Identification of the sites in collagen α-chains that bind serum anti-gelatin factor (cold-insoluble globulin)

Abstract

Anti-gelatin factor was prepared from guinea-pig and human serum by affinity chromatography on denatured type-I collagen. As shown previously, this component is related to cold-insoluble globulin. It reacted with 125I-labelled denatured collagen, and the reaction could be inhibited by preincubation with unlabelled collagenous components. In the inhibition assay comparable activities were observed for native and denatured type-I, -II, -III and -IV collagens. There was also no difference in reactivity between collagens of different species. The reactive sites in the collagen alpha-chains were located by inhibition assays on distinct CNBr- and collagenase-derived peptides. The results obtained with fragments from alpha1(I)-, alpha2- and alpha1(II)-chains indicate that the most active region is located between positions 643 and 819 of the alpha1-chain. Lower activities were found for other regions of collagen and may indicate that the factor has the potential to interact with several sites in the alpha-chains. The present data agree with observations by Kleinman, McGoodwin & Klebe [Biochem. Biophys. Res. Commun. (1976) 72, 426-432] on the specificity of a serum factor promoting the attachment of fibroblasts to collagen.

Identification and structural analysis of type I collagen sites in complex with fibronectin fragments

“…The identified site, G 778 -G 799 , lies adjacent to the MMP-1 cleavage site, in agreement with earlier data from cell culture assays (6,14,15). Interestingly, the peptide adopts a conformation in the [8][9] FnI complex similar to that of bacterial peptides in [8][9] FnI are formed at its C terminus (green dashed lines) as well as important hydrogen bonds among the residues shown.…”

“…In contrast, 6 FnI 1-2 FnII 7 FnI bound the ␣ 2 (I) peptide much more weakly that ␣ 1 (I), K d of 3 Ϯ 0.8 mM (37°C), and showed minimal perturbations. Thus, we conclude that the 2 GBD subfragments bind strongest to collagen at a site adjacent to the MMP-1 cleavage site, albeit with preference for the ␣ 1 (I) chain by 6 FnI 1-2 FnII 7 FnI. Although both subfragments retain their affinity to small collagen peptides in the context of the GBD, there is no evidence for cooperative binding to single-stranded chains.…”

“…To explore the FN-collagen interaction, we tested by NMR a series of synthetic single-stranded peptides covering ␣ 1 (I) residues G 763 -R 816 for binding the 2 subfragments of the GBD, [8][9] FnI and 6 FnI 1-2 FnII 7 FnI. The tested region of collagen spans the MMP-1 cleavage site, G 775 /I 776 , which has been shown to influence FN-collagen interactions (6). Of the FN fragments, [8][9] FnI is known to associate with ␣ 1 (I) fragments and gelatin (16,17), and 6 FnI 1-2 FnII 7 FnI binds gelatin more strongly than the shorter 6 FnI 1-2 FnII (18).…”

“…The interaction of these 2 proteins has long been demonstrated in vitro by using denatured collagen (gelatin) (6,7) and isolated collagen type I chains or chain fragments (8,9). The collagen-binding site on FN has been localized to the 42-kDa gelatin-binding domain (GBD; for an overview of FN and collagen fragments see Fig.…”

Definition of the Native and Denatured Type II Collagen Binding Site for Fibronectin Using a Recombinant Collagen System

“…A range of biochemical studies defined a 42-kDa domain containing six modules, 6 FnI 1-2 FnII 7-9 FnI, to be the gelatin-binding domain (GBD) (11)(12)(13), due to its ability to strongly and specifically bind to denatured collagen (gelatin). Two separate fragments of the GBD, 6 FnI 1-2 FnII 7 FnI and 8 -9 FnI, have been shown to independently bind denatured collagen, but with a decreased affinity (14 -17).…”