ABSTRACT
Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT.
Subject(s)
Metalloendopeptidases/chemistry , Tetanus Toxin/chemistry , Amino Acid Sequence , Botulinum Toxins, Type A/chemistry , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Molecular Sequence Data , Mutagenesis , R-SNARE Proteins , Structure-Activity Relationship , Tetanus Toxin/metabolism , Zinc/metabolismABSTRACT
Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by both BoNT/A and E at separate sites within the COOH-terminus. We now demonstrate, using toxin-insensitive mutants of SNAP-25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH-terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate-resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.
Subject(s)
Botulinum Toxins/pharmacology , Calcium-Binding Proteins , Exocytosis/drug effects , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites/drug effects , COS Cells , Chlorocebus aethiops , Drug Resistance , Growth Hormone/metabolism , Macromolecular Substances , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/genetics , PC12 Cells , Protein Binding , R-SNARE Proteins , Rats , Recombinant Fusion Proteins/metabolism , Sodium Dodecyl Sulfate/pharmacology , Synaptic Vesicles/drug effects , Synaptic Vesicles/metabolism , Synaptosomal-Associated Protein 25 , SynaptotagminsABSTRACT
Botulinum neurotoxins type A and E (BoNT/A and /E) are metalloproteases with a unique specificity for SNAP-25 (synaptosomal-associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It was proposed that this specificity is based on the recognition of a nine-residue sequence, termed SNARE motif, which is common to the other two SNARE proteins: VAMP (vesicle-associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we report on recent studies which provide evidence for the involvement of the SNARE motif present in SNAP-25 in its interaction with BoNT/A and /E by following the kinetics of proteolysis of SNAP-25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and /E to recognise SNAP-25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. We also report on studies of poisoning human neuromuscular junctions with either BoNT/A or BoNT/E and describe the unexpected finding that the time of recovery of function after poisoning is much shorter in the case of type E with respect to type A intoxication. These data are discussed in terms of the different sites of action of the two toxins within SNAP-25.
