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

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.However,no obvious fiber structure was found in the tau441 group.Accordingly,ThT binding assay showed that the fluorescence value of phosphorylated products increased(P<0.05).Moreover,the cytotoxicity of phosphorylated products increased(P<0.05).Conclusion:Recombinant human GSK-3β-His and GSK-3β-His-Bac1 proteins were successfully prepared.These two proteins have the function of catalyzing in vitro phosphorylation of tau protein at amino acids 181,217 and 404,which provided technical support for related basic research on Alzheimer’s disease.