Wu Pei-Tzu, Lin Su-Chang, Hsu Chyong-Ing, Liaw Yen-Chywan, Lin Jung-Yaw
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
FEBS J. 2006 Jul;273(14):3160-71. doi: 10.1111/j.1742-4658.2006.05325.x. Epub 2006 Jun 19.
Volvatoxin A2, a pore-forming cardiotoxic protein, was isolated from the edible mushroom Volvariella volvacea. Previous studies have demonstrated that volvatoxin A consists of volvatoxin A2 and volvatoxin A1, and the hemolytic activity of volvatoxin A2 is completely abolished by volvatoxin A1 at a volvatoxin A2/volvatoxin A1 molar ratio of 2. In this study, we investigated the molecular mechanism by which volvatoxin A1 inhibits the cytotoxicity of volvatoxin A2. Volvatoxin A1 by itself was found to be nontoxic, and furthermore, it inhibited the hemolytic and cytotoxic activities of volvatoxin A2 at molar ratios of 2 or lower. Interestingly, volvatoxin A1 contains 393 amino acid residues that closely resemble a tandem repeat of volvatoxin A2. Volvatoxin A1 contains two pairs of amphipathic alpha-helices but it lacks a heparin-binding site. This suggests that volvatoxin A1 may interact with volvatoxin A2 but not with the cell membrane. By using confocal microscopy, it was demonstrated that volvatoxin A1 could not bind to the cell membrane; however, volvatoxin A1 could inhibit binding of volvatoxin A2 to the cell membrane at a molar ratio of 2. Via peptide competition assay and in conjunction with pull-down and co-pull-down experiments, we demonstrated that volvatoxin A1 and volvatoxin A2 may form a complex. Our results suggest that this occurs via the interaction of one molecule of volvatoxin A1, which contains two amphipathic alpha-helices, with two molecules of volvatoxin A2, each of which contains one amphipathic alpha-helix. Taken together, the results of this study reveal a novel mechanism by which volvatoxin A1 regulates the cytotoxicity of volvatoxin A2 via direct interaction, and potentially provide an exciting new strategy for chemotherapy.
从食用蘑菇草菇中分离出了一种成孔性心脏毒性蛋白——草毒素A2。先前的研究表明，草毒素A由草毒素A2和草毒素A1组成，当草毒素A2与草毒素A1的摩尔比为2时，草毒素A1会完全消除草毒素A2的溶血活性。在本研究中，我们探究了草毒素A1抑制草毒素A2细胞毒性的分子机制。结果发现草毒素A1本身无毒，而且在摩尔比为2或更低时，它能抑制草毒素A2的溶血和细胞毒性活性。有趣的是，草毒素A1含有393个氨基酸残基，与草毒素A2的串联重复序列极为相似。草毒素A1含有两对两亲性α螺旋，但缺乏肝素结合位点。这表明草毒素A1可能与草毒素A2相互作用，而不与细胞膜相互作用。通过共聚焦显微镜观察发现，草毒素A1不能与细胞膜结合；然而，草毒素A1在摩尔比为2时能抑制草毒素A2与细胞膜的结合。通过肽竞争试验以及下拉和共下拉实验，我们证明草毒素A1和草毒素A2可能形成复合物。我们的结果表明，这是通过一个含有两个两亲性α螺旋的草毒素A1分子与两个各含有一个两亲性α螺旋的草毒素A2分子相互作用而发生的。综上所述，本研究结果揭示了一种新的机制，即草毒素A1通过直接相互作用调节草毒素A2的细胞毒性，并可能为化疗提供一个令人兴奋的新策略。
