AUTOLYTIC ACTIVATION OF RECOMBINANT HUMAN 72-KILODALTON TYPE-IV COLLAGENASE
AUTOLYTIC ACTIVATION OF RECOMBINANT HUMAN 72-KILODALTON TYPE-IV COLLAGENASE
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DOI：
10.1021/bi00009a011
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发表时间：
1995-03-07
期刊：
影响因子：
2.9
通讯作者：
TRYGGVASON, K
中科院分区：
文献类型：
--
作者：
BERGMANN, U;TUUTTILA, A;TRYGGVASON, K
人72 kDa IV型胶原酶（明胶酶A， MMP-2）在杆状病毒/昆虫细胞系统中表达。该酶以野生型和两种突变型产生，其中活性位点Glu（375）被Asp或Gin取代。突变蛋白的活性分别明显降低或无法检测到，从而可以对快速自激活反应进行详细分析。MMP-2很容易降解为缺乏前四个氨基酸残基的前酶形式。这种切割被证明是一个自溶过程，尽管酶活性显然不受这种截断的影响。在37℃下，在没有外部活化剂的情况下，以浓度依赖的方式实现了向活性酶形式的转化。MMP-2的激活被证明是一个循序渐进的过程，可能通过作为高度不稳定中间体的δ(1-50)形式。c端血红素样结构域在两个裂解位点被较早地去除，并且在锌结合位点内的降解使酶失活。纤连蛋白和血红素样结构域是稳定的，尽管自降解模式没有显示出任何序列特异性，除了P-1'位置的带电残基。结果表明，特定的激活剂可能不是MMP-2所必需的。
Human 72 kDa type IV collagenase (gelatinase A, MMP-2) was expressed in a baculovirus/ insect cell system. The enzyme was produced in the wild-type form and in two mutant forms, where the active site Glu(375) was substituted by Asp or Gin. The mutated proteins had strongly reduced or no detectable activity, respectively, allowing detailed analysis of rapid autoactivation reactions. MMP-2 was readily degraded to a proenzyme form lacking the first four amino acid residues. This cleavage was shown to be an autolytic process, although enzyme activity was apparently not affected by this truncation. Conversion to the active enzyme form was achieved without external activator in a concentration-dependent manner at 37 degrees C. The activation of MMP-2,was shown to be a stepwise process, probably via a Delta(1-50) form as a highly unstable intermediate. The C-terminal hemopexin-like domain is removed rather early at two cleavage sites, and degradation within the Zn-binding site inactivates the enzyme. The fibronectin- and hemopexin-like domains are stable, although the autodegradation pattern did not show any sequence specificity, except for charged residues in the P-1' position. The results indicate that a specific activator may not be essential for MMP-2.