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V Pigaga, RA Quinlan; sHSP Function in the Context of Gamma-Crystallin Folding and Aggregation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4657.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To model and investigate the process of protein aggregation during cataract formation. Methods: There is several gamma-crystallin mutations known to cause cataract. For example, R36S and R58H mutations in gammaD-crystallin result in spontaneous crystallisation of crystallins in eye lens (1), and rodent gammaB-NOP crystallin mutation might serve as a useful animal model (2). Our studies were focused on T5P mutation of gammaC-crystallin, which results in Coppock-like cataract (3). Mutant gamma-crystallin constructs were made by site directed mutagenesis after cloning of the wild type crystallins by RT-PCR. Our studies have concentrated on the T5P gammaC-crystallin.The mutant protein(s) was studied by transient transfection into human epithelial cell lines and following the expressed protein by fluorescence microscopy. Changes in protein solubility and aggregation were monitored in vitro by centrifugation, gel filtration and immunoprecipitation. In some experiments gamma-crystallins were mixed with alpha-crystallins. Results: While overexpressed wild type gammaC-crystallin remains soluble, T5P gammaC-crystallin forms aggregates in all the cell lines tested. In vitro experiments show a significant difference in protein solubility comparing wild type to mutant protein. One of the major lenticular chaperones, aB-crystallin, is able to improve the solubility of mutant gammaC-crystallin in vitro, not only by direct association, but apparently by improving the folding efficiency of the mutant. Conclusion: Our experiments provide important clues to the limits of sHSP action in a disease context - namely those steps preceding cataract. This interaction provides a model system to find additional agents capable of reversing and/or preventing cataract formation. References: (1) Pande A et al, PNAS, 98:6116-6120; (2) Klopp N et al, Genomics 52:152-158; (3) Heon E et al, Am. J. Hum. Genet., 65:1261-1267.
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