Cocklin Simon, Jost Monika, Robertson Noreen M, Weeks Stephen D, Weber Hans-Walter, Young Emily, Seal Samar, Zhang Can, Mosser Elise, Loll Patrick J, Saunders Aleister J, Rest Richard F, Chaiken Irwin M
Department of Biochemistry and A. J. Drexel Institute of Basic and Applied Protein Science, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
J Mol Recognit. 2006 Jul-Aug;19(4):354-62. doi: 10.1002/jmr.784.
Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.
最近研究表明，炭疽芽孢杆菌能分泌一种具有强大溶血/细胞溶解活性的蛋白质，被命名为炭疽溶血素O（ALO）。在本研究中，我们着手对这种潜在的炭疽毒力因子进行研究，以了解该蛋白的膜结合特性。来自炭疽芽孢杆菌的重组炭疽溶血素O（rALO35 - 512）以及两个N端截短的ALO变体（rALO390 - 512和rALO403 - 512）在大肠杆菌中过量表达并纯化至均一。通过使用小鼠和人类巨噬细胞样细胞系以及果蝇Schneider 2细胞进行细胞胆固醇耗竭实验，探究了胆固醇在ALO细胞溶解活性中的作用。用rALO35 - 512处理巨噬细胞会导致细胞因裂解而死亡，但rALO390 - 512或rALO403 - 512则不会，这种细胞溶解作用会因膜胆固醇的耗竭而被消除。果蝇细胞以麦角固醇作为主要膜固醇，对rALO介导的细胞溶解具有抗性。为了确定这种抗性的分子机制，使用Biacore技术研究了rALO与仅由POPC组成的模型膜或与包括麦角固醇在内的多种结构相似的固醇组成的模型膜之间的相互作用。rALO35 - 512和rALO403 - 512都与由POPC和胆固醇组成的模型膜有强烈结合，结合的蛋白量与胆固醇含量成正比。麦角固醇极大地减少了rALO35 - 512和rALO403 - 512的结合，而测试的其他固醇则不支持结合。rALO403 - 512与膜的相互作用表现出低纳摩尔范围内的平衡解离常数（KD），而rALO35 - 512表现出复杂的动力学，这可能是由于孔形成过程涉及多个事件。这些结果确立了胆固醇在rALO作用中的关键作用。所开发的用于测量膜环境中ALO对胆固醇识别的生物传感器方法可扩展为一个平台，用于筛选其他膜结合蛋白和肽的抑制剂。
