Sinha K, Box M, Lalli G, Schiavo G, Schneider H, Groves M, Siligardi G, Fairweather N
Department of Biochemistry, Imperial College, South Kensington, London SW7 2AY, UK.
Mol Microbiol. 2000 Sep;37(5):1041-51. doi: 10.1046/j.1365-2958.2000.02091.x.
Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy-terminal 50 kDa HC fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three-dimensional structure of HC, 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.
破伤风毒素在被内化并转运至中枢神经系统之前会与神经组织结合。破伤风毒素羧基末端50 kDa重链（HC）片段与多唾液酸神经节苷脂的结合对于这一初始细胞结合步骤至关重要。利用HC的三维结构，设计了突变体以研究单个残基在神经节苷脂结合中的作用。对突变蛋白进行了与GT1b神经节苷脂结合、与初级运动神经元结合以及在小鼠体内进行逆行运输能力的测试。获得了两类突变体：（i）那些在C末端β-三叶结构域的环区域含有缺失的突变体，其神经节苷脂和细胞结合能力大大降低，且不进行逆行运输；（ii）那些神经节苷脂结合能力降低，但保留初级神经元细胞结合和逆行运输能力的突变体。第二类包括先前与GT1b结合有关的组氨酸-1293的点突变体。我们的缺失分析与最近的结构研究完全一致，这些研究在诱变鉴定的残基紧邻区域确定了糖结合位点。这些结果表明，在不消除破伤风毒素的细胞结合和细胞内运输的情况下，神经节苷脂结合可能会严重受损。
