Hydroimidazolone modification of human αA-crystallin: Effect on the chaperone function and protein refolding ability

AlphaA-crystallin is a molecular chaperone: it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), alpha A-crystallin becomes a better chaperone. AlphaA-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of alpha A-crystallin by MGO (with 20-500 mu M) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation Of beta(L)- and gamma-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured beta(L)- and gamma-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of alpha A-crystallin to assist in the refolding of denatured MDH. However, MGO modification of chaperone-null TNS-bound alpha A-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of alpha A-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the alpha A-crystallin responsible for the chaperone function and refolding are the same in the native alpha A-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance alpha A-crystallin's ability to refold denatured proteins. (C) 2010 Elsevier B.V. All rights reserved.