摘要
目的对前期从织锦芋螺毒液中发现的芋螺毒素Tx3h进行合成鉴定和活性研究。方法通过固相多肽合成法(SPPS)人工合成了Tx3h的线性肽,采用一步氧化法对线性肽进行氧化折叠,获得了Tx3h的2个二硫键连接不同的折叠肽异构体(Tx3h-a和Tx3h-b),并对其氧化折叠条件进行了优化。通过部分还原及烷基化反应,结合MALDI-TOF-MS/MS分析,鉴定了Tx3h-a和Tx3h-b的二硫键连接方式。采用双电极电压膜片钳技术测试了Tx3h-a和Tx3h-b对α4β2和α3β2 nAChRs的抑制作用。结果通过氧化折叠条件的优化,Tx3h-a和Tx3h-b的产率分别从7.5%和9.3%提高到23.7%和22.4%。Tx3h-a和Tx3h-b的二硫键连接方式分别鉴定为I-V、II-VI、III-IV和I-VI、II-IV、III-V。在10μmol/L终浓度下,Tx3h-a和Tx3h-b对α4β2 nAChR的阻断率分别为(35.06±4.90)%、(34.85±3.02)%,对α3β2 nAChR无阻断作用。结论通过对芋螺毒素Tx3h折叠肽的合成、二硫键连接鉴定以及乙酰胆碱受体活性检测,发现二硫键连接为I-V,II-VI,III-IV和I-VI,II-IV,III-V的Tx3h折叠肽对α4β2 nAChR有更好的选择性,为其后续研究开发奠定了基础。
Objective Conotoxins is a treasure of huge drug resource with diverse structures.Conotoxin Tx3h was previously identified from Conus texile venom,but its structure and function have not been reported.Methods The linear peptide of Tx3h was synthesized by SPPS chemistry and was subsequently folded by one-step oxidation,which generated two folded isomers with different disulfide connectivities,named Tx3h-a and Tx3h-b.The oxidative folding conditions were optimized.The disulfide connectivities of Tx3h-a and Tx3h-b were characterized by partial reduction and subsequent alkylation combined with MALDI-TOF-MS/MS analysis.The inhibitory activities againstα4β2 andα3β2 nAChRs for Tx3h-a and Tx3h-b were determined by double-electrode voltage patch clamp technology.Results Through folding condition optimization,yields of Tx3h-a and Tx3h-b increased from 7.5%and 9.3%to 23.7%and 22.4%.The disulfide connectivities were characterized to be I-V,II-VI,III-IV for Tx3h-a and I-VI,II-IV,III-V for Tx3h-b.Tx3h-a and Tx3h-b displayed no blocking effect onα3β2 nAChR at 10μmol/L,while they inhibited(35.06±4.90)%for Tx3h-a and(34.85±3.02)%for Tx3h-b of ACh-evoked currents mediated byα4β2 nAChR.Conclusion We investigated the synthesis,disulfide mapping and inhibitory effects onα4β2 andα3β2 nAChRs,we found the folded Tx3h with disulfide connectivities of I-V,II-VI,III-IV and I-VI,II-IV,III-V showed better selectivity forα4β2 nAChR,which laid a foundation for further investigation of Tx3h.
作者
鞠双
颜清慧
刘思懿
张雨
罗素兰
符影
JU Shuang;YAN Qing-hui;LIU Si-yi;ZHANG Yu;LUO Su-lan;FU Ying(Key Laboratory of Tropical Biological Resources of Ministry of Education,School of Pharmaceutical Sciences,Hainan University,Haikou 570228,China;School of Medicine,Guangxi University,Nanning 530004,China)
出处
《中国海洋药物》
CAS
CSCD
2022年第4期27-35,共9页
Chinese Journal of Marine Drugs
基金
国家自然科学基金青年基金项目(81903492)
海南省自然科学基金高层次人才项目(2019RC053)资助。
