Brian A Cobb1, J Mark Petrash. 1. Departments of Ophthalmology & Visual Sciences and Genetics, Washington University School of Medicine, St. Louis, MO.
Abstract
PURPOSE: Lens lipids undergo a number of changes with age, including an overall increase in phospholipid acyl chain saturation and a decrease in length. In addition, the amount of membrane bound alpha-crystallin increases dramatically with age and with the onset of cataract. The aim of this study was to determine if a link exists between age and cataract associated changes in lens lipids and the changes in alpha-crystallin membrane association. METHODS: Protein-free lipid vesicles composed of a wide variety of synthetic and lens-derived lipid vesicles were formed by sonication. These vesicles were used with fluorescent native and recombinant alpha-crystallin conjugates in vesicle binding assays. Vesicles were collected by centrifugation and bound alpha-crystallin was quantified with fluorescence intensity measurements. RESULTS: alpha-Crystallin complexes showed remarkably similar binding profiles for all lipid vesicles tested, regardless of lipid origin, phospholipid head group, acyl chain length or saturation, and inclusion of cholesterol. In addition, recombinant alpha-crystallin complexes bind to these vesicles in a manner that is essentially indistinguishable from that of native human and bovine alpha-crystallins. Unlike alpha-crystallin binding to lens membranes containing intrinsic proteins, binding of alpha-crystallin to protein-free vesicles was very high capacity and unsaturable. CONCLUSIONS: We conclude from these data that the binding of alpha-crystallin to lens membranes is not lipid-specific. Furthermore, protein post-translational changes, such as phosphorylation, do not appear to alter alpha-crystallin binding to these vesicles. Given the linearity of the binding curves, we propose that the only limiting factor for normal alpha-crystallin membrane binding is available surface area on the bilayer. Finally, the present data suggests that increased in vivo membrane association of alpha-crystallin is not a result of lipid changes, but more likely a result of non-lipid factors such as the accumulation of high molecular weight forms of alpha-crystallin.
PURPOSE: Lens lipids undergo a number of changes with age, including an overall increase in phospholipid acyl chain saturation and a decrease in length. In addition, the amount of membrane bound alpha-crystallin increases dramatically with age and with the onset of cataract. The aim of this study was to determine if a link exists between age and cataract associated changes in lens lipids and the changes in alpha-crystallin membrane association. METHODS: Protein-free lipid vesicles composed of a wide variety of synthetic and lens-derived lipid vesicles were formed by sonication. These vesicles were used with fluorescent native and recombinant alpha-crystallin conjugates in vesicle binding assays. Vesicles were collected by centrifugation and bound alpha-crystallin was quantified with fluorescence intensity measurements. RESULTS: alpha-Crystallin complexes showed remarkably similar binding profiles for all lipid vesicles tested, regardless of lipid origin, phospholipid head group, acyl chain length or saturation, and inclusion of cholesterol. In addition, recombinant alpha-crystallin complexes bind to these vesicles in a manner that is essentially indistinguishable from that of native human and bovine alpha-crystallins. Unlike alpha-crystallin binding to lens membranes containing intrinsic proteins, binding of alpha-crystallin to protein-free vesicles was very high capacity and unsaturable. CONCLUSIONS: We conclude from these data that the binding of alpha-crystallin to lens membranes is not lipid-specific. Furthermore, protein post-translational changes, such as phosphorylation, do not appear to alter alpha-crystallin binding to these vesicles. Given the linearity of the binding curves, we propose that the only limiting factor for normal alpha-crystallin membrane binding is available surface area on the bilayer. Finally, the present data suggests that increased in vivo membrane association of alpha-crystallin is not a result of lipid changes, but more likely a result of non-lipid factors such as the accumulation of high molecular weight forms of alpha-crystallin.