Botulinum neurotoxin serotype B (BoNT/B) and
tetanus toxin (TeTx) block neuroexocytosis through selective endoproteolysis of
vesicle-associated membrane protein (VAMP). The enzymological properties of both toxins were compared for the first time in their cleavage of VAMP and various sized fragments using a sensitive chromatographic assay. The optimal substrate sizes for the
zinc-dependent
protease activities of the light chains of TeTx and BoNT/B were established using synthetic
peptides corresponding to the hydrophilic core of VAMP (30-62
amino acids in length). TeTx was found to selectively cleave the largest
peptide at a single site, Gln76-Phe77. It exhibited the most demanding specificity, requiring the entire hydrophilic domain (a 62-mer) for notable hydrolysis, whereas BoNT/B efficiently cleaved the much smaller 40-mer. Thus, an unusually long N-terminal sequence of 44
amino acids upstream of the scissile bond is required for the selective hydrolysis of VAMP by TeTx. Using the largest
peptide, BoNT/B and TeTx exhibited approximately 50% and 35%, respectively, of the activities shown toward intact VAMP,
detergent solubilized from synaptic vesicles. Given the large size of the smallest substrates, it is possible that these
neurotoxins recognize and require a three-dimensional structure. Although both toxins were inactivated by divalent
metal chelators, neither was antagonized by
phosphoramidon or ASQFETS (a substrate-related
peptide that spans the cleavage site), and TeTx was only feebly inhibited by
captopril; also, they were distinguishable in their relative activities at different pHs, temperatures, and ionic strengths. These collective findings are important in the design of effective inhibitors for both toxins, as well as in raising the possibility that TeTx and BoNT/B interact somewhat differently with VAMP.