α-Crystallin binding in vitro to lipids from clear human lenses

Abstract

The association of α-crystallin to lens membranes increases with age and cataract. Lipid compositional changes also occur with age, cataract, and diabetes. In this study we determined the influence of lipid compositional differences on the binding capacity of α-crystallin to lipid vesicles in vitro. Lipids were extracted from pools of human lenses from younger (22±4 y, n=30) and older (69±3 y, n=26) nondiabetic donors as well as from diabetics taking insulin (60±9 y, n=26) and diabetics not taking insulin (58±9 y, n=20). Diabetics were insulin dependent for an average of 6 years. Extracted lipids were extruded into large unilamellar vesicles. α-Crystallin was mixed with the lipid at 36 °C, allowed to bind for about 12 h, and centrifuged at 14 000 g. This centrifugal force was low enough to not pellet free α-crystallin but high enough to pellet the lipid and bound α-crystallin. α-Crystallin-lipid binding was characterized by comparing the amount α-crystallin in the pellets of samples with and without lipid. Protein was measured using an assay that minimized interference from lipids. Lipid composition was determined by 31P-NMR spectroscopy. The binding capacity of α-crystallin to lipids was 12, 19, 8·9, 17 μg bound/mg lipid for lens lipids extracted from younger, older, insulin-treated and nontreated diabetic donors, respectively. The amount of α-crystallin in the pellet (bound α-crystallin) was significantly lower for the lipids from the younger group of lenses, p=0·033 and insulin-treated group, p=0·006, compared with the older group of lenses. Higher binding capacity was associated with a higher relative amount of sphingolipid and lower relative amounts of phosphatidylethanolamine-related lipid and phosphatidylcholine. The binding capacity of α-crystallin to lens lipids, measured in vitro, increases with age and decreases in diabetic donors that were treated with insulin. Our data support the idea that with age and perhaps certain types of diabetes, more α-crystallin is bound to the membrane and serves as a condensation point to which other crystallins bind and then become oxidized.