Characterisation of the SNAP-25 and SNAP-23 cleavage properties of the botulinum neurotoxin-like protein from Enterococcus.

Botulinum neurotoxins (BoNT) are well-established biopharmaceuticals used to treat neuromuscular, secretory and pain conditions. They do this by blocking neurotransmitter release from neurons through the proteolysis of specific soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. Current research seeks to expand the range of BoNTs applications to other medical conditions. One possible starting point for this endeavour is the catalytic domain of the BoNT-like protein form Enterococcus faecium. This protein has been reported to cleave the BoNT resistant human SNAP-23 (hSNAP-23) isoform of SNAP-25 (synaptosomal associated protein of 25 kDa), which is the substrate of the primary BoNT pharmaceutical, BoNT serotype A1. SNAP-23 is an attractive new target because it has been reported to be involved in the excessive release of inflammatory messengers. Therefore, we characterized the substrate cleaving properties of the catalytic domain of this BoNT-like protein. Biochemical characterization showed that LC/En cleaves hSNAP-23 approximately 25 times less efficiently than SNAP-25. However, compared to LC/A, LC/En readily cleaves hSNAP-23, excelling the activity of LC/A by more than 100-fold. To lay the groundwork for later re-engineering of LC/En to tailor its enzymatic activity to hSNAP-23, we characterized the enzyme-substrate interaction through systematic mutational analysis of hSNAP-23. We found that the amino acids in the P2 and P1' positions are crucial and that the substitutions at amino acid positions 49 and 53 versus SNAP-25 largely explain hSNAP-23's 25-fold lower cleavage rate compared to SNAP-25. Therefore, the binding pockets of hSNAP-23 Lys-49 and Asn-53 in LC/En are prime targets for adapting the enzyme to increase its cleavage of hSNAP-23.