Spectroscopic analysis of pH-induced changes in the molecular features of type A botulinum neurotoxin light chain.
Spectroscopic analysis of pH-induced changes in the molecular features of type A botulinum neurotoxin light chain.
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pH 诱导的 A 型肉毒杆菌神经毒素轻链分子特征变化的光谱分析。
DOI：
10.1021/bi992729u
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Singh,BR
中科院分区：
文献类型：
--
作者：
Li,L;Singh,BR
肉毒杆菌神经毒素(BoNTs)通过阻断神经肌肉交界处的神经递质释放而导致神经瘫痪。当毒素的重链(HC)参与结合和内化时，轻链(LC)在胞吐对接/融合机制中扮演着针对目标蛋白的独特的锌内肽酶的角色。在LC转运到细胞质的过程中，它暴露在内体的低pH下。差示热变性实验表明，在低pH条件下，BONT/A LC多肽折叠发生了显著变化。此外，1-苯胺基苯磺酸(ANS)的结合揭示了在低pH条件下BONT/A LC的疏水结构域暴露。低pH诱导的结构变化(以及内肽酶活性)是完全可逆的。然而，将BONT/A LC暴露在低pH(4.7)下并没有引起结合到其活性部位的锌离子的损失。讨论了这些观察结果对活性BONT/A LC向神经细胞递送的影响。通过色氨酸荧光测量进一步分析了低pH诱导的BONT/A LC多肽折叠变化的本质。色氨酸的荧光峰位于322 nm处，在低pH值下，2.1 ns(88%)和0.6 ns(12%)两个荧光寿命分量变化不大。这些观察表明，这两个色氨酸残基被埋藏和限制在疏水环境中，很可能是BONT/A LC蛋白基质的核心不参与低pH诱导的结构变化。在两种pH条件下的近紫外圆二色谱进一步支持了这一结论。
Clostridialbotulinumneurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. While the toxin's heavy chain (HC) is involved in binding and internalization, the light chain (LC) acts as a unique Zn2+-endopeptidase against a target protein in the exocytotic docking/fusion machinery. During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate (ANS) revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low-pH-induced structural (and by implication the endopeptidase activity) changes were completely reversible. Exposure of BoNT/A LC to low pH (4.7) did not, however, evoke the loss of Zn2+bound to its active site. Implications of these observations to the delivery of active BoNT/A LC to the nerve cell are discussed. We further analyzed the nature of low-pH-induced change in the polypeptide folding of BoNT/A LC by Trp fluorescence measurements. The Trp fluorescence peak was observed at 322 nm, and the two fluorescence lifetime components estimated at 2.1 ns (88%) and 0.6 ns (12%) did not change much at low pH. These observations suggested that the two Trp residues are buried and constrained in a hydrophobic environment, and it is likely that the core of the BoNT/A LC protein matrix does not participate in the low-pH-induced structural alteration. This conclusion was further supported by the near-UV circular dichroism spectra under two pH conditions.