Synthetic study of peptide toxins for the elucidation of functions of ion channels

用于阐明离子通道功能的肽毒素的合成研究

• 批准号: 13680675
• 负责人: SATO Kazuki
• 金额: $ 2.24万
• 依托单位: Fukuoka Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680675/
• 关键词: ion channel; peptide toxin; conotoxin; cone snail toxin; peptide synthesis; chimeric analog; イモ貝

1.Syntheses of two chimeric analogs (δω and ωδ) of ω-conotoxin TxVII, an L-type calcium channel blocker, and δ-conotoxin TxVIA, a peptide toxin that slows the inactivation of sodium channels were examined. Air oxidation of a linear precursor of 8w afforded desired analog in good yield. However, that of ωδ gave complex mixture of disulfide bond isomers. Two-step selective disulfide bond formation strategy also failed to give correct product, suggesting that δω lack the residues essential for the 3D structure formation. 3D structure of δ-conotoxin TxVIA was analyzed by NMR and simulated annealing calculations. δ-Conotoxin TxVIA showed an unusual hydrophobic patch on one side of the molecule, which may play am important role in, the sodium channel binding.2.Two analogs of μ-conotoxin GIIIA, a selective blocker of muscle sodium channels, were synthesized by replacing C-terminal Ala22 with acidic Glu (A22E) and basic Lys (A22K) residues. A22E was 90-fold less active than native μGJIIA, however it showed high affinity against E765K mutant of sodium channel, indicating that C-terminal part of μGIIIA closely associates with domain II of sodium channels. A22K was also less active than native μGIIIA, suggesting that the large side chain of Lys residue may interfere the binding.3.Two peptide toxins, κ-hefutoxin 1 and 2, isolated from the venom of the scorpion Heterometrus fulvipes were chemically synthesized and confirmed to be identical to native toxins. The disulfide bond pairings were determined by enzymatic digestion. NMR studies indicated that κ-hefutoxin 1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any β-sheets. κ-Hefutoxin 1 not only blocks the voltage-gated potassium channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents.

1。的ω-核毒素TXVII的两个嵌合类似物（δΩ和ωδ）的合成，一个L型钙通道阻滞剂和δ-康型毒素TXVIA，一种肽毒素TXVIA，一种肽毒素，它减慢了减慢钠通道的失活的肽。 8W的线性前体的空气氧化物具有良好产量的所需类似物。但是，ωδ的二硫键异构体的复杂混合物。两步选择性的二硫键形成策略也未能给出正确的产物，这表明δΩ缺乏3D结构形成必不可少的残差。通过NMR和模拟退火计算分析了δ-连毒素TXVIA的3D结构。 Δ-核毒素TXVIA在分子的一侧显示出异常的疏水斑，可能在钠通道结合中起重要作用。2。µ-孔毒素GIIIA的类似物，μ-孔毒素GIIIA（肌肉钠通道的选择性阻滞剂，肌肉钠通道的选择性阻滞剂），通过替换C- terminal intrical Ala a2222222222。残差。 A22E的活性比天然μGJIIA低90倍，但是它显示出对钠通道的E765K突变体的高亲和力，这表明μGIIIA的C末端部分与钠通道的域II紧密关联。 A22K的活性也不如天然μGIIIA活跃，这表明Lys居住的大侧链可能会干扰结合。3.two辣椒，κ-脱氧蛋白1和2，是从蝎子异质体的毒液中分离出来的。通过酶消化确定二硫键配对。 NMR的研究表明，κ-脱氧蛋白1采用了两个平行螺旋的独特三维折叠，由两个没有任何β-折叠的二硫键连接。 κ-脱氧蛋白1不仅阻止了电压门控钾通道KV1.3和KV1.2，而且还会减慢KV1.3电流的激活动力学。

项目成果

Li, R.A.: "Charge conversion enables quantification of the proximity between a normally-neutral μ-conotoxin (GIIIA) site and the Na^+ channel pore"FEBS Lett.. 511. 159-164 (2002)

Li, R.A.：“电荷转换能够量化正常中性 μ-芋螺毒素 (GIIIA) 位点和 Na^+ 通道孔之间的接近程度”FEBS Lett.. 511. 159-164 (2002)

Nakamura, M., Niwa, Y., Ishida, Y., Kohno, T., Sato, K., Oba, Y., Nakamura, H.: "Modification of Arg-13 of μ-conotoxin GIIIA with piperidinyl-Arg analogs and their relation to the inhibition of sodium channels"FEBS Lett.. 503. 107-110 (2001)

Nakamura, M.、Niwa, Y.、Ishida, Y.、Kohno, T.、Sato, K.、Oba, Y.、Nakamura, H.：“用哌啶基-Arg 修饰 μ-芋螺毒素 GIIIA 的 Arg-13类似物及其与钠通道抑制的关系”FEBS Lett.. 503. 107-110 (2001)

Nakamura, M.: "Generation of polyclonal antibody against μ-Conotoxin GIIIA using an immunogen of [Cys^5]μ-conotoxin GIIIA site-specifically conjugated with bovine serum albumin"Biochem.Biophys.Res.Commun.. 290. 1037-1041 (2002)

Nakamura, M.：“使用与牛血清白蛋白位点特异性缀合的 [Cys^5]μ-芋螺毒素 GIIIA 免疫原生成针对 μ-芋螺毒素 GIIIA 的多克隆抗体”Biochem.Biophys.Res.Commun.. 290. 1037- 1041 (2002)

Nakamura, M., Oba, Y., Mod, T., Sato, K., Ishida, Y., Matsuda, T., Nakamura, H.: "Generation of polyclonal antibody against μ-Conotoxin GIIIA using an immunogen of [Cys^5]-conotoxin GIIIA site-specifically conjugated with bovine serum albumin"Biochem.Biop

Nakamura, M.、Oba, Y.、Mod, T.、Sato, K.、Ishida, Y.、Matsuda, T.、Nakamura, H.：“使用免疫原生成针对 μ-芋螺毒素 GIIIA 的多克隆抗体[ Cys^5]-芋螺毒素 GIIIA 与牛血清白蛋白位点特异性缀合“Biochem.Biop

Srinivasan, K.N., Sivaraja, V., Huys, I., Sasaki, T., Cheng, B., Kumar, T.K., Sato, K., Tytgat, J., Yu., C., San, B.C.C., Ranganathan, S., Bowie, H.J., Kini, R.M., Gopalakrishnakone: "K-Hefutoxin 1, a novel toxin from the scorpion heterometrusfidvipes wit

Srinivasan, K.N.、Sivaraja, V.、Huys, I.、Sasaki, T.、Cheng, B.、Kumar, T.K.、Sato, K.、Tytgat, J.、Yu., C.、San, B.C.C.、Ranganathan,