Bone and Joint Center, Henry Ford Hospital, Detroit, Michigan, USA.
Connect Tissue Res. 2010 Jun;51(3):230-9. doi: 10.3109/03008200903288902.
Matrix metalloproteinases (MMP)-13 activity is necessary for normal skeletal development and plays a central role in cartilage degeneration associated with osteoarthritis (OA). The studies we described here examine the interactions of the hemopexin domain of MMP-13 with proteins secreted by human chondrocytes in culture. The hemopexin domain of the MMPs and many other proteins in which this structure is found mediates protein function by forming the primary site of interaction with other proteins. We have modified a tandem affinity expression tag (hTAP) to enable efficient expression of the tagged bait protein. In this case the MMP-13 C-terminal domain (CTD) comprises hinge and hemopexin domain, and we immobilized the fusion construct on a column of agarose bound immunoglobin G. The MMP-13 CTD affinity column so generated enabled the efficient and gentle isolation of interacting proteins from the culture medium of human articular chondrocytes. TIMP1 and alpha2-macroglobulin previously shown to interact with MMP-13 as well as several proteins, fibronectin, type VI collagen and xylosyltransferase 1 and several proteoglycans, decorin, syndecan 4 and serglycin not previously recognized as interacting with MMP-13 were identified by mass spectrometry. The interaction between isolated proteins and MMP-13 CTD was verified by yeast two hybrid analysis. We also demonstrated serglycin expression by chondrocytes for the first time and its co localization with MMP-13 in a cytoplasmic granular morphology. The consequence of these interactions remains to be demonstrated, however; binding to MMP-13 suggests a role in the regulation of cartilage degradation.
基质金属蛋白酶（MMP）-13 的活性对于正常骨骼发育是必要的，并且在与骨关节炎（OA）相关的软骨退化中发挥核心作用。我们在这里描述的研究检查了 MMP-13 的血红素结合蛋白结构域与培养的人软骨细胞分泌的蛋白质之间的相互作用。MMP 和许多其他具有这种结构的蛋白质中的血红素结合蛋白结构域通过形成与其他蛋白质相互作用的主要位点来调节蛋白质功能。我们已经修改了串联亲和表达标签（hTAP），以实现标记诱饵蛋白的有效表达。在这种情况下，MMP-13 C 端结构域（CTD）包括铰链和血红素结合蛋白结构域，我们将融合构建体固定在琼脂糖结合免疫球蛋白 G 的柱上。如此产生的 MMP-13 CTD 亲和柱能够从人关节软骨细胞的培养基中有效且温和地分离相互作用的蛋白质。TIMP1 和 alpha2-巨球蛋白先前已被证明与 MMP-13 相互作用，以及几种蛋白质，纤连蛋白，VI 型胶原和木糖基转移酶 1 和几种蛋白聚糖，decorin， syndecan 4 和 serglycin 不被认为与 MMP-13 相互作用通过质谱法鉴定。通过酵母双杂交分析验证了分离蛋白与 MMP-13 CTD 之间的相互作用。我们还首次证明了软骨细胞中 serglycin 的表达及其与 MMP-13 的共定位，呈细胞质颗粒形态。然而，这些相互作用的后果仍有待证明; 与 MMP-13 的结合表明在调节软骨降解中的作用。
