Wang Lei, Liu Junliang, Ren Zhenghua, Chen Yu, Xu Anlong
Department of Pharmaceutical Engineering, College of Materials and Energy, College of Marine Science, South China Agricultural University, Guangzhou, 510642, PR China; State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, National Engineering Center of South China Sea for Marine Biotechnology, Department of Biochemistry, College of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, PR China.
State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, National Engineering Center of South China Sea for Marine Biotechnology, Department of Biochemistry, College of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, PR China.
Toxicon. 2017 Aug;134:6-13. doi: 10.1016/j.toxicon.2017.05.020. Epub 2017 May 19.
In this work, two P-superfamily conotoxins, lt9a and lt9b, were purified and characterized from the crude venom of Conus litteratus. The amino acid sequences of lt9a and lt9b were determined by the Edman degradation method. It has been suggested that both lt9a and lt9b are produced from the precursor encoded by the gene Lt9.1. During the conotoxin maturation process, different post-translational modifications occurred between lt9a and lt9b. Conotoxin lt9b was predicted to have two prolines that underwent hydroxylation and one glutamate that underwent carboxylation, while lt9a had no hydroxyproline and carboxyglutamate residue. The calculated mass weights of two P-superfamily conotoxins with three proposed disulfide bonds were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry after considering corresponding post-translational modifications. These two conotoxins showed different effects on tetrodotoxin-sensitive sodium currents. Conotoxin lt9a (300 nM) resulted in marked slowing of the tetrodotoxin-sensitive sodium current decay, a notable increase in the peak current, and an alteration in reversal potential. However, lt9b inhibits tetrodotoxin-sensitive sodium currents, and the inhibition showed a concentration-dependent with the half maximal inhibitory concentration (IC50) value of 504.04 nM, but there were no change in the activation and inactivation kinetics of currents. To the best of our knowledge, this is the first investigation of two P-superfamily conotoxins identified to act on voltage-sensitive sodium channels with different modifications.
在这项研究中，从宝石芋螺的粗毒液中纯化并鉴定了两种P-超家族芋螺毒素lt9a和lt9b。通过埃德曼降解法测定了lt9a和lt9b的氨基酸序列。研究表明，lt9a和lt9b均由基因Lt9.1编码的前体产生。在芋螺毒素成熟过程中，lt9a和lt9b发生了不同的翻译后修饰。预测芋螺毒素lt9b有两个脯氨酸发生了羟基化，一个谷氨酸发生了羧化，而lt9a没有羟脯氨酸和羧基谷氨酸残基。在考虑相应的翻译后修饰后，通过基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱法确认了具有三个假定二硫键的两种P-超家族芋螺毒素的计算质量重量。这两种芋螺毒素对河豚毒素敏感的钠电流表现出不同的作用。芋螺毒素lt9a（300 nM）导致河豚毒素敏感的钠电流衰减明显减慢，峰值电流显著增加，以及反转电位改变。然而，lt9b抑制河豚毒素敏感的钠电流，且抑制作用呈浓度依赖性，半数最大抑制浓度（IC50）值为504.04 nM，但电流的激活和失活动力学没有变化。据我们所知，这是首次对两种经鉴定作用于电压敏感钠通道且具有不同修饰的P-超家族芋螺毒素进行的研究。
