Arg26和Lys27突变对海南捕鸟蛛毒素-Ⅳ生物活性影响  

Effects of Arg26 and Lys27 Mutation on Bioactivity of HNTX-Ⅳ

作　　者：熊霞[1] 徐侠[1] 李冬玲[1] 陈平[1] 梁宋平[1] 

机构地区：[1]湖南师范大学生命科学学院,长沙410081

出　　处：《中国生物化学与分子生物学报》2005年第4期499-503,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家高技术研究发展计划项目(No.103130106)~~

摘　　要：海南捕鸟蛛毒素Ⅳ(hainantoxin-Ⅳ,HNTX-Ⅳ)是一种新型的从海南捕鸟蛛粗毒中分离纯化的作用于河豚毒素敏感型(tetrodotoxinsensitive,TTXS)钠通道阻断剂.采用2D1HNMR技术解析HNTXⅣ的空间结构为胱氨酸抑制剂结模体,为进一步阐述HNTX-Ⅳ结构与功能的关系,应用固相Fmoc方法化学合成了用丙氨酸代替海南捕鸟蛛毒素Ⅳ第26位精氨酸的单残基突变体R26AHNTXⅣ和第27位赖氨酸的单残基突变体K27AHNTXⅣ.合成的突变体用谷胱甘肽法氧化复性并通过反相高效液相色谱(RPHPLC)纯化.通过MALDITOF质谱测定突变体的分子量.通过核磁共振谱仪测定突变体的空间结构.通过全细胞膜片钳实验比较天然HNTX-Ⅳ(nHNTX-Ⅳ)和两个突变体分子的生物学活性.结果发现,nHNTX-Ⅳ的R26或K27被突变后的空间结构没有发生明显变化.R26AHNTX-Ⅳ能明显抑制TTXS钠电流,K27AHNTX-Ⅳ对TTXS钠电流无明显影响.说明第26位的精氨酸与HNTX-Ⅳ的生物学活性无关,而第27位赖氨酸则是HNTX-Ⅳ的关键残基.Hainantoxin-Ⅳ （HNTX-Ⅳ） is a novel antagonist of tetrodotoxin-sensitive sodium channels isolated from the Chinese bird spider Selenocosmia hainana Wang. The analysis of three dimensional structure of HNTX-Ⅳ, solved by two dimensional nuclear magnetic resonance （2D NMR）, reveals that it adopts an inhibitor cystine knot structural motif. To explore the relationship between structure and function of HNTX-Ⅳ, the solid-phase chemical synthesis of R26A-HNTX-Ⅳ and K27A-HNTX-Ⅳ, two mutants of HNTX-Ⅳ, where Arg-26 or Lys-27 was substituted with Ala, was carried out using the Fmoc chemistry on the Pioneer Peptide Synthesizer System. The synthetic peptides were purified and then oxidatively refolded under the optimal conditions. The synthetic mutants were analyzed by MALDI-TOF mass spectrometry for molecular weight. The conformation of oxidative refolding of mutants were determined by NMR spectroscopy. The results show that the mutants and native HNTX-Ⅳ have similar three dimensional structure. Using the whole-cell patch-clamp technique, R26A-HNTX-Ⅳ was demonstrated to inhibit tetrodotoxin-sensitive （TIX-S） sodium currents significantly and K27A-HNTX-Ⅳ failed to affect TTX-S sodium currents, indicating that Lys 27 but not Arg 26 is the key residue related to the bioactivity of HNTX-Ⅳ.

关 键 词：海南捕鸟蛛 HNTX-Ⅳ 突变体 固相多肽合成 生物活性 

分 类 号：Q25[生物学—细胞生物学] Q343