Effect of dicarbonyl-induced browning on α-crystallin chaperone-like activity: Physiological significance and caveats of in vitro aggregation assays

Abstract

α-Crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source forthe formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of α-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine α-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified α-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified α-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-1-sulphonic acid binding. In contrast, the chaperone-like function of modified α-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of α-crystallin in high proportions in the insoluble fraction. Furthermore, soluble α-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of α-crystallin.