Objective: To investigate the impacts of specific serines phosphorylation of αA Crystallin on anti-oxidation and anti-apoptotic ability of human lens epithelial cells (HLEC).
Methods: Experimental Study. Using site-directed mutagenesis, serines on specific sites (Ser45, Ser66, Ser122) were replaced respectively by aspartic acid (D) to mimic phosphorylation. Target genes were inserted into pEGFP-C1 plasmid and transfected into HLEC by Lipofectamine™ 2000. HLEC transfected with blank plasmid and plasmid over-expressing wide-type (WT) αA Crystallin were defined as control group and WT group. After H2O2 induction, Dihydroethidium (DHE) and Annexin V-PE/7AAD staining were used to detect the reactive oxygen species level and apoptotic rate. T-test, Wilcoxon-two sample test, analysis of variance and Kruskal-Wallis test were used for statistical analysis.
Results: The DHE intensity of three phosphorylation groups were 167.53±18.21, 143.54±4.78 and 143.40±30.19, which reduced significantly comparing to control group (274.42±3.71, t=9.963, P=0.001; t=34.961, P<0.01; Z=1.993, P=0.046); The same tendency were found when compared with cell group (WT) over-expressing wide-type αA Crystallin (192.67±1.47, Z=1.964, P=0.049; t=17.887, P<0.01; Z=1.964, P=0.049). After H2O2 induction for apoptosis, early apoptotic rates for three phosphorylation groups were 15.08%±0.90%, 16.59%±5.37% and 15.36%±2.62% respectively, which significantly decreased comparing with control group (41.28%±2.70%; t=21.632, P<0.01; t=8.855, t=15.450, P<0.01) and WT group (31.40%±1.64%, t=15.117, P<0.01; t=4.524, P=0.006; t=8.988, P=0.001). No significant differences were found in both measurements between the three phosphorylation groups (F=1.08, P=0.407; x2=4.345, P=0.114).
Conclusion: The Ser45, Ser66 and Ser122 phosphorylation enhanced the anti-oxidative stress and anti-apoptotic capability of HLEC.