May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Function and Limitations of Alpha Crystallins
Author Affiliations & Notes
  • V. Pigaga
    School of Biological Sciences, University of Durham, Durham, United Kingdom
  • R.A. Quinlan
    School of Biological Sciences, University of Durham, Durham, United Kingdom
  • Footnotes
    Commercial Relationships  V. Pigaga, None; R.A. Quinlan, None.
  • Footnotes
    Support  Darwin Trust
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2359. doi:
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      V. Pigaga, R.A. Quinlan; Function and Limitations of Alpha Crystallins . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2359.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Function and Limitations of α-Crystallins. Methods: Mutant γ-crystallin constructs were made by site directed mutagenesis after cloning of the wild type crystallins by RT-PCR. The mutant protein was studied by transient transfection and co-transfection with lenticular chaperones into immortalised human epithelial cell lines. In the in vitro experiments we have mixed recombinant αA-crystallin, αB-crystallin and γC-crystallins, and monitored changes in protein aggregation, solubility and interactions by centrifugation and gel filtration. Results: There are several γ-crystallin mutations known to cause cataract despite the fact, that chaperones, supposed to prevent the protein aggregation, are present in the eye lens at a very high concentrations. To investigate the possible reasons for this ineffective chaperone activity, our studies focused on the T5P mutation of γC-crystallin, which results in Coppock-like cataract (1) and the Crygbnop mutation that causes severe developmental cataract(2). Our previuos data showed the ability of the lens chaperone, αB-crystallin significantly alter protein aggregation of mutant γ-crystallins (3, 4).Now we present data, which shows, that αA-crystallin has similar effects. Transfection studies were performed and soluble and insoluble fractions compared for mutant γ-crystallins in the presence and absence of α-crystallins. Both transfection and in vitro mixing data suggest that α crystallins do change solubility of aberant protein, but only to a limited extent and not enough to prevent aggregate formation. Conclusions: Our experiments provide important clues to help define the limits of sHSP action in the context of cataract. We have established in vivo and in vitro model systems to identify additional factors needed to reverse or even inhibit the first stages of cataract formation by these γ-crystallin mutations. References: (1) Heon E et al, Am. J. Hum. Genet., 65:1261-1267. (2) Klopp N et al, Genomics, 52(2):152-8 (3) Pigaga V and Quinlan RA, ARVO 2002 (4) Sandilands A et al, EMBO J; 21(22):6005-14

Keywords: crystallins • cataract • chaperones 

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