Department of Periodontics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA.
Matrix Biol. 2011 Jan;30(1):34-42. doi: 10.1016/j.matbio.2010.09.004. Epub 2010 Oct 13.
Fibronectin (FN) purified by gelatin affinity chromatography is unstable and undergoes fragmentation. The cleavage has been ascribed to inherent autolytic protease activities as well as co-purified matrix metalloproteinases (MMP). Understanding the mechanism by which the proteolysis of FN occurs is important, because the FN fragments have biological activities that differ from those of intact FN. Having excluded contributions of other plasma-derived proteases, the present experiments demonstrated that cleavage of FN by MMP-2 to distinct fragments occurred in synergy with inherent FN activities. Limited heat treatment of FN at 56°C for 30 min inactivated the inherent protease activities sharply reducing autolysis of FN in a manner similar to that seen in the presence of serine proteinase inhibitors. Heat treatment did not alter cell attachment to FN, but significantly increased the susceptibility of FN to enzymatic cleavage by MMP-2. The carboxyl-terminal hemopexin-like domain (PEX) of MMP-2 was shown to possess critical exodomain properties required for the interactions of MMP-2 with FN, and FN was cleaved at a significantly reduced rate by an MMP-2 variant with deletion of PEX. Verifying the specificity of interactions, isolated PEX competed FN cleavage by MMP-2 in a concentration-dependent manner. These results have further elucidated the synergistic contributions of inherent autolytic serine protease-like activities and MMP-2 to fragmentation of FN and provide the rationale and basis for modified preparation and handling of FN used in biological research.
纤维连接蛋白（FN）经明胶亲和层析纯化后不稳定，会发生片段化。这种裂解归因于固有自溶蛋白酶活性以及共纯化的基质金属蛋白酶（MMP）。了解 FN 发生蛋白水解的机制很重要，因为 FN 片段具有与完整 FN 不同的生物学活性。在排除了其他血浆来源的蛋白酶的贡献后，本实验证明 MMP-2 对 FN 的裂解作用与固有 FN 活性协同发生。在 56°C 下对 FN 进行有限的热处理 30 分钟，可显著降低固有蛋白酶活性，从而使 FN 自溶急剧减少，这种方式类似于存在丝氨酸蛋白酶抑制剂的情况。热处理不会改变细胞对 FN 的附着，但会显著增加 FN 对 MMP-2 酶切的敏感性。MMP-2 的羧基末端血红素结合蛋白样结构域（PEX）被证明具有与 FN 相互作用所需的关键外显子特性，并且 MMP-2 缺失 PEX 的变体以显著降低的速率切割 FN。验证相互作用的特异性，分离的 PEX 以浓度依赖的方式竞争 MMP-2 对 FN 的切割。这些结果进一步阐明了固有自溶丝氨酸蛋白酶样活性和 MMP-2 对 FN 片段化的协同贡献，并为用于生物研究的 FN 的改良制备和处理提供了依据和基础。
