May 2005
Volume 46, Issue 13
ARVO Annual Meeting Abstract  |   May 2005
Crystallin Changes in Animal Models of A R116C Autosomal Dominant Cataract
Author Affiliations & Notes
  • C.–D. Hsu
    Ophthalmology & Visual Science, Washington Univ School of Med, Saint Louis, MO
  • J.M. Petrash
    Ophthalmology & Visual Science, Washington Univ School of Med, Saint Louis, MO
  • Footnotes
    Commercial Relationships  C. Hsu, None; J.M. Petrash, None.
  • Footnotes
    Support  NEI grants EY013897, EY02687
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 3876. doi:
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      C.–D. Hsu, J.M. Petrash; Crystallin Changes in Animal Models of A R116C Autosomal Dominant Cataract . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3876.

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

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Abstract: : Purpose: An R116C mutation of αA crystallin is associated with autosomal dominant cataract in humans. Since human lens material is not available for studies of this disease, we produced a transgenic mouse model by fusing the αA crystallin promoter with coding sequences for either wild type or R116C human αA–crystallin. Transgenic mice expressing human αA R116C develop cortical cataracts, among other defects, whereas transgenics expressing wild type human αA–crystallin are unaffected. The purpose of the present study was to evaluate whether expression of the mutant αA R116C protein caused an alteration in the solubility of endogenous mouse lens crystallins. Methods: Postnatal day 0/1 lenses from three independent αA R116C transgenic lines were analyzed by immunostaining. Transgenic lines that express the wild type human αA crystallin, as well as nontransgenic littermates, were analyzed in parallel. Tissue sections were stained using antibodies against a His epitope fused to the transgenic proteins. Additionally, immunostaining was done for ß and γ crystallins. Immunofluorescence patterns were examined by confocal microscopy. Protein solubility was probed by examining water soluble and insoluble fractions from total lens homogenates using SDS–PAGE and western blot analysis. Distribution of crystallin fractions in lens homogenates was examined by size exclusion chromatography. Western blot signals were quantified by ECLplus using a STORM phosphorimager. Results: Immunostaining for the wild type human αA transgenic protein showed a homogeneous cytoplasmic staining pattern in lens fibers. In contrast, staining for the mutant R116C αA crystallin consistently resulted in an uneven, grainy immunofluorescent pattern. A dosage effect was observed with regard to the staining homogeneity for ß– and γ–crystallins; transgenic lines that express lower amounts of the mutant R116C protein gave more homogeneous staining than the highest expressing line. Western blotting studies indicated that the insoluble protein fraction of R116C transgenic lenses contained increased levels of the ß and γ crystallins. We observed no evidence for increased proteolysis of ß– or γ–crystallins in R116C transgenic lenses. These results indicate that expression of the R116C mutant protein results in alterations in the intermolecular interactions among the major crystallin proteins. Conclusions: Expression of the human R116C αA crystallin is associated with a decrease in ß and γ crystallins solubility in lens fibers. As revealed by immunofluorescence staining patterns, this effect is related to the amount of αA R116C mutant protein expressed in the lens.

Keywords: crystallins • transgenics/knock-outs • cataract 

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