EXTRACELLULAR-MATRIX BINDING-PROPERTIES OF RECOMBINANT FIBRONECTIN TYPE II-LIKE MODULES OF HUMAN 72-KDA GELATINASE TYPE-IV COLLAGENASE - HIGH-AFFINITY BINDING TO NATIVE TYPE-I COLLAGEN BUT NOT NATIVE TYPE-IV COLLAGEN

72-kDa gelatinase/type IV collagenase is an important matrix metallopr oteinase in the degradation of basement membranes and denatured collag ens (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 fibrone ctin type II-Like modules, was expressed in Escherichia coli, Similar to full-length 72-kDa gelatinase and the type II modules in fibronecti n, the recombinant (r) fibronectin-like domain of this proteinase boun d denatured type I collagen with an apparent K-d in the micromolar ran ge, This domain, designated the collagen-binding domain (rCBD123), pos sesses at least two collagen binding sites that can each be simultaneo usly occupied, rCBD123 also avidly bound elastin and denatured types I V and V collagens, but neither native types IV and V collagens nor fib ronectin, all of which are substrates of the enzyme, Although 72-kDa g elatinase is involved in basement membrane degradation, rCBD123 also d id 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 displ aced full-length 72-kDa gelatinase bound to native type I collagen, fu rther demonstrating that the collagen binding properties of the recomb inant domain closely mimicked those of the full-length enzyme, Since r CBD123 showed reduced binding to pepsin cleaved type I collagen, eithe r or both of the collagen telopeptide ends contain recognition sites f or the 72-kDa gelatinase fibronectin-like domain, This was confirmed b y the avid binding of rCBD123 to the alpha 1(I) collagen cyanogen brom ide fragment CB2 from the NH2-terminal telopeptide. rCBD123 also bound alpha 1(I)-CB7, which encompasses the fibronectin-binding site, and t o alpha 1(I)-CB8, a fragment not bound by fibronectin, Thus, type I co llagen contains multiple binding sites for rCBD123 which are partially masked by the triple helical conformation of native collagen and full y exposed upon unfolding of the triple helix, The potential of the fib ronectin-like collagen binding domain of 72-kDa gelatinase to bind ext racellular matrix proteins may facilitate enzyme localization in conne ctive tissue matrices.