Ayyar B Vijayalakshmi, Aoki K Roger, Atassi M Zouhair
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.
Neurotoxin Research Program, Department of Biological Sciences, Allergan, Inc., Irvine, California, USA.
Infect Immun. 2015 Apr;83(4):1465-76. doi: 10.1128/IAI.00063-15. Epub 2015 Jan 26.
Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin.
肉毒杆菌神经毒素（BoNTs）对神经末梢具有独特的特异性。它们与突触前膜结合，然后转运至细胞内，在那里轻链内肽酶切割SNARE复合蛋白，破坏负责将乙酰胆碱释放到突触间隙的突触胞吐作用。这会抑制乙酰胆碱与其受体的结合，导致麻痹。结合是细胞中毒的必要事件，据信是通过重链C末端（HC）结构域发生的。随后是毒素转运并进入细胞质，这被认为是由重链N末端（HN）结构域介导的。通过使用跨越BoNT血清型A（BoNT/A）和小鼠脑突触体（SNPs）的合成肽以及来自接受BoNT/A治疗的小鼠和颈部肌张力障碍患者的抗毒素保护性抗体进行的亚分子图谱分析表明，不仅HC结构域的区域，而且HN结构域的区域也参与毒素结合过程。基于这些发现，我们表达了一种与BoNT/A区域相对应的肽，该区域包含HN结构域的729至845位残基（HN729-845）。HN729-845直接与小鼠脑SNPs结合，并显著抑制BoNT/A与SNPs的结合。这种结合涉及神经节苷脂GT1b和GD1a以及一些膜脂。该肽在1分钟内与人或小鼠神经母细胞瘤细胞结合。肽HN729-845能完全保护小鼠免受致死剂量的BoNT/A（1.05倍100%致死剂量）。在与HC结构域中967至1296位残基的肽相当的剂量下获得了这种保护活性。这些发现有力地表明，HN729-845以及由此延伸的HN结构域已完全编程并具备与神经元细胞结合的能力，并且在游离状态下甚至可以抑制毒素的结合。
