Tetanus toxin interaction with human erythrocytes. II. Kinetic properties of toxin association and evidence for a ganglioside-toxin macromolecular complex formation.
The properties of tetanus toxin interaction with human erythrocytes supplemented with disialo- and trisialo-gangliosides have been investigated. Binding of toxin is linear with time for 1 h and is 3-4-fold higher at 37 degrees C than at 4 degrees C during incubation of long duration. It exhibits saturation at toxin concentrations between 0.1 and 1 microgram/ml; however, it is nonsaturable between 1 and up to 50 micrograms/ml. It is effectively prevented by free gangliosides and antibodies or by pretreatment with sialidase but is unaffected by a number of closely related ligands including toxoid and toxin fragments. NaCl (1 M) removes a great portion (86%) of cell-associated toxin while Triton X-100 extracts an additional fraction (30%) of the salt-resistant cell-bound toxin. The residual sequestred toxin after detergent extraction is sensitive to proteolytic degradation. The trypsin-stable fraction (1.5%) is biotoxic and may be indicative of internalization of toxin. A macromolecular complex of about 700 kDa containing toxin and gangliosides has been isolated and characterized by Sephacryl S-300 gel permeation chromatography, SDS-gel electrophoresis, immunoprecipitability and biotoxicity. This complex is obtained only in ganglioside-supplemented cells and not when free 3H-labeled GD1b is reacted with 125I-labeled toxin in solution in the absence of cells. The hydrophobicity properties acquired as a result of ganglioside-toxin interaction, presumably at the cell surface, suggest a conformational change of the toxin which may enable its penetration into the bilayer.
D001667:Binding, Competitive; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D005732:Gangliosides / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009439:Neuraminidase / Q000378:metabolism; D009994:Osmolar Concentration; D013696:Temperature; D013744:Tetanus Toxin / Q000378:metabolism*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D005732:Gangliosides; D046911:Macromolecular Substances; D013744:Tetanus Toxin; D009439:Neuraminidase; D014357:Trypsin