The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain.
The IGF-binding protein (IGFBP)-5 protein contains consensus heparin binding motifs in both its carboxy (C)-terminal and central domains, although only the C-terminal site has previously been shown to be functional. We have made two chimeric IGFBP proteins by switching domains between rat IGFBP-5 and -2, named BP552 and BP522 to reflect the domains present, and a truncated rat IGFBP-5 mutant (1-168), named BP550. The ability of these proteins and wild-type (wt) IGFBPs-5 and -2 to bind to either IGFs or heparin was determined using biosensor real-time analysis and heparin ligand blotting respectively. We report that the chimeric molecules have IGF binding affinities comparable to those of the native IGFBPs from which they were derived and, as expected, the binding of BP550 to IGFs was greatly compromised. More surprising was the finding that the ability of BP552 and BP550 to bind to heparin was equivalent to that of wtIGFBP-5, whereas wtIGFBP-2 and BP522 failed to bind. These results demonstrate that the active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is active only in the absence of the C-terminal domain. We subsequently mutated two basic amino acids (R136A:R137A) in the central consensus binding sites between residues 132-140. This resulted in the loss of heparin binding for BP550, confirming the importance of these two basic amino acids in the central domain heparin binding activity. In light of these findings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix binding properties.
Song
H
H
Hannah Research Institute, Ayr, UK.
Shand
J H
JH
Beattie
J
J
Flint
D J
DJ
Allan
G J
GJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D017931:DNA Primers; D006493:Heparin / Q000378:metabolism*; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051381:Rats
D017931:DNA Primers; D018975:Insulin-Like Growth Factor Binding Protein 5; D006493:Heparin