March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Stabilization of Matastable Cataract Causing αAG98R-Crystallin by αA-Mini-Chaperone
Author Affiliations & Notes
  • Krishna Sharma
    Ophthalmology and Biochemistry, University of Missouri, Columbia, Missouri
  • Murugesan Raju
    Ophthalmology, Univ of Missouri Mason Eye Institute, Columbia, Missouri
  • Puttur Santhoshkumar
    Ophthalmology, University of Missouri-Columbia, Columbia, Missouri
  • Footnotes
    Commercial Relationships  Krishna Sharma, None; Murugesan Raju, None; Puttur Santhoshkumar, None
  • Footnotes
    Support  NIH Grant EY011981 and EY021011 and an unrestricted grant-in-aid from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3667. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Krishna Sharma, Murugesan Raju, Puttur Santhoshkumar; Stabilization of Matastable Cataract Causing αAG98R-Crystallin by αA-Mini-Chaperone. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3667.

      Download citation file:

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

  • Supplements

Purpose: : Mutant form of αA-crystallin, αAG98R, is an unstable protein with reduced chaperone activity. The mutant protein aggregates on storage and incubation at or near physiological temperature. Previous studies have shown that αA-Mini-chaperone, a peptide- DFVIFLDVKHFSPEDLTVK, representing the chaperone site in αA-crystallin functions like a molecular chaperone. The objective of the present study was to determine the effect of αA- mini-chaperone on the aggregation and chaperone activity of αAG98R-crystallin.

Methods: : Mutant and wild type αA-crystallin were expressed in E.Coli and purified following the procedures described earlier. The aggregation of αAG98R in presence and absence of αA-mini-chaperone (supplied by GenScript) during incubation was followed by examining the samples under EM. In addition, gel filtration studies and DLS analysis of the samples in a multi-angle DAWN-EOS unit was performed. The binding of chaperone peptide to αAG98R was confirmed by HPLC analysis of the αAG98R-mini-chaperone complex and use of photoactive biotinylated mini-chaperone (biotin- DFVIFLDVKH (benzoylphenylalanine) SPEDLTVK) and MS analysis.

Results: : Examination of aggregation prone αAG98R under EM showed that the oligomers begin interact with one another within 10 min of incubation at 40oC and to form initial aggregates consisting of 2 to 10 oligomers which coalesce to form larger aggregates of 10 -20 oligomers in 30 mins. We found that addition of mini-chaperone during αAG98R incubations prevented αAG98R from forming larger aggregates that precipitate with time. We also found that mini-chaperone stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystalin. Tryptic digestion and mass spectrometric analysis of αAG98R incubated with biotynylated photoactive mini-αA-crystallin showed that the mini-chaperone interacts at the 89-103 region of αAG98R.

Conclusions: : These results demonstrate that mini-chaperone recognizes the structural changes in αAG98R due to mutation, stabilizes the mutant αA-crystallin and restores the lost chaperone activity. These findings should facilitate the design of peptide chaperones to stabilize and restore chaperone activity of mutant crystallins.

Keywords: crystallins • chaperones • mutations 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.