The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation
The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation
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DOI：
10.1074/jbc.273.32.20622
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发表时间：
1998-08-07
影响因子：
4.8
通讯作者：
Overall, CM
中科院分区：
文献类型：
--
作者：
Steffensen, B;Bigg, HF;Overall, CM
重组胶原蛋白结合域（rCBD），包括三个纤连蛋白II型样模块的人明胶酶A被发现竞争的酶原形式的这种基质金属蛋白酶从细胞表面的正常人成纤维细胞培养。在伴刀豆球蛋白A处理细胞后，在rCBD存在下，明胶酶A的诱导细胞活化显著升高。因此，明胶酶A通过该结构域与细胞结合的机制方面使用细胞附着测定法进一步研究。成纤维细胞附着在rCBD包被的微孔板威尔斯孔上，其附着方式受到可溶性rCBD、针对β（1）-整联蛋白亚基而非cu-整联蛋白亚基的阻断抗体和细菌胶原酶处理的抑制。添加可溶性胶原蛋白拯救了胶原酶处理的细胞与rCBD的附着。作为辛基-β-D-硫代吡喃葡萄糖苷溶解的细胞膜提取物的配体印迹上的探针，rCBD结合140-和160-kDa的蛋白条带。它们的身份可能是既对细菌胶原酶敏感又在胃蛋白酶消化后转化为112-和126-kDa条带的前胶原链，所述条带与胶原arl（I）和α 2（I）链共迁移。具有降低的胶原亲和力的rCBD突变蛋白（Lys（263）--> Ala）显示较少的细胞附着，而肝素结合缺陷突变体（Lys（357）--> Ala）、肝素酶处理或肝素添加不改变附着。因此，明胶酶A的细胞结合机制被揭示，不涉及血红素结合蛋白COOH结构域。相反，由于涉及酶的胶原结合结构域的相互作用，形成了包含明胶酶A-天然I型胶原-β（1）-整联蛋白的附着复合物。此外，这种独特的细胞胶原结合的酶原池似乎对细胞活化是不利的。
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 beta(1)-integrin subunit but not the cu,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 arl(I) and alpha 2(I) chains. A rCBD mutant protein (Lys(263) --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys(357) --> 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-beta(1)-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.