Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin
Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin
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DOI：
10.1074/jbc.m800611200
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发表时间：
2008-07-25
影响因子：
4.8
通讯作者：
Barbieri, Joseph T.
中科院分区：
文献类型：
--
作者：
Chen, Sheng;Hall, Cherisse;Barbieri, Joseph T.
肉毒神经毒素（BoNT;血清型A-G）和破伤风神经毒素分别引起弛缓性和痉挛性麻痹。这些神经毒素是锌蛋白酶，其切割SNARE蛋白以抑制突触囊泡融合到质膜。虽然BoNT/B和破伤风神经毒素（TeNT）在相同的易断键处切割VAMP-2，但它们识别VAMP-2的机制尚不清楚。作图实验表明，VAMP-2的残基60 - 87足以被BoNT/B有效切割，VAMP-2的残基40 - 87足以被TeNT有效切割。丙氨酸扫描诱变和动力学分析确定了三个区域内的VAMP-2，被BoNT/B和TeNT识别：残基附近的易断键裂解位点（裂解区）和残基位于N-末端和C-末端区域相对于裂解区。切割区域内残基的分析显示，VAMP-2的P7、P4、P2和P1'残基处的突变对LC/B切割具有最大的抑制（>= 32倍），而VAMP-2的P7、P4、P1'和P2'残基处的突变对LC/TeNT切割具有最大的抑制（>= 64倍）。切割区域内的残基影响催化，而切割区域的N-末端和C-末端残基影响结合亲和力。因此，BoNT/B和TeNT具有相似的结构，但具有识别和切割VAMP-2的独特残基。这些研究为梭菌神经毒素如何识别其底物提供了新的见解。
Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin ( TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60 - 87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40 - 87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage ( cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (>= 32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (>= 64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.