Mapping of the Synaptosome-Binding Regions on the Heavy Chain of Botulinum Neurotoxin A By Synthetic Overlapping Peptides Encompassing the Entire Chain

Abstract

The purpose of this work was to map, on the heavy (H) chain of botulinum neurotoxin A (BoNT/A), the regions that bind to mouse brain synaptosomes (snps). We prepared 60 synthetic overlapping peptides that had uniform size and overlaps and encompassed the entire H chain (residues 499 to 1296) of BoNT/A. The ability of each peptide to inhibit the binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was studied. The binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was completely inhibited by free unlabeled BoNT/A, but not by unrelated proteins, indicating that the binding of BoNT/A to mouse brain snps was a specific event. Inhibition studies with the individual peptides showed that, on the H_N domain, inhibitory activities greater than 10% were exhibited, in decreasing order, by peptides 799–817, 659–677, 729–747, 533–551, 701–719, and 757–775. Lower inhibitory activities (between 5.6% and 8.7%) were exhibited by five other peptides, 463–481, 505–523, 519–537, 603–621 and 645–663. The remaining 18 H_N peptides had little or no inhibitory activity. In the H_C domain, peptides 1065–1083, 1163–1181 and 1275–1296 had the highest inhibitory activities (between 25% and 29%), followed (10–12% inhibitory activity) by peptides 1107–1125, 1191–1209 and 1233–1251. Two other peptides, 1079–1097 and 1177–1195, had very low (5.8% and 4.9 %) inhibitory activities. The remaining 23 H_C peptides had no inhibitory activity. Inhibition with mixtures of equimolar quantities of the most active 6 peptides of H_N, 5 of H_C or all 11 of H_N and H_C revealed that the peptides contain independent non-competing binding regions. Comparison of the locations of the snp-binding regions on the H-subunit with the regions that bind blocking mouse anti-BoNT/A Abs helped explain the protecting ability of these Abs. In the three-dimensional structure of BoNT/A, the snp-binding regions that completely coincide or significantly overlap with the antigenic regions occupy surface locations and most of them reside in the last half of the H_C domain. But some of the regions reside in the H_N domain and might play a role in the translocation event.