精氨酸修饰成纤维细胞生长因子21及修饰后蛋白体内稳定性和糖代谢调节作用的研究

Stability and Glucose Regulation of FGF21 After Modified With Arginines

成纤维细胞生长因子21(fibroblast growth factor21,FGF21)是2000年发现的一个不依赖胰岛素调节血糖的细胞因子,有望成为治疗糖尿病的候选药物.但是,野生型FGF21由于半衰期较短,在体内不稳定,从而影响其成药性.为提高FGF21的稳定性,本实验在FGF21的C端添加了2个精氨酸(arginine,Arg),命名为FGF21-2A,用Compute pI/Mw软件计算之后,等电点(isoelectric point,pI)从5.43上升到5.84,随后进行了蛋白质的表达、分离纯化、体内稳定性及糖代谢调节作用的研究.诱导表达后菌液的SDS-PAGE图经BandScan5.0分析后显示FGF21-2A的表达量相对于野生型FGF21提高了10.6%.家兔体内半衰期检测实验结果显示FGF21-2A的半衰期显著延长.GOD-POD法检测HepG2肝癌细胞葡萄糖吸收实验、糖尿病小鼠降血糖实验和肝糖原检测实验的结果证明,FGF21-2A的降糖效果得到了增强,并且持续时间相对于野生型FGF21显著延长.Real-time PCR结果发现,长期注射FGF21-2A显著提高了糖尿病小鼠肝脏内葡萄糖转运蛋白(GLUT)1和葡萄糖激酶(GK)mRNA的表达量,降低了葡萄糖-6-磷酸酶(G6P)的mRNA表达量,表明FGF21-2A调节糖代谢的机制与野生型FGF21一致.综上所述,精氨酸修饰的FGF21其蛋白质稳定性提高,进而增加了对血糖的调控效果,有望成为新型糖尿病药物.

Fibroblast growth factor-21 （FGF21）, which was discovered in 2000 as a non-insulin-dependent cytokine to regulate blood sugar, is expected to become a candidate for anti-diabetes drugs. However, the biological activity of FGF21 was influenced by its weak instability and short half-life in vivo. In this study, to improve the stability of FGF21 in vivo, we added two arginines to the C-terminus of FGF21, and named as FGF21-2A. After being calculated by the software pI/Mw, the pI of FGF21-2A reached to 5.84 from 5.43. Then we expressed and purified the mutant protein for the examination of stability and bioactivity. When we used Bandscan5.0 to analyze the supernatant of Rosetta（DE3） lysates after induction, the protein expression levels of FGF21-2A increased by 10.6%. The in vivo half-life time of FGF21-2A was significantly prolonged in pharmacokinetics experiment. And, compared to the wild-type FGF21, FGF21-2A not only had a better capacity of stimulating glucose uptake in 3T3-L1 cells in vitro and stimulating glycogen synthesis of the model mice in vivo, but also provided a significantly long lasting effect on reducing blood glucose in the type Ⅰ and type Ⅱ diabetic animals. Moreover, the results of the real-time PCR showed that, after long term treatment, FGF21-2A could increase the mRNA expression of GLUT1 and GK and reduce the mRNA expression of G6P, which suggests FGF21-2A has the same mechanism of glucose regulation with the wild-type FGF21. In conclusion, modifying FGF21 with arginines increases the protein stability, and enhances the protein bioactivity, which lays the foundation for development of drugs targeting diabetes mellitus.