RECIPROCATED MATRIX METALLOPROTEINASE ACTIVATION - A PROCESS PERFORMED BY INTERSTITIAL COLLAGENASE AND PROGELATINASE-A
RECIPROCATED MATRIX METALLOPROTEINASE ACTIVATION - A PROCESS PERFORMED BY INTERSTITIAL COLLAGENASE AND PROGELATINASE-A
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DOI：
10.1021/bi00252a007
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发表时间：
1994-12-06
期刊：
影响因子：
2.9
通讯作者：
DOCHERTY, AJP
中科院分区：
文献类型：
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作者：
CRABBE, T;OCONNELL, JP;DOCHERTY, AJP
明胶酶 A 是基质金属蛋白酶 (MMP) 家族的成员，其分泌物具有 80 个氨基酸的 N 端前肽，必须除去该前肽才能产生活性酶。在 37°C 下与等摩尔浓度的胰蛋白酶激活的间质胶原酶（另一种 MMP）一起孵育 6 小时，纯化的原明胶酶 A 被激活至最大值的 38%。活性的增加伴随着 M(r) 72 000 原明胶酶 A 裂解为以 Y-81 作为 N 末端的 M(r) 66 000 活性酶。在低浓度下，原明胶酶 A 通过非活性中间体进行加工，表明其激活是一个双相过程。胶原酶对 proE(375-->)A（原明胶酶 A 的一种突变体，不能变得活跃）的作用证实了这一点，因为在这种情况下，仅产生了以 L(38) 作为 N 末端的 M(r) 68 000 种。 Y-81 的剩余前肽氨基酸可以通过添加活性明胶酶 A 轻松去除，这表明胶原酶通过生成易于自溶激活的中间体来发挥作用。虽然相对较慢，但通过添加 100 μg/mL 肝素，激活速率可提高约 10 倍。它与胶原酶和原明胶酶 A 的 C 末端结构域结合，并且可能充当将反应物彼此靠近定位的模板。胰蛋白酶激活的胶原酶保留其前肽的 8 或 14 个氨基酸。活化的明胶酶 A 能够通过裂解 Q(80)-F-81 肽键来去除这些肽键，这一事件已被证明可以显着增加胶原酶针对纤维状胶原的活性 [Suzuki, K.、Enghild, J. J.、Morodomi, T.、Salvesen, G. 和 Nagase, H. (1990) Biochemistry 29, 10261-10270]。天然胶原蛋白的完全降解需要胶原酶和明胶酶的活性，这一事实为这种往复激活机制提供了功能基础。
Gelatinase A, a member of the matrix metalloproteinase (MMP) family, is secreted possessing an 80 amino acid N-terminal propeptide that must be removed in order to generate the active enzyme. Purified progelatinase A was activated to 38% of maximum by a 6 h incubation at 37 degrees C with equimolar concentrations of trypsin-activated interstitial collagenase (another MMP). The increase in activity was accompanied by cleavage of the M(r) 72 000 progelatinase A to the M(r) 66 000 active enzyme that has Y-81 as its N-terminus. At low concentrations, progelatinase A was processed via an inactive intermediate, suggesting that its activation is a biphasic process. This was confirmed by the action of collagenase on proE(375-->)A (a mutant of progelatinase A that cannot become active) because, in this instance, only an M(r) 68 000 species with L(38) as the N-terminus was produced. The remaining propeptide amino acids to Y-81 could be readily removed by added active gelatinase A, indicating that collagenase works by generating an intermediate that is susceptible to autolytic activation. Although relatively slow, the rate of activation could be increased approximately 10-fold by the addition of 100 mu g/mL heparin. This binds to the C-terminal domain of collagenase and progelatinase A and presumably acts as a template that positions the reactants close to one another. Collagenase activated by trypsin retains 8 or 14 amino acids of its propeptide. The activated gelatinase A was able to remove these by cleaving the Q(80)-F-81 peptide bond, an event that has been shown to significantly increase the activity of collagenase against fibrillar collagen [Suzuki, K., Enghild, J. J., Morodomi, T., Salvesen, G., and Nagase, H. (1990) Biochemistry 29, 10261-10270]. The fact that the complete degradation of native collagen requires the activities of both a collagenase and a gelatinase provides a functional basis for this reciprocated mechanism of activation.