Oligomerization of the HBV/HDV functional receptor NTCP expressed in Sf9 insect cell
Oligomerization of the HBV/HDV functional receptor NTCP expressed in Sf9 insect cell
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Sf9 昆虫细胞中表达的 HBV/HDV 功能受体 NTCP 的寡聚化
DOI：
10.1016/j.bbagen.2022.130224
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发表时间：
2022-08-12
影响因子：
3
通讯作者：
Zeng，Su
中科院分区：
文献类型：
--
作者：
Qin，Tan;Wang，Yu;Zeng，Su
牛磺胆酸钠转运多肽（NTCP，SLC 10 A1）是一种重要的胆汁酸转运蛋白，是B型和D型肝炎病毒的功能性受体。NTCP的寡聚化对于研究其与HBV preS 1肽相互作用的结构具有重要意义。采用液相色谱-质谱/质谱（LC-MS/MS）方法对重组NTCP进行功能验证。建立了定量荧光共振能量转移（FRET）方法，分析NTCP野生型（WT）和突变体之间的相互作用。免疫共沉淀（Co-IP）被用来测试NTCP变体之间的相互作用。凝胶分析显示了NTCP的单体、二聚体和低聚体条带。在青色荧光蛋白（CFP）标记的NTCP和黄色荧光蛋白（YFP）标记的NTCP之间观察到显着的FRET。CFP-和YFP-NTCP S267 F突变体之间的FRET效率低于WT。Co-IP结果表明，S267 F与WT NTCP相互作用时，共表达在cell.ConclusionDimer是主要形式的NTCP表达在Sf 9时，与洗涤剂溶解。FRET和Co-IP分析支持NTCP在Sf 9细胞中形成寡聚体。同时，对NTCP变体的FRET分析进一步阐明了NTCP寡聚化的分子机制。
BackgroundSodium taurocholateco-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.MethodsRecombinant 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.ResultsSub-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.ConclusionDimer 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 significanceOur results showed that NTCP formed oligomers in Sf9 cell. Meanwhile the FRET analysis of NTCP variants further elucidated the molecular mechanism of NTCP oligomerization.