Cloning and structure of delta-latroinsectotoxin, a novel insect-specific member of the latrotoxin family - Functional expression requires C-terminal truncation
Cloning and structure of delta-latroinsectotoxin, a novel insect-specific member of the latrotoxin family - Functional expression requires C-terminal truncation
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DOI：
10.1074/jbc.271.13.7535
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发表时间：
1996-03-29
影响因子：
4.8
通讯作者：
Usherwood, PNR
中科院分区：
文献类型：
--
作者：
Dulubova, IE;Krasnoperov, VG;Usherwood, PNR
黑寡妇蜘蛛（BWSV）（Latrodectus mactans tredecimguttatus）的毒液含有几种强效的高分子量（>110 kDa）神经毒素，这些毒素以门特异性方式引起神经递质释放。这些蛋白的分子作用机制知之甚少，因为它们的结构在很大程度上是未知的，并且它们还没有被功能性表达。（delta-LIT）是一种来自BWSV的新型昆虫特异性毒素，其含有1214个氨基酸，delta-LIT包括四个结构域：信号肽，随后是与其它latrotoxins表现出最高程度同一性的N-末端结构域，由15个锚定样重复组成的中心区域，和C-末端结构域。δ-LIT的结构域组织与其它latrotoxins的结构域组织相似，在delta-LIT基因中编码的蛋白质（类似于130 kDa）的预测分子量和表观迁移率不同于从BWSV纯化的天然delta-LIT（类似于110 kDa），这表明毒素是通过前体的蛋白水解加工产生的。纯化的天然delta-LIT的MALDI-MS显示m/z+为110916 +/-100的分子离子，表明天然delta-LIT的长度为991个氨基酸。当全长delta-LIT cDNA在细菌中表达时，蛋白质产物是无活性的，但表达一个C-含有991个残基的末端截短的蛋白质产生了一种蛋白质，该蛋白质以纳摩尔浓度在蝗虫神经肌肉接头处引起大量神经递质释放通过天然delta-LIT在蝗虫肌肉膜和人工脂质双层中形成的通道具有高Ca 2+渗透性，而通过截短的重组蛋白形成的通道则没有。
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed, This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids, delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain, The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins, The predicted molecular mass and apparent mobility of the protein (similar to 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (similar to 110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length, When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations, Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.