Proteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4). Localization of cleavage site to non-homologous region of native IGFBP-4.
Insulin-like growth factor binding protein 4 (IGFBP-4) is a 24-kDa protein that binds insulin-like growth factor 1 (IGF-1) and IGF-2 with high affinity and inhibits IGF action in vitro. We recently described a protease produced by the B104 neuronal cell line that cleaves IGFBP-4, yielding an approximate 16-kDa immunoreactive protein that binds IGFs with reduced affinity. We analyzed fragments produced by exposing pure IGFBP-4 to the protease to determine potential cleavage sites. Electrospray mass spectrometry and amino acid sequencing indicated the 16-kDa fragment spanned the NH2 terminus of native IGFBP-4 through Lys-120. There was evidence for an additional proteolytic fragment beginning at amino acid 132 and continuing to the COOH terminus. Proteolysis could be blocked by a synthetic peptide that spanned amino acids 117-126 but not by peptides that contained flanking sequences 111-120 or 125-135. Mutagenesis was used to alter the basic residue at position 120. The expressed mutant IGFBP-4 (K120A) was relatively resistant to cleavage, strongly suggesting that residues 120-121 represent the cleavage site. This region of IGFBP-4 is not homologous with other IGFBPs, explaining the apparent specificity of the protease for IGFBP-4. The 16-kDa IGFBP-4 fragment no longer inhibited IGF-1-stimulated thymidine uptake in vitro, suggesting that proteolytic processing of IGFBP-4 may have important functional consequences in vivo.
Chernausek
S D
SD
Department of Pediatrics, University of Cincinnati School of Medicine, Ohio 45229, USA.
Smith
C E
CE
Duffin
K L
KL
Busby
W H
WH
Wright
G
G
Clemmons
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D018974:Insulin-Like Growth Factor Binding Protein 4; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002352:Carrier Proteins; D017931:DNA Primers; D018974:Insulin-Like Growth Factor Binding Protein 4; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D003907:Dexamethasone; D010450:Endopeptidases; D008239:Lysine