中国生物工程杂志

Objective: To analyze the catalytic activity of GSK-3β for 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-3β expression 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-3β protein was determined by Western blot. Protein phosphorylation was conducted through GSK-3β and 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-3β protein expressed by prokaryotic virus and baculovirus was about 50 kDa and the purity of recombinant human GSK-3β protein 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-3β treatment. 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-3β and ATP was 1 μmol/L, 5 μmol/L and 3.2 mmol/L, respectively. The phosphorylation effect of GSK-3β prepared 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.