Biotin labeling and receptor analysis of huwentoxin-I
Biotin labeling and receptor analysis of huwentoxin-I
复制标题
虎纹捕鸟毒素-I的生物素标记和受体分析
DOI：
10.1039/c3ay40455b
复制
发表时间：
2013-01-01
影响因子：
3.1
通讯作者：
Wang, Xian-Chun
中科院分区：
文献类型：
--
作者：
Yan, Yi-Zhong;Liu, Hui;Wang, Xian-Chun
虎纹捕鸟蛛毒素-I（HWTX - I）是虎纹捕鸟蛛粗毒中含量最高的肽类神经毒素。这种毒素可逆地阻断离体膈神经 - 半膈肌标本的神经 - 肌肉传导，作用位点在突触前膜。为了进一步研究HWTX - I的作用机制，本研究优化了HWTX - I生物素标记的实验条件，并使用位点选择性生物素化的毒素从大鼠膈神经 - 膈肌突触的质膜中亲和纯化并鉴定其受体蛋白。实验表明，当生物素化试剂与HWTX - I在冰上以2∶1的摩尔比反应2小时，大多数HWTX - I分子被单个生物素基团标记，产率为57%。串联质谱和电生理实验表明，单个生物素标记发生在HWTX - I的Lys13残基上，并且这种生物素化没有显著影响HWTX - I的生物活性。亲和纯化的蛋白质组分经过超滤、胰蛋白酶消化并通过CapLC - MS/MS分析后，鉴定出了几种膜蛋白，包括HWTX - I可能与之相互作用的电压门控钙通道蛋白，并讨论了它们在HWTX - I作用机制中的作用。
Huwentoxin-I (HWTX-I) is the highest content peptidic neurotoxin in the crude venom of spider Selenocosmia huwena. This toxin blocks the nerve-muscle conduction of isolated phrenic nerve-hemidiaphragm preparation reversibly, with the action site in the presynaptic membrane. In order to further investigate the action mechanism of HWTX-I, the present work optimized the experimental conditions for the biotin labeling of HWTX-I and used the site-selectively biotinylated toxin to affinity purify and identify its receptor proteins from the plasma membrane in rat phrenic nerve-diaphragm synapses. The experiments demonstrated that when the biotinylation reagent reacted with HWTX-I at a molar ratio of 2 : 1 on ice for 2 h, most HWTX-I molecules were labeled by a single biotin group, with a yield of 57%. Tandem mass spectrometry and electrophysiological experiments indicated that the single biotin labeling occurred at the Lys13 residue of HWTX-I and this biotinylation did not significantly affect the bioactivity of HWTX-I. After the affinity purified protein fraction was ultrafiltered, digested with trypsin and analyzed by CapLC-MS/MS, several membrane proteins including voltage-gated calcium channel proteins that HWTX-I potentially interacted with were identified and their roles in the action mechanism of HWTX-I were discussed.