Abstract
72-kDa gelatinase/type IV collagenase is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens (gelatin). These proteolytic processes are required for pathologic tissue destruction and physiologic tissue remodeling. To investigate the molecular determinants of substrate specificity of this enzyme, a 21-kDa domain of 72-kDa gelatinase, consisting of three tandem fibronectin type II-like modules, was expressed in Escherichia coli. Similar to full-length 72-kDa gelatinase and the type II modules in fibronectin, the recombinant (r) fibronectin-like domain of this proteinase bound denatured type I collagen with an apparent Kd in the micromolar range. This domain, designated the collagen-binding domain (rCBD123), possesses at least two collagen-binding sites that can each be simultaneously occupied. rCBD123 also avidly bound elastin and denatured types IV and V collagens, but neither native types IV and V collagens nor fibronectin, all of which are substrates of the enzyme. Although 72-kDa gelatinase is involved in basement membrane degradation, rCBD123 also did not bind reconstituted basement membrane, laminin, or SPARC. Native type I collagen, which is not degraded by 72-kDa gelatinase, competed with gelatin for a shared binding site on rCBD123. rCBD123 also displaced full-length 72-kDa gelatinase bound to native type I collagen, further demonstrating that the collagen binding properties of the recombinant domain closely mimicked those of the full-length enzyme. Since rCBD123 showed reduced binding to pepsin-cleaved type I collagen, either or both of the collagen telopeptide ends contain recognition sites for the 72-kDa gelatinase fibronectin-like domain. This was confirmed by the avid binding of rCBD123 to the alpha 1(I) collagen cyanogen bromide fragment CB2 from the NH2-terminal telopeptide. rCBD123 also bound alpha 1(I)-CB7, which encompasses the fibronectin-binding site, and to alpha 1(I)-CB8, a fragment not bound by fibronectin. Thus, type I collagen contains multiple binding sites for rCBD123 which are partially masked by the triple helical conformation of native collagen and fully exposed upon unfolding of the triple helix. The potential of the fibronectin-like collagen binding domain of 72-kDa gelatinase to bind extracellular matrix proteins may facilitate enzyme localization in connective tissue matrices.
Highlights
72-kDa gelatinase/type IV collagenase is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens
Native type I collagen, which is not degraded by 72-kDa gelatinase, competed with gelatin for a shared binding site on rCBD123. rCBDl23 displaced full-length 72-kDa gelatinase bound to native type I collagen, further demonstrating that the collagen binding properties of the recombinant domain closely mimicked those of the full-length enzyme
This was confirmed by the avid binding of rCBDl23 to the al(l) collagen cyanogen bromide fragment CB2 from the NH2-terminal telopeptide. rCBDl23 bound al(l)-CB7, which encompasses the fibronectinbinding site, and to al(l)-CBS, a fragment not bound by fibronectin
Summary
Extracellular Matrix Proteins, Antibodies, and Chromatography Me· dia-Acid-soluble native type I collagen was prepared from rat tail tendons as described by Piez [36] by extraction with 0.5 M acetic acid and differential precipitation with 1.7 M NaC!. RCBD123 was incubated for 90 min at 20°C with the competing ligand at various mole ratios between 1:0 to 1:9 before loading the reaction mixture onto either gelatin-Sepharose or native type I collagen affinity columns and eluting as described. To ascribe the type I collagen binding properties of the parental 72-kDa gelatinase to a specific domain, the enzyme was bound to native type I collagen columns and, after 1 MNaCI washes, the binding competed with rCBD123 (40 fLg, 2 nmol) in chromatography buffer. RCBD123 was serially diluted in 50 mM Tris, pH 7.4, from 10,000 to 0.6 nM and incubated with 4,000 disintegrations/min native or heat-denatured [14C]glycine-Iabeled type I collagen (0.03 and 0.1 pmol, respectively) for 1 h at 20°C. Binding reactions were performed in duplicate for eight serial dilutions with appropriate controls for both native and denatured type I collagens
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