Washbourne P, Bortoletto N, Graham M E, Wilson M C, Burgoyne R D, Montecucco C
Centro CNR Biomembrane and Department of Biomedical Sciences, University of Padova, Italy.
J Neurochem. 1999 Dec;73(6):2424-33. doi: 10.1046/j.1471-4159.1999.0732424.x.
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.
神经递质从突触小泡的释放由复杂的机制介导，该机制包括V型和T型突触融合蛋白受体（SNAREs）、囊泡相关膜蛋白（VAMP）、突触结合蛋白、 syntaxin以及25 kDa的突触体相关蛋白（SNAP - 25）。它们对于神经递质的胞吐作用至关重要，因为它们是梭菌神经毒素破伤风神经毒素和肉毒杆菌神经毒素（BoNTs）的蛋白水解底物，这两种毒素分别导致破伤风和肉毒中毒。具体而言，SNAP - 25在COOH末端的不同位点被BoNT/A和E切割。我们现在使用对毒素不敏感的SNAP - 25突变体证明，这两种毒素在切割位点的特异性上有所不同。在COOH末端进行修饰后，对BoNT/E完全抗性的突变体不与VAMP结合，但仍能与VAMP和syntaxin形成耐十二烷基硫酸钠的复合物。此外，这些突变体在体内保留功能，赋予转染的PC12细胞对BoNT/E抗性的胞吐作用。这些数据提供了关于SNAP - 25 C末端结构域在胞吐作用中的功能的结构要求信息，并对体外二元相互作用对突触蛋白复合物体内功能的意义提出了质疑。
