重组人GSK-3β制备及tau磷酸化活性研究  

Preparation and Tau Phosphorylation Activity of Recombinant Human GSK-3β

作　　者：刘振武 王荷[1] 闫子迪 张莹[1] 何金生[1] LIU Zhen-wu;WANG He;YAN Zi-di;ZHANG Ying;HE Jin-sheng(College of Life Sciences and Bioengineering,Beijing Jiaotong University,Beijing 100044,China)

机构地区：[1]北京交通大学生命科学与生物工程研究院,北京100044

出　　处：《中国生物工程杂志》2023年第4期10-19,共10页China Biotechnology

基　　金：国家自然科学基金(81271417);山东省重点研发计划(2019JZZY011011)。

摘　　要：目的:表达和纯化重组人糖原合成激酶-3β(glycogen synthetic kinase-3β,GSK-3β),优化反应条件,分析GSK-3β对tau蛋白磷酸化修饰产物p-tau的生物活性。方法:构建GSK-3β原核和杆状病毒表达载体,镍亲和层析纯化,BCA法测定蛋白浓度,考马斯亮蓝染色分析纯度;免疫印迹(Western blot)鉴定GSK-3β免疫反应性;液相色谱-质谱联用(liquid chromatography-mass spectrometry,LC-MS)和斑点印迹检测GSK-3β的tau磷酸化情况;优化GSK-3β磷酸化体系中GSK-3β和三磷酸腺苷(adenosine triphosphate,ATP)的浓度;负染透射电镜(transmission electron microscopy,TEM)和硫磺素T(thioflavine-T,ThT)结合实验分析磷酸化产物形成纤维的情况;CCK8实验分析磷酸化产物的细胞毒性。结果:考马斯亮蓝染色显示,原核和杆状病毒表达的重组人GSK-3β蛋白表观分子量位于50 kDa左右,蛋白纯度分别为86%和81%;Western blot在相应位置有特异条带;LC-MS提示,经GSK-3β处理的tau蛋白有23个磷酸化位点;斑点印迹显示,兔抗pT181血清、兔抗pT217和pS404识别磷酸化的tau蛋白;优化反应条件,tau、GSK-3β和ATP的最适反应浓度分别为1μmol/L、5μmol/L和3.2 mmol/L;制备的GSK-3β对tau蛋白pT217位点磷酸化信号强于国外商品(P<0.05);TEM显示p-tau 5 d出现纤维结构,而tau未见明显的纤维结构;ThT结合实验检测到磷酸化产物的荧光值增强(P<0.05);磷酸化产物的细胞毒性增强(P<0.05)。结论:成功制备并鉴定了重组人GSK-3β,可催化tau蛋白在第181、217和404位氨基酸磷酸化,为阿尔茨海默病的基础研究提供了技术支撑。Objective:To analyze the catalytic activity of GSK-3βfor tau protein phosphorylation in vitro and the effect of phosphorylation modification on tau aggregation and cytotoxicity.Methods:Recombinant human glycogen synthetic kinase-3β(GSK-3β)was expressed and purified by prokaryotic and baculovirus expression systems.GSK-3βexpression vectors with C-terminal tag were constructed.The proteins were purified through nickel affinity chromatography,and the protein concentrations were determined by BCA kit.SDS-PAGE Coomassie bright blue staining was used to analyze the purities of proteins.The immunoreactivity of recombinant GSK-3βprotein was determined by Western blot.Protein phosphorylation was conducted through GSK-3βand recombinant human tau441 incubation in Tris-HCl solution.The concentrations of enzyme and adenosine triphosphate(ATP)were optimized.The phosphorylation of recombinant human tau441 was detected by liquid chromatograph mass spectrometer(LC-MS)and dot blot.The aggregation of phosphorylation products were determined by negative staining transmission electron microscopy(TEM)and thioflavin T(ThT)binding assay.Results:The data of SDS-PAGE showed that the apparent molecular weight of recombinant human GSK-3βprotein expressed by prokaryotic virus and baculovirus was about 50 kDa and the purity of recombinant human GSK-3βprotein was 86%and 81%,respectively.Western blot showed signal bands in corresponding positions.LC-MS analysis showed that 23 sites of tau protein were phosphorylated after GSK-3βtreatment.Dot blot showed that rabbit anti-pT181 serum,pT217 and pS404 antibodies(with priority recognition of tau181,tau217 and tau404,respectively)recognized tau441 phosphorylated in vitro.The optimal concentrations of tau,GSK-3βand ATP was 1μmol/L,5μmol/L and 3.2 mmol/L,respectively.The phosphorylation effect of GSK-3βprepared in this study on pT217 was significantly stronger than that of imported products(P<0.05).TEM images showed that fibers appeared in phosphorylated tau441 at 5 d and matured at 14 d.H

关 键 词：糖原合成激酶-3β 体外磷酸化 纤维聚集 

分 类 号：Q812[生物学—生物工程]