Cloning and activity of a novel α-latrotoxin from red-back spider venom
Cloning and activity of a novel α-latrotoxin from red-back spider venom
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DOI：
10.1016/j.bcp.2011.09.024
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发表时间：
2012-01-01
影响因子：
5.8
通讯作者：
Nicholson, Graham M.
中科院分区：
文献类型：
--
作者：
Graudins, Andis;Little, Michelle J.;Nicholson, Graham M.
欧洲黑寡妇蜘蛛Latrodectus tredecimguttatus的毒液含有几种高分子质量（110-140 kDa）的神经毒素，可诱导神经递质胞吐。其中包括一种脊椎动物特异性的latro昆虫毒素（alpha-LTX-Lt1a），导致latrodectism的临床症状，以及许多昆虫特异性的latro昆虫毒素（lit）。相比之下，尽管这些蜘蛛的中毒会引起类似的临床综合征，但人们对这些毒素在其他Latrodectus物种中的表达知之甚少。在此，我们利用串联质谱法报道了高度保守的α - ltx， α - lit和δ - lit序列标签，在生物测定引导下，用液相色谱法分离了美洲Latrodectus mactans， Latrodectus hesperus和Latrodectus hasselti毒液。尽管序列相似，研究人员发现，针对α - ltx - lt1a的抗α - ltx单克隆抗体4C4.1不能中和在离体鸡颈神经-肌肉生物测定中测试的所有其他Latrodectus毒液的神经毒性。这表明蜘蛛毒液中α - ltxs存在重要的结构差异。因此，我们克隆了澳大利亚红背蜘蛛L hasselti （α - ltx - lh1a）的α - ltx并对其进行了测序。成熟的α - ltx - lh1a氨基酸序列包含1180个残基（约132 kDa），与α - ltx - lt1a的同源性接近93%。α - ltx - lh1a由一个n端结构域和一个包含22个锚蛋白样重复序列的中心区域组成。两个furin切割位点的存在，与α - ltx - lt1a保守，表明α - ltx - lh1a来源于n端信号肽和c端前肽区域的蛋白水解切割。然而，我们发现α - ltx - lh1a在4C4.1表位上有关键的替换，这解释了单克隆抗体缺乏结合。(C) 2011爱思唯尔公司版权所有。
The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110-140 kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific alpha-latrotoxin (alpha-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved alpha-LTX, alpha-LIT and delta-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-alpha-LTX monoclonal antibody 4C4.1, raised against alpha-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve-muscle bioassay. This suggests that there are important structural differences between alpha-LTXs in theridiid spider venoms. We therefore cloned and sequenced the alpha-LTX from the Australian red-back spider L hasselti (alpha-LTX-Lh1a). The deduced amino acid sequence of the mature alpha-LTX-Lh1a comprises 1180 residues (similar to 132 kDa) with similar to 93% sequence identity with alpha-LTX-Lt1a. alpha-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with alpha-LTX-Lt1a, indicates that alpha-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that alpha-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody. (C) 2011 Elsevier Inc. All rights reserved.