Winter A, Ulrich W P, Wetterich F, Weller U, Galla H J
Institut für Biochemie, Westfälische Wilhelms-Universität, Münster, Germany.
Chem Phys Lipids. 1996 Jun 17;81(1):21-34. doi: 10.1016/0009-3084(96)02529-7.
The interaction between tetanus toxin and its fragments with gangliosides and negatively charged phosphatidylglycerols has been studied in phosphatidylcholine host membranes by protein circular dichroism measurement, calorimetry to determine lipid phase transitions, and by fluorescence spectroscopy to follow the toxin-induced pore formation by measuring the release of intravesicular entrapped dye. CD-spectroscopic secondary structure analysis showed conformational change of the toxin only in the presence of GT1b clearly demonstrating the involvement of the ganglioside headgroups for this lipid-protein-interaction. In a dot-blot analysis we showed that fragment C binds to GT1b in reconstituted vesicles and that this fragment is then accessible to a fragment C specific antibody which is only possible if fragment C is exposed at least partially on the surface of the vesicle. Our calorimetric study demonstrates the preferential binding of tetanus toxin to ganglioside GT1b. However, this protein is also able to bind to other gangliosides and also to negatively charged phospholipids causing phase separation due to electrostatic interaction. Since tetanus toxin preferentially binds short chain phosphatidylglycerol, we conclude that the protein adopts lipids with respect to charge, head group structure and chain length from the bulk phase. One consequence of this lipid-protein interaction is the ability of tetanus toxin to permeabilize lipid vesicles. Pore formation is favoured in the presence of GT1b in phosphatidylcholine membranes but only at a sufficiently high enough ganglioside content. Gangliosides others than GT1b are less effective in pore formation. In the presence of negatively charged phosphatidylglycerol tetanus toxin causes a dye release which in contrast to GT1b-containing vesicles is not saturable. We conclude that tetanus toxin acts in combination with a given number of GT1b molecules. Twenty ganglioside molecules are found to be necessary to form the stable pore. Other negatively charged lipids also cause the toxin to intercalate into the membrane but in this case the release velocity is determined by the formation of membrane defects.
通过蛋白质圆二色性测量、量热法测定脂质相变以及荧光光谱法测量囊泡内包封染料的释放来跟踪毒素诱导的孔形成，已在磷脂酰胆碱主体膜中研究了破伤风毒素及其片段与神经节苷脂和带负电荷的磷脂酰甘油之间的相互作用。圆二色光谱二级结构分析表明，仅在存在GT1b的情况下毒素才发生构象变化，这清楚地证明了神经节苷脂头部基团参与了这种脂质 - 蛋白质相互作用。在斑点印迹分析中，我们表明片段C在重构囊泡中与GT1b结合，并且该片段随后可被片段C特异性抗体识别，只有当片段C至少部分暴露于囊泡表面时才可能发生这种情况。我们的量热研究表明破伤风毒素优先与神经节苷脂GT1b结合。然而，这种蛋白质也能够与其他神经节苷脂以及带负电荷的磷脂结合，由于静电相互作用导致相分离。由于破伤风毒素优先结合短链磷脂酰甘油，我们得出结论，该蛋白质从本体相中选择电荷、头部基团结构和链长合适的脂质。这种脂质 - 蛋白质相互作用的一个结果是破伤风毒素能够使脂质囊泡通透化。在磷脂酰胆碱膜中存在GT1b时有利于孔形成，但仅在足够高的神经节苷脂含量下才会发生。除GT1b之外的神经节苷脂在孔形成方面效果较差。在存在带负电荷的磷脂酰甘油时，破伤风毒素会导致染料释放，与含GT1b的囊泡不同，这种释放是不饱和的。我们得出结论，破伤风毒素与一定数量的GT1b分子协同作用。发现形成稳定孔需要20个神经节苷脂分子。其他带负电荷的脂质也会导致毒素插入膜中，但在这种情况下，释放速度由膜缺陷的形成决定。
