Analysis of mutants of tetanus toxin H-C fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Tetanus toxin binds neuronal tissue prior to internalization and traffickin g to the central nervous system. Binding of the carboxy-terminal 50 kDa H-c fragment of tetanus toxin to polysialogangliosides is important for this i nitial cell binding step. Using the three-dimensional structure of H-c, mut ants were designed to investigate the role of individual residues in gangli oside binding. Mutant proteins were tested for binding to GT1b gangliosides , to primary motoneurons and for their ability to undergo retrograde transp ort in mice. Two classes of mutant were obtained: (i) those containing dele tions in loop regions within the C-terminal beta-trefoil domain which showe d greatly reduced ganglioside and cell binding and did not undergo retrogra de transport and (ii) those that showed reduced ganglioside binding, but re tained primary neuronal cell binding and retrograde transport. The second c lass included point mutants of Histidine-1293, previously implicated in GT1 b binding. Our deletion analysis is entirely consistent with recent structu ral studies which have identified sugar-binding sites in the immediate vici nity of the residues identified by mutagenesis. These results demonstrate t hat ganglioside binding can be severely impaired without abolishing cell bi nding and intracellular trafficking of tetanus toxin.