Liu Shihui, Bugge Thomas H, Frankel Arthur E, Leppla Stephen H
Laboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2009;539:175-90. doi: 10.1007/978-1-60327-003-8_10.
Anthrax toxin is a three-part toxin secreted by Bacillus anthracis, consisting of protective antigen (PrAg), edema factor (EF), and lethal factor (LF). To intoxicate host mammalian cells, PrAg, the cell-binding moiety of the toxin, binds to cells and is then proteolytically activated by furin on the cell surface, resulting in the active heptameric form of PrAg. This heptamer serves as a protein-conducting channel that translocates EF and LF, the two enzymatic moieties of the toxin, into the cytosol of the cells where they exert cytotoxic effects. The anthrax toxin delivery system has been well characterized. The amino-terminal PrAg-binding domain of LF (residues 1-254, LFn) is sufficient to allow translocation of fused "passenger" polypeptides, such as the ADP-ribosylation domain of Pseudomonas exotoxin A, to the cytosol of the cells in a PrAg-dependent process. The protease specificity of the anthrax toxin delivery system can also be reengineered by replacing the furin cleavage target sequence of PrAg with other protease substrate sequences. PrAg-U2 is such a PrAg variant, one that is selectively activated by urokinase plasminogen activator (uPA). The uPA-dependent proteolytic activation of PrAg-U2 on the cell surface is readily detected by western blotting analysis of cell lysates in vitro, or cell or animal death in vivo. Here, we describe the use of PrAg-U2 as a molecular reporter tool to test the controversial question of what components are required for uPAR-mediated cell surface pro-uPA activation. The results demonstrate that both uPAR and plasminogen play critical roles in pro-uPA activation both in vitro and in vivo.
炭疽毒素是由炭疽芽孢杆菌分泌的一种三部分组成的毒素，由保护性抗原（PrAg）、水肿因子（EF）和致死因子（LF）组成。为了使宿主哺乳动物细胞中毒，毒素的细胞结合部分PrAg与细胞结合，然后在细胞表面被弗林蛋白酶进行蛋白水解激活，产生活性七聚体形式的PrAg。这种七聚体作为一个蛋白质传导通道，将毒素的两个酶部分EF和LF转运到细胞胞质溶胶中，在那里它们发挥细胞毒性作用。炭疽毒素递送系统已得到充分表征。LF的氨基末端PrAg结合结构域（第1 - 254位氨基酸，LFn）足以使融合的“乘客”多肽（如铜绿假单胞菌外毒素A的ADP核糖基化结构域）在依赖PrAg的过程中转位到细胞胞质溶胶中。通过将PrAg的弗林蛋白酶切割靶序列替换为其他蛋白酶底物序列，也可以对炭疽毒素递送系统的蛋白酶特异性进行重新设计。PrAg-U2就是这样一种PrAg变体，它被尿激酶型纤溶酶原激活剂（uPA）选择性激活。通过对体外细胞裂解物进行蛋白质印迹分析，或对体内细胞或动物死亡情况进行检测，很容易检测到PrAg-U2在细胞表面的uPA依赖性蛋白水解激活。在这里，我们描述了使用PrAg-U2作为分子报告工具来测试关于uPAR介导的细胞表面pro-uPA激活需要哪些成分这一有争议问题。结果表明，uPAR和纤溶酶原在体外和体内的pro-uPA激活中都起着关键作用。
