Abstract
Recombinant peptides were designed using the C-terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli. A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC297 BoNT/A1, referred to EGFP-A2' and EGFP-RBD', exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant (KD) of EGFP-RBD' to SV2C-LD was determined to be 5.45 μM, which is 33.87- and 15.67-times smaller than the KD values for EGFP and EGFP-A2', respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBD' to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2', respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBD'. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.