GANGLIOSIDES IN PHOSPHOLIPID-BILAYER MEMBRANES - INTERACTION WITH TETANUS TOXIN

The interaction between tetanus toxin and its fragments with gangliosi des and negatively charged phosphatidylglycerols has been studied in p hosphatidylcholine host membranes by protein circular dichroism measur ement, calorimetry to determine lipid phase transitions, and by fluore scence spectroscopy to follow the toxin-induced pore Formation by meas uring the release of intravesicular entrapped dye. CD-spectroscopic se condary structure analysis showed conformational change of the toxin o nly in the presence of GTlb clearly demonstrating the involvement of t he ganglioside headgroups for this lipid-protein-interaction. Ina dot- blot analysis we showed that fragment C binds to GTlb in reconstituted vesicles and that this fragment is then accessible to a fragment C sp ecific antibody which is only possible if fragment C is exposed at lea st partially on the surface of the vesicle. Our calorimetric study dem onstrates the preferential binding of tetanus toxin to ganglioside GTl b. However, this protein is also able to bind to other gangliosides an d also to negatively charged phospholipids causing phase separation du e to electrostatic interaction. Since tetanus toxin preferentially bin ds short chain phosphatidylglycerol, we conclude that the protein adop ts lipids with respect to charge, head group structure and chain lengt h from the bulk phase. One consequence of this lipid-protein interacti on is the ability of tetanus toxin to permeabilize lipid vesicles. Por e formation is favoured in the presence of GTlb in phosphatidylcholine membranes but only at a sufficiently high enough ganglioside content. Gangliosides others than GTlb are less effective in pore formation. I n the presence of negatively charged phosphatidylglycerol tetanus toxi n causes a dye release which in contrast to GTlb-containing vesicles i s not saturable. We conclude that tetanus toxin acts in combination wi th a given number of GTlb molecules. Twenty ganglioside molecules are found to be necessary to form the stable pore. Other negatively charge d lipids also cause the toxin to intercalate into the membrane but in this case the release velocity is determined by the formation of membr ane defects.