Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3
Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3
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DOI：
10.1359/jbmr.1998.13.12.1890
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发表时间：
1998-12-01
影响因子：
6.2
通讯作者：
Gorski, JP
中科院分区：
文献类型：
--
作者：
Kremer, EA;Chen, Y;Gorski, JP
在鉴定能够产生长寿命的 50 kDa 骨酸性糖蛋白 75 (BAG-75) 片段的蛋白酶的研究过程中，观察到将基质金属蛋白酶 (MMP)-3 (基质溶素 I) 与 BAG-75 制剂一起温育会导致蛋白水解功能失活，例如，无法片段化随后添加的 I-125 标记的 BAG-75， MMP-1（间质胶原酶）也因接触 BAG-75 制剂而失活。对其机制的研究表明，BAG-75 制剂含有毫摩尔水平的无机磷酸盐，在消化条件下会形成羟基磷灰石晶体。单独的羟基磷灰石晶体以及 BAG-75-羟基磷灰石复合物中的羟基磷灰石晶体诱导 MMP-1 和 MMP-3 的活性形式和前体形式的自溶降解。羟基磷灰石存在下的自溶降解通过用肽和/或蛋白质底物测定的催化功能损失来证明，并且通过片段化成 < 10 kDa 的多肽，与羟基磷灰石一起孵育的 MMPS 的命运取决于孵育时间、游离钙浓度和晶体浓度，具体而言，羟基磷灰石诱导的自溶需要 0.5-1.0 mM 的接近生理学的游离钙浓度，自溶在存在 150 μg/ml 羟基磷灰石，其中 MMP-3 仅部分与晶体结合。然而，在较高晶体浓度下，所有输入的 MMP-3 均结合（>1000 μg/ml）也发生自溶，表明自溶可能是由结合酶介导的。羟基磷灰石的作用似乎对 MMP-1 和 MMP-3 具有特异性，因为胰凝乳蛋白酶、胰蛋白酶、木瓜蛋白酶和嗜热菌蛋白酶的催化活性在接触羟基磷灰石后保持不变。这些结果首次证明了羟基磷灰石晶体在体外的新型催化作用，并提供了分子间、自溶、钙离子依赖性、基质金属蛋白酶特异性降解机制的初步生化特征。
In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin I) with preparations of BAG-75 led to inactivation of proteolytic function, e,g,, an inability to fragment I-125-labeled BAG-75 added subsequently, MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations, Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of < 10 kDa, The fate of MMPS incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals, Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM, Autolysis was maximal in the presence of 150 mu g/ml hydroxyapatite where MMP-3 was only partially bound to crystals, However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 mu g/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.