Effect of cross-linking on the chaperone-like function of alpha crystallin.
Alpha crystallin can function as a molecular chaperone in suppressing the heat-induced aggregation of other crystallins and proteins. During cataractogenesis, alpha-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 beta L-crystallin as the target protein and the percentage of alpha-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 beta L-crystallin aggregation at 56 degrees C. The bovine cortical WISS with 50% immunoreactive alpha-crystallin showed 62% of the chaperone-like activity displayed by native alpha-crystallin. The WISS from bovine lens nucleus and human lenses with 17% and 5% immunoreactive alpha-crystallin showed 19% and 4% chaperone-like activity compared to native alpha-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 alpha-crystallin can be altered by non-disulfide cross-linking, native alpha-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 alpha-crystallin was effective in inhibiting the aggregation of beta L-crystallins at 56 degrees C, but required a two- to five-fold higher concentration than the native alpha-crystallin. alpha-Crystallin with higher degree of cross-linking showed lower chaperone-like activity. alpha-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 alpha-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 alpha-crystallin decreases its chaperone-like function.
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212, USA.
K K
KK
Sharma
B J
BJ
Ortwerth
D000368:Aged; D000369:Aged, 80 and over; D000375:Aging; D000818:Animals; D002386:Cataract / Q000378:metabolism; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003459:Crystallins / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D007096:Imidoesters / Q000494:pharmacology; D007908:Lens, Crystalline / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary
D003432:Cross-Linking Reagents; D003459:Crystallins; D007096:Imidoesters; D004120:Dimethyl Suberimidate; C007471:dimethyl dithiobispropionimidate