Institute for Biophysics, Department of Nanobiotechnology, University of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria.
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand.
Toxins (Basel). 2020 Apr 3;12(4):226. doi: 10.3390/toxins12040226.
Cytolytic toxin (Cyt) is a toxin among insecticidal proteins. Cyt toxin directly interacts with membrane lipids for cytolytic action. However, low hemolytic activity is desired to avoid non-specific effects in mammals. In this work, the interaction between Cyt2Aa2 toxin and model lipid bilayers mimicking the erythrocyte membrane was investigated for Cyt2Aa2 wild type (WT) and the T144A mutant, a variant with lower hemolytic activity. Quartz crystal microbalance with dissipation (QCM-D) results revealed a smaller lipid binding capacity for the T144A mutant than for the WT. In particular, the T144A mutant was unable to bind to the phosphatidylcholine lipid (POPC) bilayer. However, the addition of cholesterol (Chol) or sphingomyelin (SM) to the POPC bilayer promoted binding of the T144 mutant. Moreover, atomic force microscopy (AFM) images unveiled small aggregates of the T144A mutant on the 1:1 sphingomyelin/POPC bilayers. In contrast, the lipid binding trend for WT and T144A mutant was comparable for the 1:0.4 POPC/cholesterol and the 1:1:1 sphingomyelin/POPC/cholesterol bilayers. Furthermore, the binding of WT and T144A mutant onto erythrocyte cells was investigated. The experiments showed that the T144A mutant and the WT bind onto different areas of the erythrocyte membrane. Overall the results suggest that the T144 residue plays an important role for lipid binding.
细胞溶素毒素（Cyt）是杀虫蛋白中的一种毒素。Cyt 毒素直接与膜脂相互作用，发挥细胞溶作用。然而，为了避免在哺乳动物中产生非特异性效应，希望具有较低的溶血活性。在这项工作中，研究了模拟红细胞膜的模型脂质双层与 Cyt2Aa2 毒素野生型（WT）和溶血活性较低的 T144A 突变体之间的相互作用。石英晶体微天平耗散（QCM-D）结果表明，T144A 突变体的脂质结合能力小于 WT。特别是，T144A 突变体不能结合磷脂酰胆碱脂质（POPC）双层。然而，向 POPC 双层添加胆固醇（Chol）或鞘磷脂（SM）促进了 T144 突变体的结合。此外，原子力显微镜（AFM）图像显示 T144A 突变体在 1:1 鞘磷脂/POPC 双层上形成小聚集体。相比之下，WT 和 T144A 突变体在 1:0.4 POPC/胆固醇和 1:1:1 鞘磷脂/POPC/胆固醇双层上的脂质结合趋势相当。此外，还研究了 WT 和 T144A 突变体在红细胞上的结合。实验表明，T144A 突变体和 WT 结合在红细胞膜的不同区域。总体而言，结果表明 T144 残基在脂质结合中起重要作用。
