Larry Takemoto

Previous theoretical and experimental studies have predicted that the loss of weak protein interactions between α- and γ-crystallins could result in a decrease in the transparent properties of the aging lens. α-Crystallins were prepared from the nucleus of old bovine lens, and γ-crystallins were prepared from whole fetal bovine lens or from the nucleus of old bovine lens. The possible interactions of old α-crystallins with either old γ-crystallins or fetal γ-crystallins were quantitated at equilibrium using microequilibrium dialysis. The amount of each γ-crystallin species in the “full” versus “empty” chambers was determined by reverse phase chromatography to obtain a binding ratio (full/empty). A binding ratio greater than 1.00 is indicative of a α-crystallin/γ-crystallin interaction. Within experimental error (±2X standard deviation), there were no interactions between aged γ-crystallins with aged α-crystallins while there were significant interactions between some of the fetal γ-crystallins with aged α-crystallins. In the aged bovine lens, when transparency is known to decrease, there is no detectable interaction of γ-crystallins with α-crystallins as measured by microequilibrium dialysis. The results are consistent with the hypothesis that short-range, weak, attractive interactions between α- and γ-crystallins are necessary for maximum transparency of the lens.

Purpose: Quantitate the interaction of mutant (R116C) and wildtype human alphaA crystallins with actin. Methods: AlphaA crystallins, expressed in a recombinant system, were purified, followed by passage through an actin affinity column. Results: Binding of mutant alphaA crystallin was significantly less than binding of wildtype alphaA crystallin. Conclusions: The R116C mutation of alphaA crystallin found in human cataracts binds less to the cytoskeletal component actin. Since both alphaA crystallin and actin are necessary for proper development of the lens, decreased binding of the mutant protein to actin may perturb normal differentiation processes of lens cells which are necessary for transparency.

One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.

Purpose. Determine if gammaS crystallin is preferentially deamidated in the high molecular weight aggregate fraction of old human lenses. Methods. The high molecular weight aggregate and lower mole...

To identify and quantitate deamidation of a specific asparagine residue of gammaS-crystallin that preferentially undergoes deamidation during the process of human senile cataractogenesis. Reverse phase chromatography, together with synthetic peptide standards, was used to resolve the amidated and deamidated forms of asparagine-143 in the gammaS-crystallin sequence 131-145 from total tryptic digests of the central, nuclear region of human cataractous and normal lenses. Identities of the resolved peptides co-eluting with synthetic peptide standards were confirmed by mass spectral analysis. The synthetic peptide standards were also used to quantitate the amount of deamidation occurring in individual cataractous and normal lenses. In all lenses analyzed, there was greater deamidation of asparagine-143 in cataractous lenses, as compared with age-matched normal lenses. The results demonstrate, for the first time, that increased deamidation of a specific asparagine residue is present in proteins from the human cataractous lens.