TY - JOUR
T1 - Analysis of mutants of tetanus toxin H-C fragment: ganglioside binding, cell binding and retrograde axonal transport properties
AU - Sinha, K
AU - Box, M
AU - Lalli, G
AU - Schiavo, G
AU - Schneider, H
AU - Groves, M
AU - Siligardi, G
AU - Fairweather, N
PY - 2000
Y1 - 2000
N2 - Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy-terminal 50 kDa H-c fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three-dimensional structure of H-c, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C-terminal beta-trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine-1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar-binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.
AB - Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy-terminal 50 kDa H-c fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three-dimensional structure of H-c, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C-terminal beta-trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine-1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar-binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.
UR - http://www.scopus.com/inward/record.url?scp=0033812830&partnerID=8YFLogxK
U2 - 10.1046/j.1365-2958.2000.02091.x
DO - 10.1046/j.1365-2958.2000.02091.x
M3 - Article
VL - 37
SP - 1041
EP - 1051
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 5
ER -