Gangliosides in phospholipid bilayer membranes: interaction with tetanus toxin.
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.
Winter
A
A
Institut für Biochemie, Westfälische Wilhelms-Universität, Münster, Germany.
Ulrich
W P
WP
Wetterich
F
F
Weller
U
U
Galla
H J
HJ
D002942:Circular Dichroism; D005732:Gangliosides / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008567:Membranes, Artificial; D010539:Permeability; D013744:Tetanus Toxin / Q000737:chemistry*
D005732:Gangliosides; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D013744:Tetanus Toxin; C016481:trisialoganglioside GT1