用SDSL-EPR技术测量LSECtin-CRD与甘露糖结合过程中的位点运动性变化

Mobility Analysis of Residue Sites in LSECtin-CRD After Binding with Mannose Using SDSL-EPR

目的:应用定点自旋标记-电子顺磁共振(SDSL-EPR)技术检测肝脏及淋巴结窦内皮细胞C型凝集素(LSECtin)的糖基识别结构域CRD上不同位点氨基酸的运动特性及其与甘露糖结合过程中的构象变化。方法:通过SOE-PCR方法对LSECtin-CRD引入半胱氨酸点突变,构建p ET28b-Tat-LSECtin-CRD载体,可溶性表达LSECtin-CRD突变体蛋白;对突变位点进行自旋标记;EPR检测标记后样品的EPR波谱;用SS-FDDM软件对EPR波谱进行模拟和解析,获得旋转相关时间τc,研究其构象特点及变化。结果:在CRD的Ca^(2+)结合位点和配体结合功能区构建了5个突变体蛋白;LSECtin-CRD与Ca^(2+)和甘露糖结合后,4个位点的τc值明显升高,表明CRD结构域整体运动性降低;5个突变位点运动性差异较大,其中A258位点运动性变化最大,可能是参与糖基结合的关键位点。结论:SDSL-EPR技术能够有效研究LSECtin-CRD参与糖基结合过程中的构象运动性,研究结果证明了LSECtin-CRD在与糖基结合后整体运动性受限。

Objective：To analysis mobility of single residue sites in the carbohydrate recognition domain（CRD）of liver and lymph node sinusoidal endothelial cell C-type lectin（LSECtin） and investigate conformational changes of CRD in binding process with mannose using site directed spin labeling electron paramagnetic resonance（SDSL-EPR） technology.Methods：Selected amino acids in LSECtin-CRD were mutated to Cys by sequence overlapped extension PCR（SOE-PCR）,and pET28b-Tat-LSECtin-CRD were constructed to express LSECtin-CRD mutants in supernatants.Cys were spin labeled with nitroxides.EPR spectra of mutants were detected and simulated and analyzed by spectrum simulation-Fourier deconvolution distance measurement（SS-FDDM） software for obtaining rotational correlation time τc,thereby studying the conformational changes of LSECtin-CRD.Results：5 mutated proteins with selected residue site in Ca2＋ and ligands binding area were obtained.After binding with Ca2＋ and mannose,τc of 4 LSECtin-CRD sites were increased indicating the global mobility of CRD were decreased.The mobility of 5 mutated sites had great difference,and the mobility of site A258 changed remarkably suggesting that A258 could be key site in carbohydrate binding.Conclusion：SDSL-EPR could effectively study the mobility of single residue sites in LSECtin-CRD in binding process with mannose.The results demonstrated that global mobility of LSECtin-CRD decreased after binding with carbohydrate.