April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Mini-chaperone Modulates The Impaired Stability of Cataract Causing Mutant αAG98R Crystallin
Author Affiliations & Notes
  • Murugesan Raju
    University of Missouri, Columbia, Missouri
  • Puttur Santhoshkumar
    University of Missouri, Columbia, Missouri
  • Kavi Thakkar
    University of Missouri, Columbia, Missouri
  • Krishna Sharma
    Ophthalmology and Biochemistry,
    University of Missouri, Columbia, Missouri
  • Footnotes
    Commercial Relationships  Murugesan Raju, None; Puttur Santhoshkumar, None; Kavi Thakkar, None; Krishna Sharma, None
  • Footnotes
    Support  NIH Grant EY11981 and EY021011 and an unrestricted grant-in-aid from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4742. doi:
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      Murugesan Raju, Puttur Santhoshkumar, Kavi Thakkar, Krishna Sharma; Mini-chaperone Modulates The Impaired Stability of Cataract Causing Mutant αAG98R Crystallin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4742.

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

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Purpose: : The cataract causing mutation G98R in human αA-crystallin (αAG98R) is associated with reduced oligomeric stability. The objective of the present study was to determine the effect of mini-chaperone on the stability of αAG98R crystallin. Mini-chaperone is derived from αA-crystallin (αA70-88), functions like a molecular chaperone.

Methods: : G98R mutant protein and wild type αA-crystallin were expressed in E.Coli cells and purified by chromatography method. The chaperone property of αAG98R was measured using ADH aggregation assay. The mutant protein showing chaperone activity forms larger light scattering aggregates after one hr incubation with client protein. To test whether mini-chaperone prevents αAG98R-ADH from light scattering aggregate formation, we added mini-chaperone at 70 min during assay and continued monitoring light scattering at 360nm. In other experiments αAG98R and wild-type αA-crystallin (0.5mg/ml) were incubated in presence or absence of mini-chaperone (10 µg) at 40oC. The samples were analyzed by gel filtration column connected to a multi-angle light scattering instrument.

Results: : We found that mini-chaperone stabilized the αAG98R-ADH complex as soon as it was added into the assay mixture indicating that mini-chaperone increases the stability of αAG98R-ADH complex. During gel filtration the αAG98R eluted as two peaks, one at the expected oligomer elution region and a second peak, corresponding to the dissociated subunits indicating impaired oligomer stability of the mutant protein. We recovered only 7.1 µg mutant protein at the oligomeric peak with an average molar mass of 2.3 x106 out of 75 µg due to precipitation when mutant protein was incubated for 1 hr. However, in the presence of mini-chaperone, we recovered significantly increased amount (59.2 µg out of 75 µg) of mutant protein with average molar mass 3.2 x106 indicating that mini-chaperone prevented the precipitation of αAG98R protein. Consistent with increase in molar mass the hydrodynamic radius (Rh) of the stabilized αAG98R increased from 15.31nm to 16.46nm. Unlike the αAG98R, the molar mass of αA-wild-type protein did not change in presence of mini-chaperone at 40oC.

Conclusions: : Our study shows that the interaction of mini-chaperone with mutant αAG98R protein prevented the aggregation of unstable αAG98R-crystallin. Additionally, the data suggest that the mini-chaperone also stabilizes the aggregation prone crystallin-client protein complexes.

Keywords: chaperones • cataract • mutations 

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