Palacios-Ortega Juan, García-Linares Sara, Åstrand Mia, Al Sazzad Md Abdullah, Gavilanes José G, Martínez-del-Pozo Álvaro, Slotte J Peter
Departamento de Bioquímica y Biología Molecular I, Universidad Complutense , 28040 Madrid, Spain.
Biochemistry, Faculty of Science and Engineering, Åbo Akademi University , 20500 Turku, Finland.
Langmuir. 2016 Apr 12;32(14):3476-84. doi: 10.1021/acs.langmuir.6b00082. Epub 2016 Mar 29.
Sticholysin II (StnII) is a pore-forming toxin that uses sphingomyelin (SM) as the recognition molecule in targeting membranes. After StnII monomers bind to SM, several toxin monomers act in concert to oligomerize into a functional pore. The regulation of StnII binding to SM, and the subsequent pore-formation process, is not fully understood. In this study, we examined how the biophysical properties of bilayers, originating from variations in the SM structure, from the presence of sterol species, or from the presence of increasingly polyunsaturated glycerophospholipids, affected StnII-induced pore formation. StnII-induced pore formation, as determined from calcein permeabilization, was fastest in the pure unsaturated SM bilayers. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/saturated SM bilayers (4:1 molar ratio), pore formation became slower as the chain length of the saturated SMs increased from 14 up to 24 carbons. In the POPC/palmitoyl-SM (16:0-SM) 4:1 bilayers, SM could not support pore formation by StnII if dimyristoyl-PC was included at 1:1 stoichiometry with 16:0-SM, suggesting that free clusters of SM were required for toxin binding and/or pore formation. Cholesterol and other sterols facilitated StnII-induced pore formation markedly, but the efficiency did not appear to correlate with the sterol structure. Benzyl alcohol was more efficient than sterols in enhancing the pore-formation process, suggesting that the effect on pore formation originated from alcohol-induced alteration of the hydrogen-bonding network in the SM-containing bilayers. Finally, we observed that pore formation by StnII was enhanced in the PC/16:0-SM 4:1 bilayers, in which the PC was increasingly unsaturated. We conclude that the physical state of bilayer lipids greatly affected pore formation by StnII. Phase boundaries were not required for pore formation, although SM in a gel state attenuated pore formation.
嗜硫菌素II（StnII）是一种成孔毒素，它以鞘磷脂（SM）作为靶向膜的识别分子。StnII单体与SM结合后，几个毒素单体协同作用寡聚形成功能性孔道。目前对StnII与SM结合以及随后的孔道形成过程的调控还不完全清楚。在本研究中，我们研究了源自SM结构变化、甾醇种类存在或多不饱和甘油磷脂增加导致的双层膜生物物理性质如何影响StnII诱导的孔道形成。通过钙黄绿素通透实验测定，StnII诱导的孔道形成在纯不饱和SM双层膜中最快。在1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）/饱和SM双层膜（摩尔比4:1）中，随着饱和SM链长从14个碳增加到24个碳，孔道形成变得更慢。在POPC/棕榈酰-SM（16:0-SM）4:1双层膜中，如果二肉豆蔻酰磷脂酰胆碱与16:0-SM以1:1化学计量比包含在内，SM就无法支持StnII形成孔道，这表明毒素结合和/或孔道形成需要游离的SM簇。胆固醇和其他甾醇显著促进StnII诱导的孔道形成，但效率似乎与甾醇结构无关。苯甲醇在增强孔道形成过程方面比甾醇更有效，这表明对孔道形成的影响源于醇诱导的含SM双层膜中氢键网络的改变。最后，我们观察到在PC/16:0-SM 4:1双层膜中，随着PC不饱和程度增加，StnII诱导的孔道形成增强。我们得出结论，双层膜脂质的物理状态极大地影响了StnII诱导的孔道形成。虽然处于凝胶态的SM会减弱孔道形成，但孔道形成并不需要相界。
