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R.H. Nagaraj, B. Liu, T. Oya–Ito, D.G. Smith, H.A. Pilch; Methylglyoxal–modification enhances antiapoptotic properties of alpha–crystallin . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3967.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have recently demonstrated that methylglyoxal (MGO), an Î±–dicarbonyl compound present in the lens can chemically modify Î±–crystallin and enhance its chaperone function (Nagaraj et al., Biochemistry, 42: 10746–10755, 2003). In this study, we have investigated effect of MGO modification on the antiapoptotic properties of Î±–crystallin. Methods: Bovine lens Î±–crystallin was incubated with or without 25 mM MGO for one week at 37 0C and pH 7.4 and conjugated with Alexa–350 fluorophore. Alexa–350 conjugated or unconjugated Î±–crystallin (50 Î¼g) was encapsulated in BioPORTER liposome and incubated with human lens epithelial cells (HLE B–3) for 4 hrs. The cells were then incubated with 1ÂµM staurosporine (STS) for 3 hrs. Apoptotic cells (Annexin V positive) were quantified by flow cytometry. Effect of transfer of native or MGO–modified Î±–crystallin on reactive oxygen species (ROS) and glutathione (GSH) content was determined. Cytochrome c content was estimated by Western blotting. Effect on caspase–8 mediated activation of capase–3 was also investigated. Results: BioPORTER–mediated transfer of Î±–crystallin (Alexa–350 conjugated) into HLE B–3 cells was evident by the results of fluorescence microscopy, Western blotting with anti–Î±–crystallin antibody or anti–argpyrimidine antibody, and ELISA. We achieved approximately 7–8% of crystallin transfer into cells by this method. STS treatment resulted in 24 Â± 0.6% apoptotic cells, but after transfer of Î±–crystallin (not conjugated with Alexa–350) apoptotic cells were considerably reduced (14.9 Â± 0.6%). Transfer of MGO–modified Î±–crystallin further decreased apoptotic cells (6.9 Â± 0.8%). GSH content dramatically decreased in cells incubated with STS alone, transfer of native and MGO–modified Î±–crystallin inhibited GSH loss by 17 and 20%, respectively. Transfer of native or MGO–modified Î±–crystallin had no significant effect on cytochrome c content. ROS content was significantly lower in cells transferred with MGO–modified Î±–crystallin when compared to cells that were treated with STS alone. Caspase–8 mediated activation of caspase–3 was inhibited by Î±–crystallin, but the effect was more pronounced with MGO–modified Î±–crystallin. Conclusions: Our data suggest that Î±–crystallin becomes a better antiapoptotic protein after modification by MGO, which may be due to multiple mechanisms, and may involve enhancement of chaperone function, reduction in oxidative stress and prevention of caspase–8–mediated caspase–3 activation.
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