Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25 for proteolysis.
Botulinum neurotoxins type A and E (BoNT/A and BoNT/E) are metalloproteases with a unique specificity for SNAP-25 (synaptosome-associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It has been suggested that this specificity is directed through the recognition of a nine residue sequence, termed SNARE motif, that 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 analyse the involvement of the four copies of the SNARE motif present in SNAP-25 in its interaction with BoNT/A and BoNT/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 BoNT/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. Also, a non-neuronal isoform of SNAP-25, Syndet, is shown to be sensitive to BoNT/E, but not BoNT/A, whilst the SNAP-25 isoforms from Torpedo marmorata and Drosophila melanogaster were demonstrated not to be substrates of these metalloproteases.
Washbourne
P
P
Centro C.N.R. Biomembrane and Dipartimento di Scienze Biomediche, Università di Padova, Padua, Italy.
Pellizzari
R
R
Baldini
G
G
Wilson
M C
MC
Montecucco
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D050825:Synaptosomal-Associated Protein 25; D014101:Torpedo; D033921:Vesicular Transport Proteins
D029721:Drosophila Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C494208:SNAP25 protein, human; D050600:SNARE Proteins; C494207:Snap25 protein, Drosophila; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E