Poulain B, Wadsworth J D, Shone C C, Mochida S, Lande S, Melling J, Dolly J O, Tauc L
Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
J Biol Chem. 1989 Dec 25;264(36):21928-33.
The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof. In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step. At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably to common ecto-acceptor via the H2 region. In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction. In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals. Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals. In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons. Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release. This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.
利用完整的肉毒杆菌神经毒素（BoNT）、其重链（HC）或轻链（LC）及其蛋白水解片段，研究了BoNT的结合、内化及对递质释放的抑制作用。在海兔神经元中，将温度降至10摄氏度可防止外源性施加A型或E型BoNT时乙酰胆碱释放的阻断，这是因为中毒在膜结合步骤被阻止。在此低温下，A型HC、H2（由HC的N端组成）或H2L（H2与LC通过二硫键连接）可拮抗A型或E型BoNT的神经麻痹作用，表明后者通过H2区域与共同的胞外受体饱和结合。相比之下，H2L在小鼠神经肌肉接头处无法抵消BoNT诱导的麻痹。与这种物种差异一致，与天然BoNT不同，在哺乳动物外周或中枢神经末梢中未检测到125I标记的H2L的饱和结合。可能更严格的结构要求是毒素在抑制小鼠神经肌肉突触处神经传递比海兔神经末梢更有效的基础。在进一步鉴定毒素中的功能域时，发现未加工的E型BoNT单链形式在细胞外或细胞内应用于海兔神经元时无效。值得注意的是，将后者浴用在预先注射了HC而非H2L或LC的神经元上会导致释放阻断。这表明单链形式可以内化，并且其抑制作用不仅需要LC，还需要加工后的HC；一致地，E型BoNT的蛋白水解形式是有活性的。
