HBV/HDV 功能性受体 NTCP 在 Sf9 昆虫细胞中的寡聚化。
Oligomerization of the HBV/HDV functional receptor NTCP expressed in Sf9 insect cell.
机构信息
Department of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
Department of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
出版信息
Biochim Biophys Acta Gen Subj. 2022 Nov;1866(11):130224. doi: 10.1016/j.bbagen.2022.130224. Epub 2022 Aug 6.
BACKGROUND
Sodium taurocholate co-transportering polypeptide (NTCP, SLC10A1) is a vital bile acid transporter and the functional receptor of hepatitis B and D virus. The oligomerization of NTCP is important for the structural study of its interaction with HBV preS1 peptide.
METHODS
Recombinant NTCPs were expressed in Sf9 host cell using baculoviruses. Function of recombinant NTCP was verified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. A quantitative fluorescence resonance energy transfer (FRET) method was established to analyze the interaction between NTCP wild type (WT) and mutants. Co-immunoprecipitation (Co-IP) was used to test the interaction between NTCP variants.
RESULTS
Sub-cellular location of recombinant NTCPs varies with the modification of NTCP. Bands of monomer, dimer and oligomers were shown in gel analysis of NTCP. Significant FRET was observed between cyan florescence protein (CFP) tagged NTCP and yellow florescence protein (YFP) tagged NTCP. FRET efficiency between CFP- and YFP-NTCP S267F mutants was lower than WT. Co-IP results showed that S267F interacts with WT NTCP when co-expressed in cell.
CONCLUSION
Dimer is the predominant form of NTCP expressed in Sf9 when solubilized with detergent. FRET and Co-IP analysis support that NTCP forms oligomers in Sf9 cell.
GENERAL SIGNIFICANCE
Our results showed that NTCP formed oligomers in Sf9 cell. Meanwhile the FRET analysis of NTCP variants further elucidated the molecular mechanism of NTCP oligomerization.
背景
牛磺胆酸钠共转运蛋白（NTCP，SLC10A1）是一种重要的胆汁酸转运蛋白，也是乙型肝炎和丁型肝炎病毒的功能性受体。NTCP 的寡聚化对于研究其与 HBV preS1 肽相互作用的结构至关重要。
方法
使用杆状病毒在 Sf9 宿主细胞中表达重组 NTCP。通过液相色谱-质谱/质谱（LC-MS/MS）方法验证重组 NTCP 的功能。建立了定量荧光共振能量转移（FRET）方法来分析 NTCP 野生型（WT）和突变体之间的相互作用。共免疫沉淀（Co-IP）用于测试 NTCP 变体之间的相互作用。
结果
重组 NTCP 的亚细胞定位随 NTCP 的修饰而变化。凝胶分析显示 NTCP 存在单体、二聚体和多聚体条带。青色荧光蛋白（CFP）标记的 NTCP 和黄色荧光蛋白（YFP）标记的 NTCP 之间观察到明显的 FRET。CFP-和 YFP-NTCP S267F 突变体之间的 FRET 效率低于 WT。Co-IP 结果表明，当共表达时，S267F 与 WT NTCP 相互作用。
结论
当用去污剂溶解时，Sf9 中表达的 NTCP 主要形式为二聚体。FRET 和 Co-IP 分析支持 NTCP 在 Sf9 细胞中形成寡聚体。
一般意义
我们的结果表明 NTCP 在 Sf9 细胞中形成寡聚体。同时，NTCP 变体的 FRET 分析进一步阐明了 NTCP 寡聚化的分子机制。
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