The Use of Intact Protein Mass Spectrometry to Determine the Enzymatic Target of Novel Botulinum Neurotoxins Type F

摘要

1. Botulism is a disease which is caused by exposure to any one of the highly toxic proteins known as botulinum neurotoxins (BoNTs). BoNT are composed of a heavy chain, which binds to receptors on the neuron, and a light chain that is a protease. 2. Botulinum neurotoxins are currently classified into seven serotypes, labeled A-G. BoNT/A,/C, and /E cleave SNAP-25 (synaptosomal-associated protein) whereas BoNT/B, /D, /F, and /G cleave synaptobrevin-2 (also known as VAMP-2). 3. Our laboratory previously reported that all BoNT subtypes within the same serotype cleaved their respective substrate in the same location (1). At the time that this work was reported, only four subtypes of BoNT/F were known. 4. In 2010, three new subtypes of BoNT/F were reported based on phylogenetic analysis of botulinum neurotoxin type F genes (2). It was noted that both BoNT/F5 and /F7 were highly divergent in their gene and amino acid sequences compared to the other BoNT/F subtypes (see Table 1 in the results section). 5. BoNT/F5 has an amino acid identity of only 69.9% with /F1 at the holotoxin level, and BoNT/F7 is 73.7% identical to /F1. The amino acid identity (47.3%) observed in the light chain between BoNT/F5 and /F1 is even more pronounced as is the identity between BoNT/F7 and /F1 (63.3%). 6. BoNT/F5 and /F7 did not cleave peptide substrates based upon the sequence of synaptobrevin-2 in the same location as all other BoNT/F. 7. BoNT/F5 and /F7 cleave full-length recombinant synaptobrevin-2 with the cleavage site identified by mass spectrometry.