Differential Temperature-dependent Chaperone-like Activity of αA- and αB-crystallin Homoaggregates

Abstract

␣-Crystallin, a heteromultimeric protein made up of ␣A-and ␣B-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of ␣-crystallin can enhance its chaperone-like activity severalfold. The two subunits of ␣-crystallin have extensive sequence homology and individually display chaperonelike activity. We have investigated the chaperone-like activity of ␣A-and ␣B-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, ␣B-crystallin shows significant protective ability even at subphysiological temperatures, at which ␣A-crystallin or heteromultimeric ␣-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of ␤ L -crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quenching by acrylamide shows that the tryptophans in ␣B-crystallin are more accessible than the lone tryptophan in ␣A-crystallin even at 25°C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on ␣B-crystallin. Circular dichroism studies show some tertiary structural changes in ␣A-crystallin above 50°C. ␣B-crystallin, on the other hand, shows significant alteration of tertiary structure by 45°C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, ␣B-crystallin is much more sensitive to temperaturedependent structural perturbation than ␣A-or ␣-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of ␣-crystallin and indicate different roles for the two proteins both in ␣-crystallin heteroaggregate and as separate proteins under stress conditions. ␣-Crystallin is a major protein of the mammalian lens and constitutes as much as 50% of its dry weight. Studies over the past few years have shown that ␣-crystallin is expressed in several nonlenticular tissues such as heart, brain, and kidney, and its expression is enhanced severalfold during stress and disease conditions (1-6). ␣-Crystallin is shown to have homology with small heat shock proteins (7-10). Horwitz (11) shows that ␣-crystallin can prevent the thermal aggregation of ␤-and ␥-crystallins and a few other proteins like a molecular chaperone. Demonstration of chaperone-like activity of ␣-crystallin has provided an excellent opportunity to investigate the mechanistic aspects of chaperone function in general and the role of ␣-crystallin under stress conditions in particular. It is possible that, in the lens, ␣-crystallin may chaperone the formation of the transparent and appropriately ...

Domain Swapping in Human αA and αB Crystallins Affects Oligomerization and Enhances Chaperone-like Activity

“…In order to probe the molecular mechanism of the chaperone-like activity and its enhancement upon structural perturbation, we have been studying ␣-crystallin and its constituent subunits. Our recent study on the ␣A and ␣B homoaggregates showed that, despite high sequence homology, these proteins differ in their stability, chaperone-like activity, and the temperature dependence of this activity (8). This study also indicated different roles for the two proteins in the ␣-crystallin heteroaggregate in the eye lens and as separate proteins in non-lenticular tissues.…”

Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster

“…This difference is not caused by their mode of purification, as demonstrated with His-Hsp22 purified under native conditions and with urea. Small Hsps of other organisms have also been reported to show differences in their chaperone activity; among others, this is the case for Xenopus laevis Hsp30C and Hsp30D (Abdulle et al 2002), for mammalian ␣A-crystallin and ␣B-crystallin (Datta and Rao 1999;Van Boekel et al 1999;Reddy et al 2000) and recently for mammalian Hsp22 (Chowdary et al 2004) and S. cerevisiae Hsp42 and Hsp26 (Haslbeck et al 2004). For example, despite the high degree of sequence homology and structural similarity of ␣A-and ␣B-crystallin, ␣A-crystallin is more efficient in preventing thermal aggregation of proteins whereas ␣B-crystallin performs better against reduction-induced aggregation of proteins (Datta and Rao 1999).…”

α-Crystallin-Type Heat Shock Proteins: Socializing Minichaperones in the Context of a Multichaperone Network

“…Advances in this direction have centered on the lenticular ␣-crystallins. Recombinant ␣B-crystallin was shown to be a better chaperone than ␣A-crystallin, in particular at physiological temperatures (57,311). Optimal chaperone activity probably is sacrificed for stability reasons in the naturally occurring 3:1 stoichiometry of ␣A-and ␣B-crystallins.…”