INVESTIGATING THE ROLE OF THE MEMBRANE LOCALIZATION DOMAIN (MLD) IN BOTULINUM NEUROTOXIN A (BoNT/A) DURATION OF ACTION

Abstract

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are among the most potent biological toxins known to humans and are the causative agents of botulism, a severe neuroparalytic disease 1. Among the BoNT serotypes (A-G), BoNT/A1 is distinguished by its high potency and prolonged duration of action, whereas BoNT/A3 exhibits reduced potency and a significantly shorter duration 2,3. Preliminary observations suggest that BoNT/A1 and BoNT/A3 may differ in the intracellular localization of their Light Chain (LC) proteases: BoNT/A1 LC predominately localizes to the plasma membrane, while BoNT/A3 LC is distributed throughout the cytosol and on intracellular vesicles 4.

A previously identified region of low sequence homology between BoNT/A1 and BoNT/A3, termed the Low Homology Domain (LHD), contains a subregion implicated in membrane association, designated the Membrane Localization Domain (MLD) 5,6. To investigate the role of the MLD in intracellular trafficking and functional persistence, we engineered recombinant chimeric BoNT LCs and holotoxins by exchanging the LHD or MLD between BoNT/A1 and BoNT/A3. Using LC encoding mRNA transfection or holotoxin intoxication in human induced pluripotent stem cell (hiPSC)-derived motor neurons, we evaluated SNAP-25 cleavage as a functional readout of BoNT activity. Duration of action of holotoxins was further assessed in an in vivo mouse model.

Our results demonstrate that the MLD is essential but not sufficient for the differences in potency and duration of action between BoNT/A1 and BoNT/A3.