May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Dissociated Subunits of A-crystallinG98R Exhibit Chaperone-Like Activity
Author Affiliations & Notes
  • M. Raju
    Ophthalmology, Mason Eye Institute Univ of MO, Columbia, Missouri
  • P. Santhoshkumar
    Ophthalmology, Mason Eye Institute Univ of MO, Columbia, Missouri
  • K. K. Sharma
    Ophthalmology, Mason Eye Institute Univ of MO, Columbia, Missouri
  • Footnotes
    Commercial Relationships  M. Raju, None; P. Santhoshkumar, None; K.K. Sharma, None.
  • Footnotes
    Support  EY11981, EY 14795, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4506. doi:https://doi.org/
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    • Get Citation

      M. Raju, P. Santhoshkumar, K. K. Sharma; Dissociated Subunits of A-crystallinG98R Exhibit Chaperone-Like Activity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4506. doi: https://doi.org/.

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

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Abstract

Purpose: : Recently, we observed that cataract causing mutant G98R alpha A-crystallin dissociated into subunits upon dilution of the protein. The present study was undertaken to explore the chaperone function of dissociated subunits of G98R αA-crystallin.

Methods: : Substitution of Glycine with arginine at 98 residue in human αA-crystallin was accomplished by site directed mutagenesis. The recombinant protein was expressed in E .coli cells and purified by chromatographic techniques. Purified G98R protein was diluted into 1mg/ml in buffer, pH 5.8 and incubated at 37oC for 1hr. The dissociated subunits were recovered after filtration through 50 kDa cutoff filter. These proteins were analyzed by SDS-PAGE and Mass Spectrometry and characterized by multi-angle light scattering and circular dichroism spectroscopy. The chaperone-like activity of mutant G98R crystallin subunits was assessed by citrate synthase (CS) and alcohol dehydrogenase (ADH) aggregation assays.

Results: : The size exclusion chromatography of αA-G98R in TSK-3000 showed that the mutant protein dissociates into monomeric mass. The amount of the monomeric form increased with decreasing pH. At about pH 5.8, nearly all of the αA-G98R dissociated into monomers. The dissociated subunits were less stable than the oligomer. The circular dichroism studies showed no difference in the secondary structure between dissociated subunits of αA-G98R and the oligomer. The subunits of G98R exhibited chaperone-like activity during thermal aggregation assay with ADH and CS. The dissociated subunits of αA-G98R also suppressed aggregation of urea denatured ADH in a refolding assay.

Conclusions: : The present study shows that αA-G98R subunits have chaperone-like activity and oligomer state is not a pre-requisite for chaperone-like activity of αA-crystallin.

Keywords: crystallins • chaperones • protein structure/function 
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