Selective association of crystallins with lens 'native' membrane during dynamic cataractogenesis

Abstract

Plasma membrane with its associated extrinsic proteins was isolated from normal and cataractous rat lenses by centrifugation of the total water insoluble fraction from homogenized lenses on a discontinuous sucrose gradient. Membrane, which we call "native" membrane, was recovered mainly from the 25/45% sucrose interface. Development of the experimental U18666A cataract resulted in plasma membrane shifting to higher density (the 50/55% sucrose fraction) and great increases in the urea soluble protein content of the lens. At early stages of cataract development, most of the increased urea soluble protein was membrane associated, presumably as extrinsic protein. With advancing cataract, most of the urea soluble protein appeared in an essentially membrane-free pellet fraction. The urea soluble protein associated with the cataract membrane was shown by combined IEF, SDS-PAGE, Western blotting, amino acid compositional analysis and protein sequence determinations to be mainly composed of modified alpha- and beta-crystallins. Alpha A-crystallin truncated by not more than 27 residues from the carboxyl terminus plus beta b1 crystallin truncated by 49 residues from the amino terminus were conclusively identified. In addition to beta b1, a population of six alpha-crystallin derived polypeptides were specifically enriched in the cataract membrane fraction. Four of these six alpha-crystallins appear to be truncated from their carboxyl terminus, a modification which should have increased their hydrophobicity. The pellet fraction, which accumulated in the lens nucleus as the cataract advanced, was enriched in urea soluble gamma-crystallin derived polypeptides. We suggest that protein insolubilization in this experimental cataract involves the selective and tight association of principally modified alpha-crystallins to the fiber cell plasma membrane.

Evidence for Distinct Cholesterol Domains in Fiber Cell Membranes from Cataractous Human Lenses

“…Although a certain basal level of crystallin association with fiber cell membranes occurs in normal lenses, massive binding of crystallins to lens membranes occurs in human cataracts (65). Interestingly, similar crystallin binding effects were observed for lens membranes isolated from rats treated with U18666A (66,67), suggesting that changes in membrane lipid composition were related to crystallin-membrane binding. Tang et al (68) recently demonstrated that increasing the cholesterol content of model membranes enriched with sphingomyelin attenuated the binding of ␣-crystallin; however, lens membrane-crystallin binding properties associated with cholesterol domains have not been examined.…”

Lipids and the ocular lens

“…It may be relevant to cataractogenesis that as ␣ -crystallin becomes denatured, it binds more deeply into the membrane ( 128,134 ). The association of ␣ -crystallin with the membrane increases with age and cataract, as does light scattering ( 130,140,141 ). The increased association is thought not to be due to posttranslational modifi cations of ␣ -crystallin.…”

Characterization of α-Crystallin-Plasma Membrane Binding

“…The prevailing explanation for these observations is that the amount of soluble ␣-crystallin available to bind partially denatured proteins becomes depleted with age, and the resulting high molecular mass aggregates slowly become insoluble. Interestingly, the amount of crystallin protein, especially ␣-crystallin, bound to the membrane also increases dramatically with increasing age and/or cataract formation (29,30). It could be that the progressive insolubilization of ␣-crystallin is due, in part, to increased membrane binding that is also associated with aging and cataract formation.…”