Madshus I H, Stenmark H, Sandvig K, Olsnes S
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
J Biol Chem. 1991 Sep 15;266(26):17446-53.
Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin.
在通过聚合酶链反应扩增DNA模板后，于体外制备了由白喉毒素和蛋白A的重复Fc结合结构域组成的融合蛋白。这些融合蛋白能与包被有抗体的Vero细胞紧密结合。一种含有全长白喉毒素的融合蛋白在比单独的白喉毒素更低的浓度下就具有毒性，这显然是由于其结合效率更高。融合蛋白的酶部分在暴露于低pH值时会穿过表面膜。与天然白喉毒素一样，该融合蛋白在低pH值下形成阳离子选择性通道。过量的未标记白喉毒素会抑制源自放射性标记融合蛋白的蛋白酶保护片段的形成。此外，下调白喉毒素受体的条件会降低细胞对融合蛋白的敏感性，这支持了需要天然白喉毒素受体的观点。在低于18摄氏度的温度下，融合蛋白的毒性会大幅降低，而天然白喉毒素则不存在温度阻断现象。布雷菲德菌素A可保护Vero细胞免受融合蛋白的侵害，但不能保护其免受白喉毒素的侵害。结果表明，即使在毒素由替代结合部分结合的情况下，高效的毒素转运也需要白喉毒素受体，并且这表明融合蛋白的细胞内转运途径与白喉毒素不同。
