Peptide Substrate Specificity and Properties of the zinc‐endopeptidase Activity of Botulinum Type B Neurotoxin

Abstract

Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle-associated membrane protein (VAMP). A synthetic peptide of h u m d r a t VAMP-2 [ VAMP-2-(60-94)] is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane-associated protein (at the Gln76-Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin. Cleavage of the VAMP-2 peptide was demonstrated by both botulinum type B neurotoxin (K, = 3.3X10-4M) and by its purified light subunit (K, = 3.5X10-4M). The endopeptidase displayed a pH optimum of 7.0-7.5 and was inhibited by greater than 0.2 M NaCl and greater than 0.05 M sodium phosphate. Neurotoxin which had been inactivated by dialysis against EDTA could be re-activated by incubation with various divalent cations, notably Znz+ and Cu2+. The substrate specificity of botulinum type B neurotoxin was studied using various analogues of VAMP-2 (60-94). The neurotoxin cleaved selectively to the N-terminal side of phenylalanine and tyrosine; no activity was observed with either leucine, valine or alanine in the P: position. The properties of the P, amino acid were less critical; the neurotoxin cleaving the Cterminus of glutamine, asparagine and alanine. A substrate analogue with valine in the P, position corresponding to the sequence of rat VAMP-1 was not cleaved. The rate of cleavage of a substrate analogue representing the sequence of human VAMP-1, however, was more than twofold that of the VAMP-2 peptide. The properties and substrate specificity of botulinum type B neurotoxin suggest that the toxin represents a novel class of endopeptidase which requires a specific peptide substrate conformation for the expression of proteolytic activity.

Botulinum Neurotoxin Serotype F: Identification of Substrate Recognition Requirements and Development of Inhibitors with Low Nanomolar Affinity

“…In the BoNT F minimum substrate, 27 of the required residues are on the N-terminal side of the scissile bond, while only seven are needed on the C-terminal side. The latter situation is similar to that of the BoNT A substrate, with only five residues C-terminal to the cleavage site (31), but differs from that of BoNT B, where 18 residues are needed (29).…”

Substrate residues N‐terminal to the cleavage site of botulinum type B neurotoxin play a role in determining the specificity of its endopeptidase activity

“…Even larger substrates (>50 residues) are required by tetanus toxin and botulinum type A neurotoxin. Surprisingly, in a study of the peptide substrate specificity of BoNT/B only one VAMP residue at the cleavage site (Phe77) was found to be critical to the endopeptidase activity of the toxin [14]. These data suggest that determinants other than the cleavage site sequence are required to account for the neurotoxin's highly specific proteolytic activity.…”

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

“…The strongest cleavage defect in vitro resulted from the S75F mutation at the P2 position; Ϸ1,500-fold excess B-LC was required to achieve 50% cleavage. Prior studies have shown that S75A and S75T substitutions, which are more conservative than our S75F substitution, exert modest defects on the rate of Sb fragment cleavage in vitro (9). The strong cleavage defect conferred by S75F leads us to suggest that B-LC does not recognize Sb when it contains residues with large and͞or aromatic side chains at the P2 position.…”

“…Additional Sb residues are probably required for efficient B-LC recognition. Indeed, we were surprised when amino acid substitutions did not appear at the PЈ1 position (F73) of Snc2͞Sb͞Snc2 by yeast selection; Sb PЈ1 residue (F77) was implicated previously in B-LC recognition (9). Absence of PЈ1 substitutions in our yeast genetic selection can be explained by the fact that when we constructed an F73A substitution in Snc2͞Sb͞Snc2 and expressed it in yeast, the mutant SNARE did not support cell growth (data not shown).…”

“…To probe LC substrate specificity, cell-free assays have been developed, employing purified endoprotease and soluble SNARE protein fragments to probe cleavage of mutated SNARE sequences. The model emerging from these studies is one where cleavage site residues and residues positioned in the SNARE motif, a conserved 9-to 10-residue element, are important for LC substrate recognition (4,5,(8)(9)(10)). An alternative approach to elucidate substrate specificity comes from the threedimensional structure of serotype A-LC in complex with a SNAP-25 protein fragment (6,7).…”