STEPWISE ACTIVATION MECHANISMS OF THE PRECURSOR OF MATRIX METALLOPROTEINASE-3 (STROMELYSIN) BY PROTEINASES AND (4-AMINOPHENYL)MERCURIC ACETATE
STEPWISE ACTIVATION MECHANISMS OF THE PRECURSOR OF MATRIX METALLOPROTEINASE-3 (STROMELYSIN) BY PROTEINASES AND (4-AMINOPHENYL)MERCURIC ACETATE
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DOI：
10.1021/bi00476a020
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发表时间：
1990-06-19
期刊：
影响因子：
2.9
通讯作者：
SALVESEN, G
中科院分区：
文献类型：
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作者：
NAGASE, H;ENGHILD, JJ;SALVESEN, G
用动力学和序列分析的方法研究了人基质金属蛋白酶3前体(proMMP3/prostromellysin)被蛋白酶和(4-氨基苯基)醋酸汞(APMA)激活的机制。中性粒细胞弹性蛋白酶、血浆激肽释放酶、纤溶酶或胰凝乳酶在37℃孵育原基质金属蛋白酶-3。C导致His82-Phe83键断裂，形成MR=45000的基质金属蛋白酶-3。由于这一键不太可能被这些蛋白酶切割，因此推测激活的蛋白酶对原基质金属蛋白酶-3的初始攻击会产生一种中间形式，然后被加工成更稳定的形式MR=45000。为了验证这一假设，我们将proMMP3与这些丝氨酸蛋白酶在最小化MMP3作用的条件下孵育。这导致了MR=53000的主要中间体和MR=49000和47000的两种次要形式的积累。人中性粒细胞弹性蛋白酶产生的53000个MR中间体是由Val35-Arg36键裂解而来的，而血浆激肽释放酶裂解Arg36-Arg37和Lys38-Asp39键以及胰凝乳素产生的Phe34-Val35键都位于前肽的中间附近。这些中间体转化为完全活性的45000 mR形式的基质金属蛋白酶-3的结果是中间体的双分子反应。APMA产生的类似的短寿命中间体MR=46000是Glu68-Val69键分子内断裂的结果，然后通过MMP3的分子间反应将其转化为稳定的MR=45000的MMP3。但基质金属蛋白酶-3不能激活基质金属蛋白酶原-3。这些结果表明，蛋白水解酶去除NH2末端34-38个残基或APMA去除68个残基是激活原基质金属蛋白酶-3的关键步骤。对前肽的这种初始处理允许在天然前基质金属蛋白酶-3中被蛋白质分解阻碍的组氨酸82-Phe83键被激活的中间体正确定位以进行切割，从而产生稳定的45000 MR基质金属蛋白酶-3。这种逐步激活的过程使原基质金属蛋白酶-3可以被具有不同底物特异性的各种蛋白酶激活。
The mechanisms of activation of the precursor of human matrix metalloproteinase 3 (proMMP-3/prostromelysin) by proteinases and (4-aminophenyl)mercuric acetate (APMA) were investigated by kinetic and sequence analyses. Incubation of proMMP-3 with neutrophil elastase, plasma kallikrein, plasmin, or chymotrypsin at 37.degree. C resulted in the formation of MMP-3 of Mr = 45000 by cleaving of the His82-Phe83 bond. Since this bond is unlikely to be cleaved by these proteinases it was postulated that an initial attack of an activator proteinase on proMMP-3 creates an intermediate form, which is then processed to a more stable form of Mr = 45000. To test this hypothesis proMMP-3 was incubated with these serine proteinases under conditions that minimize the action of MMP-3. This led to the accumulation of major intermediates of Mr = 53000 and two minor forms of Mr = 49000 and 47000. The 53000 Mr intermediate generated by human neutrophil elastase resulted from cleavage of the Val35-Arg36 bond, whereas plasma kallikrein cleaved the Arg36-Arg37 and Lys38-Asp39 bonds and chymotrypsin the Phe34-Val35 bond, all of which are located near the middle of the propeptide. Conversion of these intermediates to the fully active 45000 Mr form of MMP-3 resulted from a bimolecular reaction of the intermediates. A similar short-lived intermediate of Mr = 46000 generated by APMA was a result of the intramolecular cleavage of the Glu68-Val69 bond, and it was then converted to a stable MMP-3 of Mr = 45000 by a intermolecular reaction of MMP-3. However, MMP-3 failed to active proMMP-3. These results indicate the removal of the NH2-terminal 34-38 residues by proteinases or 68 residues by APMA is the crucial step for activation of proMMP-3. This initial processing of the propeptide allows the His82-Phe83 bond, which is hindered from proteolysis in native proMMP-3, to be correctly oriented for cleavage by activated intermediates, thereby producing stable 45000 Mr MMP-3. This stepwise activation process allows proMMP-3 to be activated by various proteinases with distinct substrate specificities.