May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Identification of the Interaction Between Alpha Crystallin and GRIFIN
Author Affiliations & Notes
  • K. A. Barton
    Ophthalmology & Visual Science, Washington Univ Sch of Medicine, Saint Louis, Missouri
  • C.-D. Hsu
    Ophthalmology & Visual Science, Washington Univ Sch of Medicine, Saint Louis, Missouri
  • J. Petrash
    Ophthalmology & Visual Science, Washington Univ Sch of Medicine, Saint Louis, Missouri
  • Footnotes
    Commercial Relationships  K.A. Barton, None; C. Hsu, None; J. Petrash, None.
  • Footnotes
    Support  NIH Grants EY13897, EY05856, P30EY02687, T32EY013360 and by an unrestricted grant to the Department of Ophthalmology & Visual Sciences from RPB.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4096. doi:https://doi.org/
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    • Get Citation

      K. A. Barton, C.-D. Hsu, J. Petrash; Identification of the Interaction Between Alpha Crystallin and GRIFIN. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4096. doi: https://doi.org/.

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

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Abstract

Purpose: : Previous in vitro studies suggest that α-crystallin interacts with various proteins in their native state but the biological importance of these interactions is not well defined. The goal of this study is to characterize molecular interactions involving α-crystallin in the intact lens.

Methods: : Transgenic mouse lenses designed to over-express a polyHistidine-tagged αA crystallin were subjected to mild crosslinking to fix any in vivo protein complexes. Histidine tagged αA crystallin and any interacting proteins were co-purified by metal affinity chromatography under denaturing conditions and were identified by tandem mass spectrometry. To validate the cross-linking studies, we measured interactions between α-crystallin and substrate proteins using calf lens α-crystallin as well as oligomeric complexes reconstituted from recombinant human αA and/or αB subunits.

Results: : Treatment of transgenic lenses with cross-linking agents captured proteins that interact with α-crystallin under native conditions. Epitope-tagged α-crystallin, and its binding partners, were isolated in the presence of 6 M urea and identified by tandem mass spectrometry. In addition to several crystallins that have been shown by others to interact with α-crystallin, we identified GRIFIN (galectin-related inter-fiber protein) as a novel binding partner to α-crystallin. Equilibrium binding analysis with native bovine α-crystallin revealed an affinity of 6.5 ± 0.8 µM at a stoichiometry of 0.25 ± 0.01 α-crystallin subunit per GRIFIN monomer. Up to an 8-fold enhancement of binding was observed when GRIFIN and α-crystallin were incubated in the presence of 3mM ATP.

Conclusions: : The equilibrium binding and chemical cross-linking results suggest that some interactions likely occur between GRIFIN and α-crystallin in the mouse lens. Binding interactions between α-crystallin and GRIFIN may be regulated by ATP levels.

Keywords: crystallins • chaperones • proteomics 
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