Research On The Relationship Of Botulinum Neurotoxin Type A And Fibroblast Growth Factor Receptor 3

Botulinum neurotoxin(BoNT)is produced as a potent toxin by the anaerobic Clostridium botulinum.It causes relaxation paralysis by blocking nerve-muscle synaptic transmission,which is the most poisonous substance as known.Seven serotypes(A to G)of botulinum toxin are currently known,among them,A,B,E and F cause human natural intoxication.According to the Dual-receptor model of botulinum toxin,the receptors of BoNT/A are gangliosie GT1 b and synaptic vesicle protein SV2 C.At present,the characterization of the receptor binding region is clear.In 2013,Brigitte et al found that fibroblast growth factor receptor 3(FGFR3)was a newly discovered receptor of BoNT/A.When toxins combined with FGFR3,will triggered endocytosis,but FGFR3 and BoNT/A binding region,it plays a role in botulism and the correlation with two other receptors are unclear.This study focused on identifying the FGFR3 binding site of BoNT/A and virulence differences among different subtypes of BoNT/A,the relationship among the three receptors by means of truncated mutation,peptide library analysis,molecular docking,sequence alignment,construction of mutants and animal experiments.Aimed to expound the mechanism of botulism and to lay the theory foundation for botulinum toxin vaccine and the neutralizing antibody.Expression and purification of the receptor FGFR3 and the receptor binding region of the BoNT/A(heavy chain C-terminus,BoNT/AHc)and preparation of FGFR3-BoNT/AHc crystals;expression and purification of the truncated fragment BoNT/A HCN and HCC through indirect Enzyme linked immunosorbent assay(ELISA),determined that the binding site of FGFR3 and BoNT/A was located in the HCN region.Then,it was determined by peptide library screening that small peptides 20,21,and 37 had strong binding ability to FGFR3.Analysis of the spatial position of these three peptides by software,revealed that the three small peptides were spatially adjacent and formed a concave surface.Based on peptide library screening results,the potential binding sites of BoNT/A and receptor FGFR3 were predicted using Discovery Studio,obtain two models Pose14 and Pose155.Through the point mutatipn finally identification of the direct interaction binding sites(amino acid residues Ser902,Thr963,Asn966,Asn971,Glu982,Gln988,Asp1058,Gly1059 and Arg1061).We also investigated the Causes of Virulence difference in subtypes of BoNT/A and obtained the following conclusions.First,the activities of BoNT/A1,BoNT/A2,and BoNT/A4 were indentified by animal analysis the three subtype toxins were A1> A2> A4.By comparison of the amino acid sequences,found that the LC and HC domains of A1 and A4 varied enormously.The light chain A4 L was expressed and purified,the activities of A1 and A4 light chains were compared by light chain endopeptidase activity assay and it was found that there was no significant difference between them.The differences in the HC domains were analyzed by constructing the A1 and A4 chimeras.Mouse bioassay experiments showed that the chimera containing the BoNT/A1 Hc domain was active.It reveals that A1 and A4 are different on HC.The differences in the HCN sequence are greater than the HCC in A1 and A4 amino acid sequence alignments,and the sequence of the 37 th peptide on the HCN domain is also significantly different.Small peptide No.37 was predicted the binding domain of receptor FGFR3,and it was speculated that the virulence difference of subtypes of BoNT/A was caused by the binding ability of receptor FGFR3,which should be further verified by experiments.The study of the relationship between the three receptors of BoNT/A proceed from the following two aspects.On the one hand,mutated the known receptor binding site(GT1b/SV2C)on BoNT/A,the animal activity suggested that the receptors GT1 b and SV2 C were related to virulence;On the other hand,we prepared mouse monoclonal antibodies of anti-SV2 C and FGFR3.With the antibody blocking the receptor SV2C/FGFR3 in the mouse activity assay,we found that the receptors SV2 C and FGFR3 are associated with virulence,in addition,when the receptors SV2 C and FGFR3 were blocked at the same time the degree of virulence reduction did not increase in the degree of neutralization;In the cell viability assay,no antibody toxin SNAP25197 was detected after blocking the receptor SV2C/FGFR3 alone,which is consistent with the results of animal activity experiments,indicating that all three receptors are related to virulence and the relationship between the three receptors remains to be further studied.