Alpha-crystallin Racemization And Its Role In Human Age-related Cataract

Age-related cataract is the most common reason of blindness. At present, surgery is still the only effective way to treat cataract. Therefore, it is of great importance to research on the prevention or non-surgical intervention of cataract.Racemization is one of the most important protein post-translational modifications. Previous studies showed that partial L-amino acid in lens protein transferred into D-amino acid through racemization during the ageing process, leading to structural turnover of crystallins. The aggregated high-molecular-weight (HMW) protein accumulated in the nuclear and cortical region of lens, gradually damaging the normal function of crystallins to maintain the transparency of lens. The effect and participation of racemization in age-related changes of crystallins, and the associated mechanism in the cataractogenesis needs further study.In this study, we mainly focused on the most important structural protein—— a-crystallin. Firstly, we used reversed phase high performance liquid chromatography to determine the rate of racemization, the most abundant post-translational modifications in lens. Secondly, we researched on the effect of racemization on lens membrane fluidity and proteins in natural-aged lens and simulated aging models.In summary, to fully assess the influence of a-crystallin racemization on age-related cataract, we developed an improved method to detect racemization in lens crystallin, and successfully established a simulated ageing model with lens epithelial cells. The observation of racemization will be of great use in the future study of cataract prevention and non-surgical intervention.Part une Racemization Analysis of a-crystallin in Human Age-related Cataract Lens and Lens Epithelial CellsPurpose:To optimize the experimental protocol of reversed phase high performance liquid chromatography (RP-HPLC). To analysis the racemization in human age-related cataract (ARC) lens and lens epithelial cells (LECs).Methods:(1) Natural ageing:After isolating a-crystallin using gel filtration chromatography from the nuclear region of transparent and ARC lens, the samples were treated with o-phtalaldehyde and N-tertbutyloxycarbonyl-L-cysteine for pre-column derivatization before RP-HPLC analysis. To measure the racemization in a-crystallin, calculate the D/L ratio according to the peak area in each gragh. Human ARC lens were classified by Lens Opacities Classification System I11 (LOCS Ⅲ), ARC NC2-3 and ARC NC5-6.(2) Ageing model:Lens epithelial cells heated at 50℃ were used as ageing model. Incubated under 50℃ for Omin、30min and 60min, likewise the protein samples were processed with isolation, purification, acidolysis, pre-column derivatization and racemization analysis.Results:We applied an improved protocol for RP-HPLC analysis to better isolate D/L-amino acids. We found that, (1) as to natural ageing, a-crystallin racemization rate in the normal control group is the lowest,14.55%±0.16% in average. ARC NC2-3 group was 15.3%±0.28% in average, and ARC NC5-6 group ranked the highest with an average value of 5.52%±0.19%. Moreover, a-crystallin racemization rates in two ARC group were significantly higher than the control group (P<0.05). In addition, ARC patients with a higher nuclear color (NC) grading and older age, had an increasing tendency in a-crystallin racemization rate. (2) As to the ageing model, LECs showed a higher a-crystallin racemization rate with the elongation of heating.Conclusion:Both of natural ageing and ageing model showed a increasing tendency of α-crystallin racemization.Part Two Influence of α-crystallin Racemization on Lens Membrane Fluidity and its Function as Molecular ChaperonePurpose:Compare the influence of α-crystallin racemization on lens membrane fluidity and its function as molecular chaperone using the ageing model.Methods:Laurdan staining and two-photon confocal microscopy were applied to analyze the lens membrane fluidity of each group with different heating time. Immunofluorescence staining were used to detect the expression and location of αA-crystallin (CRYAA) in human lens epithelial cells (hLECs). The percentage of relative absorbency (or turbidity) difference between α-crystallin and target protein (β-crystallin or γ-crystallin) were calculated as the activity of α-crystallinas molecular chaperon.Results:Along with heating, racemized CRYAA gradually accumulated around the nuclear and lens membrane fluidity increased. Both of the α-crystallin isolated from the nuclear region of normal and ARC lens can prevent β-and γ-crystallin from heat aggregation. The unheated and heated hLECs showed the same protective effect as above. However, the α-crystallin from ARC lens and heated hLECs showed a relatively lower protective effect on β-and γ-crystallin.Conclusion:The increase of α-crystallin racemization may induce the increase of lens membrane fluidity and the decrease of its normal function as the molecular chaperone.Part Three Influence of Intervened α-crystallin Racemization on Lens Membrane Fluidity and its Function as Molecular ChaperonePurpose:To test and verify the influence of α-crystallin racemization on lens membrane fluidity and its function as molecular chaperone by intervening α-crystallin racemization in the ageing model.Methods:Specific small interfering RNAs (siRNAs) were screened and transfected into human lens epithelial cells to surpress the expression of PCMT1 encoding PIMT. Western Blot was used for transfection efficiency analysis. RP-HPLC were used to analyze the α-crystallin racemization in four groups:control, control+PIMT siRNA, heat, heat+PIMT siRNA. The percentage of relative absorbency (or turbidity) difference between α-crystallin and target protein (β-crystallin or γ-crystallin) were calculated as the activity of α-crystallinas molecular chaperon.Results:The racemization rate of α-crystallin were increased in hLECs after transfected with PIMT siRNA sequences. It not only weaken its regulative effect on lens membrane fluidity, but also attenuated its function as molecular chaperone.Conclusion:According to cytology experiments, α-crystallin racemization is likely to be an important mechanism during lens ageing, as it plays a role in the regulation of lens membrane fluidity and molecular chaperone activity.