载脂蛋白A-I对基质金属蛋白酶-2的抑制作用机理-《集美大学学报（自然科学版）》

文章信息/Info

Title:: Inhibition Mechanism of Apolipoprotein A-I on Matrix Metalloproteinase-2

Author(s):: WANG Yujia; LI Wanyu; CAI Yuchen; ZHANG Lingjing; WENG Ling; SUN Lechang; CAO Minjie; (College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,China)

Keywords:: matrix metalloproteinase-2; Hypophthalmichthys molitrix; apolipoprotein A-I; endogenous inhibitor; molecular docking

摘要:: 为了分离纯化鲢鱼基质金属蛋白酶-2（matrix metalloproteinases-2，MMP-2）的内源性抑制剂，克隆并表达了鲢鱼MMP-2的催化结构域（rHm-MMP-2c），并将其作为筛选内源性抑制剂的靶标酶。通过60 ℃加热、50%～90%的硫酸铵分级沉淀、DEAE-Sepharose和Phenyl-Sepharose柱层析等手段，从鲢鱼肌肉中纯化了一种MMP-2内源性抑制剂，该抑制剂相对分子质量为28 ku，能有效抑制rHmMMP-2c在4 ℃冷藏过程中对I型胶原的降解。质谱鉴定结果表明，纯化的抑制剂为载脂蛋白A-I（Apo A-I）。抑制动力学显示，Apo A-I对rHm-MMP-2c呈现竞争性抑制，抑制常数Ki为1.31 μmol/L，半抑制浓度（IC50）为3.33 μmol/L。分子对接结果显示，酶和抑制剂结合的主要作用力为氢键相互作用，抑制原因可能是Apo A-I与MMP-2纤连蛋白结合区结合形成了空间位阻，阻碍了底物与酶的结合。

Abstract:: In order to identify the endogenous inhibitor of MMP-2 in silver carp (Hypophthalmichthys molitrix),the catalytic domain of MMP-2(rHm-MMP-2c) was cloned,expressed in Escherichia coli and purified as a target enzyme to screen endogenous inhibitor.By heating at 60 ℃,50%-90% ammonium sulfate fractionation and column chromatographies of DEAE-Sepharose and Phenyl-Sepharose,an endogenous inhibitor of MMP-2 was purified from silver carp muscle.The molecular weight of the inhibitor was 28 ku,and it effectively inhibited the degradation of type I collagen by rHm-MMP-2c in the process of storage at 4 ℃.Mass spectrometry analysis showed that the purified inhibitor was apolipoprotein A-I(Apo A-I),which inhibited rHm-MMP-2c in a reversible competitive mode with half inhibitory concentration of 3.33 μmol/L and inhibition constant(Ki)of 1.31 μmol/L,respectively.Molecular docking showed that hydrogen bond was the main interaction force and Apo A-I binds to the rHm-MMP-2c fibronectin II binding domain to form steric hindrance,blocking the substrate binding to the MMP-2.