College of Biological Science and Agriculture, Qiannan Normal University for Nationalities, Duyun, Guizhou, 558000, China.
Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Science, South China Normal University, Guangzhou, Guangdong, 510631, China.
Protein Expr Purif. 2020 Jul;171:105629. doi: 10.1016/j.pep.2020.105629. Epub 2020 Mar 19.
Matrix metalloproteinases (MMPs) are evolutionarily conserved extracellular matrix proteinases. Genetic analysis of the Drosophila MMPs, Mmp1 and Mmp2, in vivo reveal that they play vital roles in tissue remodeling. Although the catalytic domain (CD) undertakes most MMP functions, few studies have sought to demonstrate the biochemical properties of the CDs of fly MMPs. Here, we identified the overexpression, purification, and refolding of the CDs of Drosophila Mmp1 and Mmp2 for biochemical studies. Zymography assays and substrate degradation analysis showed that both Mmp1-CD and Mmp2-CD were able to digest casein, gelatin, fibronectin, collagen (types I, IV, and V), while Mmp2-CD showed much higher degradation activity compared with Mmp1-CD. Moreover, human collagen III could be degraded by Mmp1-CD but not Mmp2-CD, and rat collagen I and laminin could be degraded by Mmp2-CD but not Mmp1-CD, suggesting that Drosophila Mmp1 and Mmp2 might have overlapping yet distinct substrate specificity. Using synthetic fluorescent substrates, we further demonstrated that the enzymatic activity of Mmp1-CD and Mmp2-CD could be inhibited by human tissue inhibitors of metalloproteinases (TIMPs). These results reveal the context of the cooperative yet distinct roles of Mmp1 and Mmp2 in tissue remodeling.
基质金属蛋白酶（MMPs）是进化上保守的细胞外基质蛋白酶。对果蝇 MMPs（Mmp1 和 Mmp2）的体内遗传分析表明，它们在组织重塑中发挥着至关重要的作用。尽管催化结构域（CD）承担了大多数 MMP 功能，但很少有研究试图证明果蝇 MMPs 的 CDs 的生化特性。在这里，我们鉴定了 Drosophila Mmp1 和 Mmp2 的 CDs 的过表达、纯化和重折叠，用于生化研究。酶谱分析和底物降解分析表明，Mmp1-CD 和 Mmp2-CD 都能够消化酪蛋白、明胶、纤维连接蛋白、胶原蛋白（I、IV 和 V 型），而 Mmp2-CD 与 Mmp1-CD 相比表现出更高的降解活性。此外，人胶原蛋白 III 可被 Mmp1-CD 降解，但不能被 Mmp2-CD 降解，而大鼠胶原蛋白 I 和层粘连蛋白可被 Mmp2-CD 降解，但不能被 Mmp1-CD 降解，提示果蝇 Mmp1 和 Mmp2 可能具有重叠但又不同的底物特异性。使用合成荧光底物，我们进一步证明 Mmp1-CD 和 Mmp2-CD 的酶活性可被人基质金属蛋白酶抑制剂（TIMPs）抑制。这些结果揭示了 Mmp1 和 Mmp2 在组织重塑中协同但又不同的作用的背景。
