Characterization of αA-crystallin from high molecular weight aggregates in the normal human lens

Purpose: Lens alpha-crystallins, composed of two subunits of alphaA- and alphaB-crystallin, form low molecular weight (LMW) water soluble aggregates with an average molecular mass of approximately 800 kDa. In the intact lens, some of the alpha-crystallins are associated with even larger high-molecular-weight (HMW) aggregates which are thought to be precursors of components found in the water insoluble fraction. Although the mechanism of HMW aggregation and insolubilization are not known, the process is considered to be related to cataract formation. The purpose of the present study is to compare alphaA-crystallins from HMW and LMW forms in order to help understand the mechanism of insolubilization. Methods: HMW and LMW alphaA-crystallins were isolated from lenses of 50 year old and 2 year old human donors and compared with respect to chaperone activity and fluorescence. We also analyzed isomerization and racemization of Asp-58 and Asp-151 residues in alphaA-crystallin from HMW and LMW fractions. Results: The chaperone activity of HMW alphaA-crystallin was lower than that of LMW alphaA-crystallin. Fluorescence spectra indicated that HMW alphaA-crystallin was more hydrophobic than that of LMW. Isomerization of normal alpha-linkage to abnormal beta-linkage at both Asp-58 and Asp-151 residues markedly increased in HMW alphaA-crystallin compared with that of LMW alphaA-crystallin. The D/L ratio of beta-Asp-58 of either HMW or LMW forms were higher than 1.0, showing that inversion occurred in this site. In addition, the D/L ratio of the Asp-151 residue from HMW alphaA-crystallin was significantly lower than that of the LMW form. Conclusions: These results were qualitatively the same as those previously found in alphaA-crystallin from total proteins of cataractous versus normal lenses, suggesting that changes in the native structure of alphaA-crystallin associated with the HMW fraction of normal lenses reflect the same changes that occur to a greater degree in total proteins of human cataractous lens.