Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells

Abstract

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is clinically used because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have high degree of amino acid sequence similarity in light chain (LC) (96%), whereas their N‐and C‐terminal heavy chain (H_N and H_C) differ by 13%. LC acts as a zinc‐dependent endopeptidase, H_N as the translocation domain, and H_C as the receptor‐binding domain. BoNT/A2 and BoNT/A1 had similar potency in mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify domains responsible for these characteristics, H_N of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in E. coli, chimeric and wild‐type BoNT/As were purified as single‐chain proteins and activated by conversion to disulfide‐linked dichains. Toxicities of recombinant wild‐type and chimeric BoNT/As were similar, but it dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP‐25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP‐25 cleavage activity. BoNT/A2 H_N is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.