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

Authors

• William H. Tepp, University of Wisconsin - Madison
• Marite Bradshaw, University of Wisconsin - Madison
• Alexander P. Gardner, Medical College of Wisconsin - Milwaukee
• Rebecca L. Kaufman, University of Wisconsin - Madison
• Joseph T. Barbieri, Medical College of Wisconsin - Milwaukee
• Sabine Pellett, University of Wisconsin - Madison

Document Type

Article

Publication Date

3-2023

Publisher

MDPI

Source Publication

International Journal of Molecular Sciences

Source ISSN

1661-6596

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.

Comments

Published version. International Journal of Molecular Sciences, Vol. 24, No. 6 (2023). DOI. This article is © The Authors, Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Alexander Gardner was affiliated with Medical College of Wisconsin, Milwaukee at the time of publication.

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Recommended Citation

Tepp, William H.; Bradshaw, Marite; Gardner, Alexander P.; Kaufman, Rebecca L.; Barbieri, Joseph T.; and Pellett, Sabine, "Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain" (2023). College of Nursing Faculty Research and Publications. 1211.
https://epublications.marquette.edu/nursing_fac/1211