Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain

Abstract

Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a β-strand peptide (556–564) and glycan-N559 within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D1141 and N1142) within the β-peptide binding interface and one amino acid variant (R1292) located near the SV2C glycan-N559. Introduction of BoNT/A4 β-strand peptide variant (D1141 and N1142) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N559 variant (D1141, N1142, and R1292) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N559 variant (G1292) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 β-strand peptide variants (G1141, S1142, and G1292) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C β-peptide and -glycan-N559 interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C β-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C563.

Embracing the Versatility of Botulinum Neurotoxins in Conventional and New Therapeutic Applications

“…The significant differences between BoNT/A3 and BoNT/A4 in binding affinity and cleavage efficiency particularly affects their S1’ subsite recognition [ 146 ]. Further functional and modelling studies indicated that in rodent models, the disruption of HCc-V2C β-peptide and -glycan-N559 interactions mediates low BoNT/A4 potency, while in human motor neurons, the disruption of HCc-SV2C β-peptide alone mediates low BoNT/A4 potency due to species-specific variation at the SV2C site [ 147 ].…”

Recent Developments in Vaccine Design: From Live Vaccines to Recombinant Toxin Vaccines

“…However, studies have shown that the LC and H N domains of BoNTs are more immunogenic than the HC domains [ 66 , 110 ], suggesting that optimal vaccines should include these domains. Other studies have demonstrated that even if the H C is modified to lack ganglioside binding, it can still stimulate protective immunity in outbred mice [ 105 , 106 , 107 , 111 ], as well as that catalytic activity of the BoNT/LC can be diminished by introducing two to three single amino acid mutations that prevent coordination of the zinc in the LC [ 74 , 112 , 113 ]. This led to the idea of genetically inactivated holotoxin vaccines that are not able to produce toxicity but maintain their overall structure and immunogenicity.…”