Steffensen B, Bigg H F, Overall C M
Faculty of Dentistry and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 1998 Aug 7;273(32):20622-8. doi: 10.1074/jbc.273.32.20622.
Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.
包含人明胶酶A的三个类纤连蛋白II型模块的重组胶原结合域（rCBD）被发现可在培养的正常人成纤维细胞表面与这种基质金属蛋白酶的酶原形式竞争。在用伴刀豆球蛋白A处理细胞后，在rCBD存在的情况下，明胶酶A的诱导细胞活化显著升高。因此，使用细胞附着试验进一步研究了该结构域与细胞结合的明胶酶A的机制方面。成纤维细胞以一种被可溶性rCBD、β1整合素亚基的阻断抗体（但不是α2整合素亚基的阻断抗体）以及细菌胶原酶处理所抑制的方式附着于包被有rCBD的微孔板孔。添加可溶性胶原可挽救胶原酶处理的细胞与rCBD的附着。作为对辛基-β-D-硫代葡萄糖苷溶解的细胞膜提取物的配体印迹的探针，rCBD结合了140 kDa和160 kDa的蛋白条带。它们的身份可能是前胶原链，对细菌胶原酶敏感，并且在胃蛋白酶消化后转化为与胶原α1(I)和α2(I)链共迁移的112 kDa和126 kDa条带。具有降低的胶原亲和力的rCBD突变蛋白（Lys263→Ala）显示出较少的细胞附着，而肝素结合缺陷突变体（Lys357→Ala）、肝素酶处理或添加肝素并未改变附着。因此，揭示了一种明胶酶A的细胞结合机制，该机制不涉及血红素结合蛋白COOH结构域。相反，由于涉及该酶的胶原结合结构域的相互作用，形成了一种包含明胶酶A-天然I型胶原-β1整合素的附着复合物。此外，这种独特的细胞结合的胶原结合酶原池似乎对细胞活化具有抗性。
