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.研究了l型钙通道阻滞剂ω-conotoxin TxVII和减缓钠通道失活的肽毒素δ-conotoxin TxVIA两种嵌合类似物（δω和ωδ）的合成。对8w的线性前驱体进行空气氧化，可获得所需的模拟物，产率高。而ωδ给出了复杂的二硫键异构体混合物。两步选择性二硫键形成策略也未能给出正确的产物，这表明δω缺乏三维结构形成所必需的残基。通过核磁共振和模拟退火计算分析了δ- concontoxin TxVIA的三维结构。δ-Conotoxin TxVIA分子的一侧有一个不寻常的疏水性斑块，这可能在钠离子通道结合中起重要作用。以酸性Glu （A22E）和碱性Lys （A22K）残基取代c端Ala22，合成了肌钠通道选择性阻滞剂μ- concontoxin GIIIA的两个类似物。A22E对钠通道突变体E765K具有较高的亲和力，表明μGJIIA的c端与钠通道结构域II密切相关。A22K的活性也低于天然μGIIIA，这表明Lys残基的大侧链可能会干扰其结合。用化学方法合成了从异角蝎毒液中分离得到的两种肽毒素κ-河豚毒素1和2，证实其与天然毒素相同。二硫键对通过酶切测定。核磁共振研究表明，κ-河狐毒素1采用独特的三维折叠，由两个二硫桥连接的两个平行螺旋，没有任何β-片。κ-河福毒素1不仅阻断了电压门控钾通道Kv1.3和Kv1.2，而且减慢了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.: "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., 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)

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,

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