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 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 determ...
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Veröffentlicht in: | The Journal of biological chemistry 1995-05, Vol.270 (19), p.11555-11566 |
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Zusammenfassung: | 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.270.19.11555 |