Effect of cross-linking on the chaperone-like function of alpha crystallin

Abstract

Alpha crystallin can function as a molecular chaperone in suppressing the heat-induced aggregation of other crystallins and proteins. During cataractogenesis, α-crystallin becomes a water-insoluble, high-molecular-weight, cross-linked aggregate. To determine whether the chaperone activity of alpha crystallin is lost during this age-related modification, extracts were prepared by sonication of water-insoluble proteins isolated from aged bovine lenses and human cataract lenses. All the preparations were tested for chaperone-like activity using βL-crystallin as the target protein and the percentage of α-crystallin in water-insoluble sonicated supernatant (WISS) was determined by slot blot immunoassay. The WISS from bovine as well as human lenses were still effective in protecting βL-crystallin aggregation at 56°C. The bovine cortical WISS with 50% immunoreactive α-crystallin showed 62% of the chaperone-like activity displayed by native α-crystallin. The WISS from bovine lens nucleus and human lenses with 17% and 5% immunoreactive α-crystallin showed 19% and 4% chaperone-like activity compared to native α-crystallin. Prior treatment of the WISS of both bovine and human lenses with dithiothreitol resulted in nearly 50% increase in chaperone-like activity suggesting possible loss of chaperone-like activity due to disulfide cross-links. To see if the chaperone-like activity of α-crystallin can be altered by non-disulfide cross-linking, native α-crystallin isolated from bovine lenses was cross-linked with dimethylsuberimidate (DMS) and dimethyl 3,3′-dithiobispropionimidate (DTBP) and tested for chaperone-like activity. The DMS cross-linked α-crystallin was effective in inhibiting the aggregation of βL-crystallins at 56°C, but required a two- to five-fold higher concentration than the native α-crystallin. α-Crystallin with higher degree of cross-linking showed lower chaperone-like activity. α-Crystallin cross-linked with DTBP, a cleavable cross-linking agent, also showed a 80% loss in chaperone-like activity. However, when the DTBP cross-linked α-crystallin was treated with dithiothreitol to cleave the cross-links there was a 50% recovery in the chaperone-like activity. These data suggest that the age-related cross-linking, which restricts the molecular flexibility of α-crystallin decreases its chaperone-like function.