Steffensen B, Wallon U M, Overall C M
Faculty of Dentistry, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1995 May 12;270(19):11555-66. doi: 10.1074/jbc.270.19.11555.
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.
72 kDa明胶酶/IV型胶原酶是一种在基底膜和变性胶原蛋白（明胶）降解中起重要作用的基质金属蛋白酶。这些蛋白水解过程对于病理性组织破坏和生理性组织重塑是必需的。为了研究该酶底物特异性的分子决定因素，在大肠杆菌中表达了由三个串联的纤连蛋白II型样模块组成的72 kDa明胶酶的21 kDa结构域。与全长72 kDa明胶酶和纤连蛋白中的II型模块相似，该蛋白酶的重组（r）纤连蛋白样结构域以微摩尔范围内的表观解离常数（Kd）结合变性的I型胶原。该结构域被命名为胶原结合结构域（rCBD123），具有至少两个可同时被占据的胶原结合位点。rCBD123还能强烈结合弹性蛋白以及变性的IV型和V型胶原，但不结合天然的IV型和V型胶原以及纤连蛋白，而这些都是该酶的底物。尽管72 kDa明胶酶参与基底膜降解，但rCBD123也不结合重组基底膜、层粘连蛋白或富含半胱氨酸的酸性分泌蛋白（SPARC）。不被72 kDa明胶酶降解的天然I型胶原与明胶竞争rCBD123上的一个共享结合位点。rCBD123还能取代结合在天然I型胶原上的全长72 kDa明胶酶，进一步证明重组结构域的胶原结合特性与全长酶的特性非常相似。由于rCBD123与胃蛋白酶切割的I型胶原的结合减少，I型胶原的一个或两个胶原肽末端都含有72 kDa明胶酶纤连蛋白样结构域的识别位点。这通过rCBD123与来自NH2末端肽的α1（I）胶原溴化氰片段CB2的强烈结合得到证实。rCBD123还结合α1（I）-CB7，其包含纤连蛋白结合位点，以及α1（I）-CB8，一个不被纤连蛋白结合的片段。因此，I型胶原含有多个rCBD123的结合位点，这些位点在天然胶原的三螺旋构象中部分被掩盖，而在三螺旋解折叠时完全暴露。72 kDa明胶酶的纤连蛋白样胶原结合结构域结合细胞外基质蛋白的潜力可能有助于酶在结缔组织基质中的定位。
