Shimokawa K, Jia L G, Wang X M, Fox J W
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Arch Biochem Biophys. 1996 Nov 15;335(2):283-94. doi: 10.1006/abbi.1996.0509.
The expression in human embryonic kidney (HEK 293) cells of the recombinant zymogen form (pro-) of the Crotalus atrox hemorrhagic metalloproteinase, atrolysin E, is presented. The nascent protein is comprised of pre-, pro-, proteinase-, spacer-, and disintegrin domains. The biochemical characterization of the recombinant zymogen is described along with its activation by C. atrox crude venom and other hemorrhagic toxins. Unlike the zymogen forms of the matrix metalloproteinases, pro-atrolysin E is not activated by the organomercurial, (4-aminophenyl)mercuric acetate. Pro-atrolysin E could be enzymatically activated by C. atrox crude venom, PMSF-inhibited crude venom, atrolysin A, and atrolysin E itself. There is no evidence of autoactivation. Using two polyclonal antibodies directed against the proteinase domain and the disintegrin domain of atrolysin E, the proteolytic processing of the recombinant protein by atrolysin A was followed. The first cleavage of pro-atrolysin E by atrolysin A removes the pro-domain. The second proteolysis step removes the disintegrin domain to produce the proteinase/spacer protein. These studies have identified potential activators of snake venom pro-metalloproteinases in crude venom and suggest a general scheme for the activation and processing of venom pro-metalloproteinases by the endogenous, active metalloproteinases.
本文展示了重组的响尾蛇（Crotalus atrox）出血性金属蛋白酶阿特罗溶素E（atrolysin E）的酶原形式（pro-）在人胚肾（HEK 293）细胞中的表达。新生蛋白由前肽、酶原、蛋白酶、间隔区和去整合素结构域组成。文中描述了重组酶原的生化特性及其被响尾蛇粗毒和其他出血毒素激活的情况。与基质金属蛋白酶的酶原形式不同，前阿特罗溶素E不会被有机汞化合物醋酸（4-氨基苯基）汞激活。前阿特罗溶素E可被响尾蛇粗毒、苯甲基磺酰氟抑制的粗毒、阿特罗溶素A和阿特罗溶素E自身酶促激活。没有自动激活的证据。使用两种针对阿特罗溶素E蛋白酶结构域和去整合素结构域的多克隆抗体，追踪了阿特罗溶素A对重组蛋白的蛋白水解过程。阿特罗溶素A对前阿特罗溶素E的首次切割去除了前结构域。第二步蛋白水解去除了去整合素结构域，产生蛋白酶/间隔区蛋白。这些研究确定了粗毒中蛇毒金属蛋白酶原的潜在激活剂，并提出了内源性活性金属蛋白酶激活和加工蛇毒金属蛋白酶原的一般方案。
