DIFFERENCES IN THE PROTEASE ACTIVITIES OF TETANUS AND BOTULINUM-B TOXINS REVEALED BY THE CLEAVAGE OF VESICLE-ASSOCIATED MEMBRANE-PROTEIN AND VARIOUS SIZED FRAGMENTS

Botulinum neurotoxin serotype B (BoNT/B) and tetanus toxin (TeTx) bloc k neuroexocytosis through selective endoproteolysis of vesicle-associa ted membrane protein (VAMP). The enzymological properties of both toxi ns were compared for the first time in their cleavage of VAMP and vari ous sized fragments using a sensitive chromatographic assay. The optim al substrate sizes for the zinc-dependent protease activities of the l ight chains of TeTx and BoNT/B were established using synthetic peptid es 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 specifici ty, requiring the entire hydrophilic domain (a 62-mer) for notable hyd rolysis, whereas BoNT/B efficiently cleaved the much smaller 40-mer. T hus, 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 similar to 50% and 35%, respectively, of the activities shown toward intact VA MP, detergent solubilized from synaptic vesicles. Given the large size of the smallest substrates, it is possible that these neurotoxins rec ognize 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; a lso, they were distinguishable in their relative activities at differe nt pHs, temperatures, and ionic strengths. These collective findings a re important in the design of effective inhibitors for both toxins, as well as in raising the possibility that TeTx and BoNT/B interact some what differently with VAMP.