摘要
为了进一步探究第11位氨基酸的性质对LvIA靶点结合活性的影响,设计了LvIA的2个新型突变体[D11R]LvIA和[D11H]LvIA,即用2个碱性氨基酸-精氨酸(R)和组氨酸(H)分别替换原来的酸性氨基酸D。先人工合成了这2个新突变体的线性肽,然后采用2步氧化法进行折叠,以获得在第1位和第3位半胱氨酸(Cys 1~3)、第2位和第4位半胱氨酸(Cys 2~4)之间定点连接形成二硫键。经高效液相色谱分离纯化和质谱鉴定,合成了含有Cys(1~3, 2~4)二硫键连接方式的多肽,其分子质量正确,纯度在95%以上。利用双电极电压钳电生理学技术对这2种突变体与α3β2 nAChR的结合活性进行了检测。结果发现,当该位点的氨基酸性质由酸性转换为碱性后,对LvIA的活性影响巨大,直接导致对α3β2 nAChR的阻断活性丧失。[D11R]LvIA和[D11H]LvIA的活性与野生型LvIA相比分别降低了574.38%和408.62%。由此表明,第11位氨基酸的酸碱性对LvIA的活性至关重要。
snail toxin LvIA is a small peptide targeting α3β2 acetylcholine receptor(nAChR), which was found in the South China Sea snail. The structure and function of LvIA were previously analyzed in our laboratory. After alanine scanning mutation it was found that the mutant [D11A] LvIA, which was replaced by alanine(A) with aspartic acid at the site 11 of LvIA, maintained its activity against α3β2 nAChR. In order to further explore the effect of the properties of amino acid at the position 11 on the binding activity of LvIA target, two new mutants of LvIA [D11R] LvIA and [D11H] LvIA were designed, namely, two basic amino acids-arginine(R) and histidine(H) were used to replace the original acid amino acid D, respectively. The linear peptides of the two new mutants were synthesized and then folded by a two-step oxidation method to obtain a disulfide bond between the first and third cysteines(Cys 1-3) and the second and fourth cysteines(Cys2-4). The peptides containing Cys(1-3, 2-4) disulfide bond were successfully synthesized by high performance liquid chromatography(HPLC) and mass spectrometry. The synthesized peptides were correct in molecular weight and their purity was above 95%. The binding activity of these two mutants to α3β2 nAChR was detected by two-electrode voltage clamp electrophysiology. The results showed that when the amino acid properties of the site were changed from acidic to alkaline, LvIA activity was greatly affected, resulting in direct loss of α3β2nAChR blocking activity. The activity of [D11R] LvIA and [D11H] LvIA was 574.38% and 408.62% lower than that of wild-type LvIA, respectively. This suggests that the acidity and basicity of the 11th amino acid is crucial to the activity of LvIA. These results provide some reference for the optimization design and modification of LvIA in the future, based on which it is expected to obtain more specific peptide tools targetingα3β2 nAChR, which can provide a better pharmacological molecular probe for the study of the structure,function and distribution of an α3β2 nAChR receptor.
作者
李浩楠
杨奕帅
长孙东亭
朱晓鹏
罗素兰
LI Haonan;YANG Yishuai;ZHANG SUN Dongting;ZHU Xiaopeng;LUO Sulan(Department of Pharmacy,Sanya City Womenfolk&Infant Health Care Hospital,Shanghai Children’s Medical Center,Sanya,Hainan 572000;Ministry of Education Key Laboratory of Tropical Biological Resources/Haikou Key Lab for Marine Drug/School of Life Sciences and Pharmacy,Hainan University,Haikou,Hainan 570228,China)
出处
《热带生物学报》
2023年第1期1-7,共7页
Journal of Tropical Biology
基金
三亚市科技计划项目(2021GXYL42)
国家自然科学基金项目(81872794)。
