芋螺毒素Lv68的一步氧化及其对乙酰胆碱受体的阻断活性

One-step Oxidative Folding and nAChR Blocking Activity of Conotoxin Lv68

本研究通过固相合成法合成芋螺毒素Lv68线性肽,研究并优化了其主要异构体的一步氧化折叠条件,并通过电压膜片钳技术对主异构体产物进行了多种乙酰胆碱受体亚型(α3β4,α6β4,α4β2,α9α10,α1β1γδ,α1β1δεnAChRs)的活性测试,旨在为以烟碱型乙酰胆碱受体为靶点的先导药物分子的发现奠定基础,同时为该类其他芋螺毒素的氧化折叠方法、生物活性研究以及深入开发利用提供参考和借鉴。结果表明,芋螺毒素Lv68的一步法氧化折叠最优条件为反应体系组成为0.1 mol·L^−1 Tris-HCl,1 mmol·L^−1 EDTA,GSH/GSSG 1 mmol·L^−1/1 mmol·L^−1,线性肽浓度为50μmol·L^−1,反应时间为2 h,产率高达18.14%,Lv68折叠肽在1μmol·L^−1浓度下对α9α10 nAChR具有较好的阻断作用(阻断率约80%)。本研究明确了芋螺毒素Lv68的一步氧化折叠条件,证实了Lv68对α9α10乙酰胆碱受体亚型具有阻断活性,拓宽了含半胱氨酸框架Ⅲ的M超家族芋螺毒素的作用靶点范围。

Conotoxin Lv68 is a new M superfamily conotoxin cloned from the cDNA of Conus lividus from Hainan.It consists of 15 amino acid residues,and contains 6 cysteine,and the cysteine framework is-CC-C-CCC-(type Ⅲ).Linear peptide of conotoxin Lv68 was synthesized by using Solid Phase Peptide Synthesis.The one-step oxidative conditions of disulfide bond folding for the major folded products of Lv68 were studied and optimized.Bioactivities of the major folded products were assayed on a series of nicotinic acetylcholine receptor(nAChRs)subtypes(α3β4,α6β4,α4β2,α9α10,α1β1γδandα1β1δε)by using the voltage patch clamp technology.The Lv68 produced maximum folding yield(18.14%)under the optimized folding conditions including reaction system composition(0.1 mol·L^−1 Tris-HCl,1 mmol·L^−1 EDTA,GSH/GSSG 1 mmol·L^−1/1 mmol·L^−1),linear peptide concentration(50μmol·L^−1),and reaction time(2 h).A medium blocking effect(80%inhibition)onα9α10 nAChR subtype was detected for the Lv68 at the concentration of 1μmol·L^−1.These results confirmed the optimized folding conditions of conotoxin Lv68 and its activity to blockα9α10 nAChR,which broadened the target range of the M superfamily conotoxins containing cysteine frameworkⅢ.The results not only lay the foundation for the further development of Lv68 as an analgesic drug lead targetingα9α10 nAChR,but also provide a reference and guidance for the oxidative folding,biological activity research and further development and utilization of other M-conotoxins containing cysteine frameworkⅢ.