Washbourne P, Pellizzari R, Baldini G, Wilson M C, Montecucco C
Centro C.N.R. Biomembrane and Dipartimento di Scienze Biomediche, Università di Padova, Padua, Italy.
FEBS Lett. 1997 Nov 24;418(1-2):1-5. doi: 10.1016/s0014-5793(97)01328-8.
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.
A型和E型肉毒杆菌神经毒素（BoNT/A和BoNT/E）是金属蛋白酶，对SNAP-25（25 kDa的突触体相关蛋白）具有独特的特异性，SNAP-25是神经胞吐机制的一种重要蛋白质成分。有人提出，这种特异性是通过识别一个九残基序列来实现的，该序列被称为SNARE基序，它是另外两种SNARE蛋白（囊泡相关膜蛋白VAMP和 syntaxin）所共有的，而这两种蛋白是其他六种梭菌神经毒素仅有的已知底物。在这里，我们通过跟踪缺失SNARE基序的SNAP-25突变体的蛋白水解动力学，分析了SNAP-25中存在的四个SNARE基序拷贝在其与BoNT/A和BoNT/E相互作用中的作用。我们发现，一个基序拷贝就足以让BoNT/A和BoNT/E识别SNAP-25。虽然靠近切割位点的基序拷贝显然参与了识别，但在其缺失的情况下，其他更远端的基序拷贝也能够支持蛋白水解。此外，SNAP-25的一种非神经元异构体Syndet被证明对BoNT/E敏感，但对BoNT/A不敏感，而电鳐和果蝇的SNAP-25异构体被证明不是这些金属蛋白酶的底物。
