P ; Docherty, K ; Clark, A . / Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro. In: European Journal of Biochemistry. 2000 ; Vol. 267. pp. 4998-5004.
@article{1b2f0543848c48fa8ee4e71f856ec5b9,
title = "Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro",
abstract = "Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.",
keywords = "PC2, PC3, processing, proinsulin, pro-islet amyloid polypeptide, PANCREATIC-ISLETS, BETA-CELLS, B-CELLS, 7B2, LOCALIZATION, PURIFICATION, PROINSULIN, EXPRESSION, MATURATION, CLEAVAGE",
author = "Higham, {C E} and Hull, {R L} and L Lawrie and Shennan, {K I J} and Morris, {J F} and Birch, {N P} and K Docherty and A Clark",
year = "2000",
language = "English",
volume = "267",
pages = "4998--5004",
journal = "European Journal of Biochemistry",
issn = "0014-2956",
publisher = "Wiley-Blackwell",

}

TY - JOUR

T1 - Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro

AU - Higham, C E

AU - Hull, R L

AU - Lawrie, L

AU - Shennan, K I J

AU - Morris, J F

AU - Birch, N P

AU - Docherty, K

AU - Clark, A

PY - 2000

Y1 - 2000

N2 - Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.

AB - Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.

KW - PC2

KW - PC3

KW - processing

KW - proinsulin

KW - pro-islet amyloid polypeptide

KW - PANCREATIC-ISLETS

KW - BETA-CELLS

KW - B-CELLS

KW - 7B2

KW - LOCALIZATION

KW - PURIFICATION

KW - PROINSULIN

KW - EXPRESSION

KW - MATURATION

KW - CLEAVAGE

M3 - Article

VL - 267

SP - 4998

EP - 5004

JO - European Journal of Biochemistry

JF - European Journal of Biochemistry

SN - 0014-2956

ER -