Departament de Bioquímica i Biologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Catalunya, Spain.
Laboratory of Cellular and Molecular Neurobiology, Department of Pathology and Experimental Therapeutics, School of Medicine, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain; Biomedical Research Institute of Bellvitge (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain; Institut de Neurociències, Universitat de Barcelona, Spain.
Biochim Biophys Acta Biomembr. 2019 Jan;1861(1):161-169. doi: 10.1016/j.bbamem.2018.08.003. Epub 2018 Aug 16.
Epsilon toxin (Etx) from Clostridium perfringens is synthesized as a very low-active prototoxin form (proEtx) that becomes active upon proteolytic activation and has the capacity to cross the blood-brain barrier (BBB), thereby producing severe neurological effects. The identity and requirements of host receptors of Etx remain a matter of controversy. In the present study, we analysed the binding of proEtx or Etx to liposomes containing distearoylphosphatidylcholine (DSPC), cholesterol and sulfatide, or alternatively to detergent-solubilized lipids, using surface plasmon resonance (SPR). We also tested the influence of calcium on Etx or proEtx binding. Our findings show that the presence of sulfatide in liposomes increases both Etx and proEtx binding, and Etx binding is enhanced by calcium. These results were corroborated when SPR was conducted with immobilized toxin, since detergent-solubilized sulfatide increases its binding to Etx in the presence of calcium, but not to proEtx. Moreover, binding affinity is also affected, since the treatment of liposomes with sulfatase causes the dissociation rate constants (K) in both proEtx and Etx to increase, especially in the case of proEtx in the presence of calcium. In addition, protein-lipid overlay assays corroborated the calcium-induced enhancement of Etx binding to sulfatide, and to lipids extracted from sulfatide-enriched rat brain lipid rafts. In conclusion, the present work highlights the role of sulfatide as an important element in the pathophysiology of Etx and reveals the influence of calcium in the interaction of Etx, but not of proEtx, with the target membrane.
来自梭状芽孢杆菌的ε 毒素（Etx）最初合成时是一种低活性的原毒素形式（proEtx），经蛋白水解激活后具有穿透血脑屏障（BBB）的能力，从而产生严重的神经效应。Etx 的宿主受体的身份和要求仍然存在争议。在本研究中，我们使用表面等离子体共振（SPR）分析了 proEtx 或 Etx 与含有二硬脂酰基磷脂酰胆碱（DSPC）、胆固醇和神经节苷脂的脂质体，或替代地与去污剂溶解的脂质的结合。我们还测试了钙对 Etx 或 proEtx 结合的影响。我们的研究结果表明，脂质体中神经节苷脂的存在增加了 Etx 和 proEtx 的结合，并且钙增强了 Etx 的结合。当 SPR 与固定化毒素一起进行时，这些结果得到了证实，因为在钙存在的情况下，去污剂溶解的神经节苷脂增加了其与 Etx 的结合，但不能与 proEtx 结合。此外，结合亲和力也受到影响，因为用神经节苷脂酶处理脂质体导致 proEtx 和 Etx 的解离速率常数（K）增加，尤其是在钙存在的情况下。此外，蛋白脂质覆盖测定法证实了钙诱导的 Etx 与神经节苷脂以及从富含神经节苷脂的大鼠脑脂筏中提取的脂质结合增强。总之，本工作强调了神经节苷脂作为 Etx 病理生理学中重要元素的作用，并揭示了钙对 Etx 与靶膜相互作用的影响，但对 proEtx 没有影响。
