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

doi:10.13523/j.cb.2211025

China Biotechnology

2023, Vol. 43

Issue (4): 10-19 DOI: 10.13523/j.cb.2211025

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

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

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Abstract

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.

Key words： Glycogen synthesi kinase-3β Phosphorylation in vitro Fiber aggregation

Received: 13 November 2022 Published: 04 May 2023

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	Zhen-wu LIU
	He WANG
	Zi-di YAN
	Ying ZHANG
	Jin-sheng HE

Cite this article:

LIU Zhen-wu, WANG He, YAN Zi-di, ZHANG Ying, HE Jin-sheng. Preparation and Tau Phosphorylation Activity of Recombinant Human GSK-3β. China Biotechnology, 2023, 43(4): 10-19.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2211025 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I4/10

Fig.1 Construction and identification of prokaryotic express vector pET30a-GSK-3β-His (a) Schematic diagram of vector construction (b) Double digestion analysis of pET30a-GSK-3β-His. M: DNA marker; Lane 1: pET30a-GSK-3β-His digested with Kpn I and Nde I

Fig.2 Purification and identification of recombinant human GSK-3β protein in prokaryotic express system (a) SDS-PAGE analysis of the purified GSK-3β (b) Western blot analysis of the purified GSK-3β with anti-GSK-3β antibody (c) Western blot analysis of the purified GSK-3β with anti-His-tag antibody

Fig.3 Construction and identification of recombinant baculovirus genome (a) Schematic diagram of vector construction (b) Gel electrophoresis of PCR products. M: DNA marker; Lane 1: PCR products of target gene including kozak sequence (c) Double digestion of vector and target gene. Lane 1: pFastBac1 digested with Xba I and Kpn I; Lane 2: Target gene digested with Xba I and Kpn I (d) Identification of pFastBac1-GSK-3β-His by double enzyme digestion. Lane 1: pFastBac1-GSK-3β-His digested with Xba I and Kpn I (e) Identification of rBacmid-GSK-3β-His by PCR. Lane 1: PCR products of rBacmid-GSK-3β-His

Fig.4 Purification and identification of recombinant human GSK-3β-His-Bac1 protein expressed in baculovirus (a) SDS-PAGE analysis of the purified GSK-3β-His-Bac1 (b) Western blot analysis of the purified GSK-3β with anti-GSK-3β antibody (c) Western blot analysis of the purified GSK-3β with anti-His-tag antibody

Fig.5 Mass spectrometry analysis of phosphorylation sites of p-tau (shadow marks)

Fig.6 Analysis of phosphorylation site and efficiency of tau catalyzed by GSK-3β (a) Identification of GSK-3β catalyzed tau phosphorylation in vitro via immunoblot (b) Comparison of different GSK-3β catalytic effects. GSK-3β-Bac1CS represents commercialized baculovirus expressed GSK-3β, GSK-3β-proCB represents commercialized prokaryotic expressed GSK-3β

Fig.7 Optimization of ATP concentration in phosphorylation reaction system (a) Dot blot analysis of p-tau products with different concentrations of ATP (b) Quantitative analysis of fluorescence values in (a)

Fig.8 Optimization of GSK-3β concentration in phosphorylation reaction system (a) Dot blot analysis of p-tau products with different GSK-3β concentrations (b) Quantitative analysis of fluorescence values in (a)

Fig.9 Comparison of several GSK-3β catalytic effects on tau phosphorylation in vitro (a) (c) Phosphorylation of tau pT181 site (b) (d) Phosphorylation of tau pT217 site

Fig.10 GSK-3β catalyzed tau phosphorylation was prone to fiber formation (a) GSK-3β catalyzed tau phosphorylation to form fibers in vitro under TEM (fibers are indicated by arrows, scale bar = 200 nm) (b) Comparison of fiber length formed after tau phosphorylation (c) Comparison of fiber length at 5 d (d) ThT fluorescence assays to evaluate GSK-3β catalyzed p-tau aggregation

Fig.11 GSK-3β catalyzed p-tau aggregates were toxic to cells (a) (b) Cytotoxicity analysis of p-tau in different concentrations. Viability of cells after 24 h treatment of 0.25~1mmol/L of p-tau or tau protein. n=3, ^* P<0.05, ^** P<0.01

Table 1 Lists of main parameters of recombinant GSK-3β in this study


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