摘要
半乳糖凝集素-3 (galectin-3, Gal-3)属于β半乳糖苷酶结合凝集素家族,具有特异性结合半乳糖的特性。通过C端糖蛋白结合区(carbohydrate recognition domain, CRD), Gal-3可结合糖基化胰岛素受体(insulin receptor,IR)的半乳糖苷链,从而抑制IR信号通路,导致胰岛素抵抗,被视为治疗胰岛素抵抗和2型糖尿病的潜在药物作用靶点。本研究根据Gal-3结合糖基化蛋白半乳糖苷链的特性,设计了一种简便的Gal-3抑制剂筛选模型。在大肠杆菌表达Gal-3蛋白,经纯化后,用异硫氰酸荧光素(fluorescein isothiocyanate, FITC)修饰,获得Gal-3-FITC。Gal-3-FITC自发绿色荧光,与表面表达有大量糖蛋白的人胰腺癌细胞(PANC-1)孵育后, PANC-1细胞带有荧光信号。若待测化合物有Gal-3抑制活性,则该化合物可降低Gal-3-FITC与细胞的结合,从而降低PANC-1细胞荧光信号。通过荧光信号变化可评价Gal-3抑制剂的抑制强度。进一步研究表明,该筛选模型简易稳定,具有良好重复性, Z’因子在0.7和0.85之间。本研究采用FITC标记Gal-3的方式在PANC-1细胞上用荧光强度反映Gal-3和糖蛋白的结合水平,构建了Gal-3抑制剂的高通量筛选模型。
Galectin-3(Gal-3) belongs to the galectin family and is specific in binding β-galactoside.Through its C-terminal domain,Gal-3 binds to the galactoside group of the glycosylated insulin receptor(IR) and inhibits IR signaling pathway,which leads to the insulin resistance.Thus,Gal-3 is a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes.Here we report a simple Gal-3 screening model based on the property that Gal-3 binds to the galactoside.We expressed and purified human Gal-3 in Escherichia coli(E.coli),and labeled it with fluorescein isothiocyanate(FITC) in vitro.After incubating FITC labeled Gal-3(Gal-3-FITC)with PANC-1 cells,which express glycosylated membrane protein,PANC-1 cells started to show green fluorescent signal due to the Gal-3-FITC binding to the glycosylated membrane protein.Gal-3 inhibitor disrupts the binding of Gal-3-FITC and PANC1 cells,subsequently leads to the decrease of the fluorescent signal in PANC-1 cells.We can evaluate the inhibitory efficiency of Gal-3 inhibitors through measurement of the fluorescent signal.Further studies show this model is simple,stable,and repeatable with a Z’ factor between 0.7 and 0.85.In sum,we have successfully established an in vitro high-throughput screening model for Gal-3 inhibitors.
作者
马春晓
邢肖伟
侯少聪
何淑旺
颜世强
李平平
MA Chun-xiao;XING Xiao-wei;HOU Shao-cong;HE Shu-wang;YAN Shi-qiang;LI Ping-ping(State Key Laboratory of Bioactive Substance and Function of Natural Medicines,Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China;Shandong DYNE Marine Biopharmaceutical Co.,Ltd.,Weihai 264333,China)
出处
《药学学报》
CAS
CSCD
北大核心
2023年第1期156-161,共6页
Acta Pharmaceutica Sinica
基金
国家自然科学基金资助项目(81770800)
中国医学科学院医学与健康科技创新工程(2021-I2M-1-016)
中国医学科学院中央级公益性科研院所基本科研业务费(2018RC350004)
北京高校卓越青年科学家计划项目(BJJWZYJH01201910023028)。
