Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.
Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.
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DOI：
10.1016/s0021-9258(19)49547-1
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发表时间：
1992-08
期刊：
影响因子：
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通讯作者：
Rafael Fridman;Thomas R. Fuerst;Robert E. Bird;Matti Hoyhtya;M. Oelkuct;Sue Kraus;D. Komarek
中科院分区：
文献类型：
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作者：
Rafael Fridman;Thomas R. Fuerst;Robert E. Bird;Matti Hoyhtya;M. Oelkuct;Sue Kraus;D. Komarek
72-kDa明胶酶/IV型胶原酶，一种被认为在转移和血管生成中起作用的金属蛋白酶，与金属蛋白酶组织抑制剂-2（TIMP-2）（一种有效的酶活性抑制剂）形成非共价化学计量复合物。为了确定负责TIMP-2结合的72-kDa明胶酶的区域，使用聚合酶链反应技术构建了一系列酶的NH 2-和COOH-末端缺失。在重组牛痘病毒哺乳动物细胞表达系统（Vac/T7）中表达全长和截短的酶。纯化终止于残基425（Δ 426-631）和454（Δ 455-631）的两种截短的酶。像全长重组72 kDa明胶酶，这两个COOH-末端截短的酶被激活与有机汞和消化明胶和天然胶原IV型。与全长酶相比，Δ 426-631和Δ 455-631酶对TIMP-2抑制的敏感性较低，需要10 mol TIMP-2/mol酶来实现酶活性的最大抑制。只有当使用过量摩尔浓度的抑制剂时，激活的而不是潜伏形式的Δ 426-631和Δ 455-631蛋白才与TIMP-2形成复合物。我们还表达了205个氨基酸的COOH末端片段，delta 1-426，并发现它结合TIMP-2。此外，缺少酶原的NH 2-末端78个氨基酸（δ 1-78）的72-kDa明胶酶的截短形式保留了结合TIMP-2的能力。这些研究表明，72 kDa明胶酶缺乏COOH-末端结构域保留完整的酶活性，但获得降低的敏感性TIMP-2抑制。这些数据表明，无论是活性位点和COOH-末端尾巴的72 kDa明胶酶独立和合作参与TIMP-2的结合。
The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.