Primary Study On The Molecular Basis Underlying The Interaction Between Botulinum Neurotoxin And Its Receptor And Their Application

Botulinum neurotoxins (BoNTs) are extremely potent bacterial protein toxins. The active form of the BoNTs is structurally organized into heavy and light chains linked by a disulphide bond. The heavy chain plays dual role in the toxic action, cell surface binding (binding domain), and translocation across membranes (translocation domain).the toxins binds to the surface of plasma membrances,and followed by receptor-mediated endocytosis and translocation across the endosome membrane, and then exert its toxicity. Among them, First step in the action of BoNTs is specific binding to the receptors on the surface of neurons; this is the key step to initiate toxicity. The C-terminal portion of the heavy chain (Hc) is the receptor binding domain, Synaptotagmins II (syt II) is the specific protein receptor of BoNTs newly found, but the molecular basis underlying the interaction between BoNTs and the receptor have been poorly established. Determination of key molecular structure in the interaction between toxin and target cells is very helpful to molecular mechanism of botulism.In this study, we focus on the key step to interact between BoNTs and receptors. Firstly, mimic epitope peptides were designed and synthesized with bioinformatics and computer-aided molecular modeling design method. And then the animal test demonstrated that the mimic epitope peptides could induce protective immune-responses in Balb/C mice against the challenge. Additionally, specific binding peptides of mimic epitope peptides were selected by phage display, which also showed specific protection against BoNTs. We identified the key epitopes on the receptor binding domain by above works. BoNT/A is the domain of 1110-1172 and 1260-1295, but BoNT/B is 1183-1291. Secondly, after expression in E.coli and purification, recombinant protein receptor (syt II) of BoNTs could bind specifically with BoNT/B and its Hc domain. The affinity value was 2.99×10^-7M measured by analysis of SPR of Biacore. And then, the molecular basis underlying the interaction between BoNT/B and its receptor were 1235-1291 established by the interaction with mimic epitope peptides. Meanwhile, according to the amino acid sequence of luminal domain of Syt II, two overlapping receptor peptides were synthesized, and the residues G37-K50 of Syt II was found critical for binding of BoNT/B by molecular biology method.Finally, it was demonstrated that recombinant syt II possesses the inhibition activity against BoNTs by cell and mice assay. Our experiment data proved the double-receptor concept of BoNTs, also showed gangliosides can facilitate syt II -BoNT/B interaction, the mice survival rate is 60%, but gangliosides themselves could not provide any protection. And anti-BoNT/B ScFv was chosen and linked with syt II to construct recombinant ScFv-Syt II-LD, the fused protein might be developed as an inhibitor of the toxin by specific targeting of double-function to improve antagonistic effect against toxins.In summary, our work has established the molecular basis underlying the interaction between BoNT/B and its receptor. These result will be of great help for the illumination of BoNTs pathogenic mechanism and further recognition of molecular structure of the receptor binding domain, also can provide definite targets for drug screening against BoNTs.