Botulinum A Like Type B and Tetanus Toxins Fulfils Criteria for Being a Zinc‐Dependent Protease

Abstract

Although botulinum neurotoxin (BoNT) types A and B and tetanus toxin (TeTx) are specific inhibitors of transmitter release whose light chains contain a zinc-binding motif characteristic of metalloendoproteases, only the latter two proteolyse synaptobrevin. Chelation of zinc or its readdition at high concentration hindered blockade of neuromuscular transmission by BoNT/A and B, indicating that type A also acts via a zinc-dependent mechanism. Such treatments prevented proteolysis of synaptobrevin II in rat brain synaptic vesicles by BoNT/B and TeTx but only the activity of the latter was antagonised appreciably by ASQFETS, a peptide spanning their cleavage site. The toxin's neuroparalytic activities were attenuated by phosphoramidon or captopril, inhibitors of certain zinc requiring proteases. However, these agents were ineffective in reducing the toxins' degradation of synaptobrevin except that a high concentration of captopril partially blocked the activity of TeTx but not BoNT/B, as also found for these drugs when tested on synaptosomal noradrenaline release. These various criteria establish that a zinc-dependent protease activity underlies the neurotoxicity of BoNT/A, a finding confirmed at motor nerve endings for type B and TeTx. Moreover, the low potencies of captopril and phosphoramidon in counteracting the toxins' effects necessitate the design of improved inhibitors for possible use in the clinical treatment of tetanus or botulism.

Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics

“…Moreover, the H C moiety of diphtheria toxin can be replaced with the binding domain from tetanus toxin without disruption of the acidification-dependent translocation of its enzymatic chain into cells (38). In contrast, the LC-H N moieties with an intact disulfide bridge are crucial for productive translocation (12,39,40). Noncovalent interactions are also involved in maintaining LC-H N interactions that appear to be important for structural stability, as a chimera consisting of the LC of BoNT/A linked to the HC of BoNT/E was poorly expressed, relatively insoluble, and susceptible to proteolysis (data not shown).…”

Recombinant Forms of Tetanus Toxin Engineered for Examining and Exploiting Neuronal Trafficking Pathways

“…After equilibration, TeTx, a recombinant sample (or buffer as a control) was added and toxin-induced neuromuscular paralysis was determined as the time taken for nerve-evoked muscle contraction to decrease to 10% of original value, as described previously (9,27). For competition studies, conditions designed to minimize toxin uptake into the nerve endings were used.…”

Activation of TRPV1 Mediates Calcitonin Gene-Related Peptide Release, Which Excites Trigeminal Sensory Neurons and Is Attenuated by a Retargeted Botulinum Toxin with Anti-Nociceptive Potential

“…BOTOX offers the advantages of having a much longer action and greater potency in blocking the release of transmitters and inflammatory/pain mediators via cleavage of SNAP-25, a 206-residue SNARE protein essential for neuroexocytosis (de Paiva et al, 1993). However, additional investigations are required to find a molecular basis for its beneficial effects in some migraineurs (Silberstein et al, 2000;Mauskop, 2002), which are not consistently observed (for review, see Naumann et al, 2008).…”