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Reevaluation of the Role of Gangliosides as Receptors for Tetanus Toxin
Author(s) -
Critchley David R.,
Habig William H.,
Fishman Peter H.
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb02852.x
Subject(s) - toxin , chemistry , tris , chromatography , membrane , sodium dodecyl sulfate , biochemistry , gel electrophoresis
Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 m M NaCl, 50 m M Tris‐HCl (pH 7.4) than in 25 m M Tris‐acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris‐saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100‐fold higher in Trissaline compared to Tris‐acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125 I‐labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order G Dlb ≅ G Tlb ≅ G Q1b > G D2 > G D3 ≫ G D1a ≅ G M1 . Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris‐saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low‐salt and ‐pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.