| 鞠双,颜清慧,刘思懿,等.芋螺毒素Tx3h的合成鉴定和活性的研究[J].中国海洋药物,2022,41(4):27-35. |
| 芋螺毒素Tx3h的合成鉴定和活性的研究 |
| Synthesis, Identification and Activity of Conotoxin Tx3h |
| 投稿时间：2022-05-07 修订日期：2022-06-01 |
| DOI： |
| 中文关键词: 富含二硫键的芋螺毒素 二硫键连接 一步氧化 乙酰胆碱受体 |
| English Keywords:Disulfide-rich Conotoxin Disulfide Mapping One-step oxidation nAChRs |
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| 中文摘要: |
| 目的 芋螺毒素是一类结构多样、数量庞大的海洋药源宝库。研究前期从织锦芋螺毒液中发现的芋螺毒素Tx3h，它的的结构和功能未见报道。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的两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10 μmol/L终浓度下，Tx3h-a和Tx3h-b对α4β2 nAChR的阻断率分别为(35.06 ± 4.90) %和(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。 |
| English Summary: |
| Abstract: Objective Conotoxins is a treasure of huge drug resource with diverse structures. A conotoxin Tx3h was previously identified from Conus texile venom, but no record of its structure and function have 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 was 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 against α4β2 and α3β2 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 on α3β2 nAChR at 10 μmol/L, while they inhibited (35.06 ± 4.90) % for Tx3h-a and (34.85 ± 3.02) % for Tx3h-b of ACh-evoked currents mediated by α4β2 nAChR. Conclusion We investigated the synthesis, disulfide mapping and inhibitory effects on α4β2 and α3β2 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 for α4β2 nAChR, which lay a foundation for further investigation of Tx3h. |
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