提高O-糖基化水平对CHO-K1细胞突变型tau蛋白过度磷酸化的影响

Effect of increased O-GlcNAcylation on the hyperphosphorylation induced by overexpression of mutant tau protein on CHO-K1 cells

目的观察提高O-糖基化水平对CHO-K1细胞中突变型R406W tau蛋白过度磷酸化及细胞轴突转运功能的影响。方法分别将突变型PSG-5 R406W tau质粒和PSG-5空载体质粒转染CHO-K1细胞。将成功转染PSG-5 R406W tau质粒的CHO-K1细胞分为实验组与对照组,分别加入20μM的NAG-Ae及等量的DMEM/F12培养基,转染PSG-5空载体的细胞为空载体组,培养12 h。用Western blot法检测tau蛋白表达;免疫印迹法检测实验组与对照组细胞tau蛋白的糖基化及磷酸化水平;荧光漂白后恢复技术(FRAP)检测三组细胞漂白后荧光恢复的速度以表示细胞轴突转运速度。结果转染空载体的细胞未见tau蛋白表达,转染PSG-5 tau R406W的细胞tau蛋白呈高表达。实验组与对照组细胞O-糖基化水平分别为1.30±0.09、0.59±0.04(P<0.05)。实验组tauThr205、tauS-er396、tauThr212位点磷酸化水平分别为0.44±0.21、0.98±0.01、0.44±0.19,对照组分别为1.11±0.38、1.34±0.22、0.79±0.05,两组相比,P均<0.05。10、40、70 s时实验组荧光强度分别为262.00±12.50、300.00±10.00、461.66±10.40,对照组分别58.33±10.40、131.66±11.54、208.33±14.43,空载体组分别为514.00±10.14、888.33±11.06、1188.00±10.60,三组相比,P均<0.05。结论提高CHO-K1细胞的O-糖基化水平有助于减轻突变型tau蛋白的过度磷酸化水平,改善细胞轴突转运功能。

Objective To research the effects of increased O-GlcNAcylation on hyperphosphorylation of mutant R406W tan protein and function of axonal transport on CHO-K1. Methods CHO-K1 were used to establish the cell, which were line overexpressing mutant PSG-5 R406W tan or PSG-5 vector plasmid. CHO-K1 cells, which were successfully transfected plasmid PSG-5 R406W tau, were divided into experimental and control groups, treated with 20 μM NAG-Ae and the same amount of DMEM/F12 culture medium respectively, cultured for 12 h. Western blot were performed to detect the changes of O-GlcNAeylation and phosphorylation of tau protein of cells transfected with PSG-5 R406W tan and fluores- cence recovery after photobleaching （FRAP） were used to observe the changes of axonal transport in living cell of three groups. Results Endogenous tau protein were not found in cells transfected with PSG-5 vector, however, tan protein was highly expressed in cells transfected with PSG-5 R406W tan. O-GlcNAcylation level of experimental group and control group were 1.30 ± 0.09, 0.59 ±0. 04 （ P 〈 0.05 ）. At the sites of tan Thr205, tauSer396, tauThr212, phosphorylation lev- el of experimental group were 0.44 ± 0.21, 0.98 ± 0.01, 0.44± 0.19 and the control group were 1.11 ± 0.38, 1.34 ± 0. 22, 0. 79 ± 0.05 （ all P 〈 0.05 ）. The fluorescence intensity at 10 s, 40 s, 70 s in experimental group they were 262.00 ± 12.50, 300.00 ± 10.00, 461.66 ± 10.40 respectively; in control group they were 58.33 ± 10.40, 131.66 ± 11.54, 208.33 ± 14.43 ; in cells transfected with PSG - 5 vector group they were 514.00 ± 10.14, 888.33 ± 11.06, 1188.00 ± 10.6 0 （ all P 〈 0.0 5 ） . Conclusion Increasing O - GlcNAcylation of CHO - K 1 cells may contribute to alteration of tan protein phosphorylation and improvement of axonal transport ability.