Evans E R, Sutton J M, Gravett A, Shone C C
Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, Wilts. SP4 0JG, UK.
Toxicon. 2005 Sep 15;46(4):446-53. doi: 10.1016/j.toxicon.2005.06.006.
The botulinum neurotoxin endopeptidases appear to recognise their intracellular protein substrates via two distinct sites: the cleavage site sequence and a 'recognition site' motif. In the present study phage display has been employed to generate a library of vesicle-associated membrane protein (VAMP2) variants in which the toxin recognition motif (part of the SNARE motif ELDDRADA) has been modified. VAMP (1-94) was displayed on the surface of M13 bacteriophage and this fragment was recognised and cleaved by botulinum neurotoxin type B (BoNT/B). A phage-displayed library was constructed in which six residues of the recognition domain (VAMP residues 63-68; wild-type sequence LDDRAD) were randomised, and a selection method established for identifying cleaved VAMP variants. Sequence analysis of 24 clones revealed that 5 contained two acidic residues although none corresponded to the native sequence. Cleavage was reduced compared to wild-type VAMP, and cleavage of mutants containing no acidic residues was also observed. The data are discussed in relation to the substrate recognition mechanism of BoNT/B.
切割位点序列和一个“识别位点”基序。在本研究中，噬菌体展示技术被用于生成一个囊泡相关膜蛋白（VAMP2）变体文库，其中毒素识别基序（SNARE基序ELDDRADA的一部分）已被修饰。VAMP（1-94）展示在M13噬菌体表面，该片段被B型肉毒杆菌神经毒素（BoNT/B）识别并切割。构建了一个噬菌体展示文库，其中识别结构域的六个残基（VAMP残基63-68；野生型序列LDDRAD）被随机化，并建立了一种用于鉴定被切割的VAMP变体的筛选方法。对24个克隆的序列分析表明，5个克隆含有两个酸性残基，尽管没有一个与天然序列相对应。与野生型VAMP相比，切割减少，并且还观察到不含酸性残基的突变体的切割。结合BoNT/B的底物识别机制对这些数据进行了讨论。
