Structural determinants of the specificity for synaptic vesicle-associated membrane protein/synaptobrevin of tetanus and botulinum type B and G neurotoxins.
Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic vesicle-associated membrane protein (VAMP)/synaptobrevin, an essential protein component of the vesicle docking and fusion apparatus. VAMP contains two copies of a nine-residue motif, also present in SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin, the two other substrates of clostridial neurotoxins. This motif was suggested to be a determinant of the target specificity of neurotoxins. Antibodies raised against this motif cross-react among VAMP, SNAP-25, and syntaxin and inhibit the proteolytic activity of the neurotoxins. Moreover, the various neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin recognition was probed by site-directed mutagenesis. Substitution of acidic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These results suggest that the two copies of the motif have a tandem association in the VAMP molecule.
Pellizzari
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R
Centro Consiglio Nazionale delle Ricerche di Biomembrane and Dipartimento di Scienze Biomediche, Università di Padova, Via Trieste 75, Padova, Italy.
Rossetto
O
O
Lozzi
L
L
Giovedi'
S
S
Johnson
E
E
Shone
C C
CC
Montecucco
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism; D003429:Cross Reactions; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D005089:Exocytosis; D007158:Immunologic Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D011994:Recombinant Proteins; D050600:SNARE Proteins; D013329:Structure-Activity Relationship; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin / Q000737:chemistry* / Q000378:metabolism; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D033921:Vesicular Transport Proteins; D010450:Endopeptidases; D001905:Botulinum Toxins