海南捕鸟蛛毒素-IV突变体的固相合成和生理活性分析  

Solid-phase Synthesis and Biological Characterization of S12A-HNTX-IV and R29A-HNTX-IV: Two Mutants of Hainantoxin-IV

作　　者：徐侠[1] 熊霞[1] 李冬玲[1] 肖玉成[1] 王贤纯[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：《生物工程学报》2005年第1期92-96,共5页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划项目 (No .10 3-13-0 1-0 6)。~~

摘　　要：海南捕鸟蛛毒素_IV(HNTX_IV)是从我国海南捕鸟蛛粗毒中分离出的一种TTX_敏感型的钠离子通道阻断剂 ,由 35个氨基酸残基组成 ,含 3对二硫键。为了研究HNTX_IV结构与功能的关系 ,用芴甲氧羰基 (Fomc)固相多肽合成方法合成了用丙氨酸 (Ala)替代HNTX_IV第 12位丝氨酸 (Ser12 )的突变体S12A_HNTX_IV和替代第 2 9位精氨酸 (Arg2 9)的突变体R2 9A_HNTX_IV。合成的突变体经谷胱甘肽法氧化复性和纯化后 ,分别用MALDI_TOF质谱进行分子量鉴定 ,用一维核磁共振波谱法分析空间结构的变化 ,膜片钳电生理方法分析生物学活性。结果表明 ,Ser12和Arg2 9被Ala突变后没有明显影响分子的空间结构 ,S12A_HNTX_IV的生物学活性与天然HNTX_IV的相近 ,提示Ser12与HNTX_IV的生物学活性无关或关系不大 ;而R2 9A_HNTX_IV的生物学活性下降了 15 5倍 ,说明Arg2 9是与HNTX_IV生物学活性相关的关键残基之一。推测R2 9A_HNTX_IV活性的降低是由于Ala替代Arg后改变了HNTX_IV与受体作用的位点 ,而不是由于毒素分子整体空间结构变化所致。Hainantoxin_IV (HNTX_IV) purified from the venom of the spider Selenocosmia hainana is a potent antagonist that acts on tetrodotoxin_sensitive (TTX_S) sodium channels. It is a 35_residue polypeptide and includes three disulfide bridges. In order to investigate the structure_function relationship of HNTX_IV, two mutants (S12A_HNTX_IV and R29A_HNTX_IV) of HNTX_IV in which Ser12 and Arg29 were replaced by Ala respectively, were synthesized by solid_phase Fmoc chemistry, followed by oxidative refolding of purified peptides under the optimal conditions. The synthetic mutants were analyzed by MALDI_TOF mass spectrometry, nuclear magnetic resonance spectroscopy (NMR) and electrophysiological experiments for molecular weight, conformation and physiological activity, respectively. The results show that the mutants and native HNTX_IV (nHNTX_IV) have almost identical three_dimensional structures. The bioactivity level of S12A_HNTX_IV is also about the same as that of nHNTX_IV, suggesting that Ser12 does not play any important role for the bioactivity of this toxin. The bioactivity of R29A_ HNTX_IV is reduced by at last 155 times, indicating that Arg29 is a key residue relative to the bioactivity of HNTX_IV. It is presumed that the decrease in activity of R29A_HNTX_IV is due to the changes of the property in the binding site rather than the change in the basic conformation of the molecule.

关 键 词：海南捕鸟蛛毒素-IV 固相多肽合成 突变体 结构与功能 

分 类 号：Q51[生物学—生物化学]