April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Lens Crystallin Aggregation and Insolubilisation: Role of A-Crystallin-Dervied Peptide
Author Affiliations & Notes
  • K. Sharma
    Ophthalmology and Biochemistry, University of Missouri, Columbia, Missouri
  • P. Santhoshkumar
    Ophthalmology, University of Missouri-Columbia, Columbia, Missouri
  • M. Raju
    Ophthalmology, Univ of Missouri Mason Eye Institute, Columbia, Missouri
  • Footnotes
    Commercial Relationships  K. Sharma, None; P. Santhoshkumar, None; M. Raju, None.
  • Footnotes
    Support  NIH Grant EY11981 and an Unrestricted grant-in-aid from Research to Prevent Blindness (RPB)
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 5805. doi:
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      K. Sharma, P. Santhoshkumar, M. Raju; Lens Crystallin Aggregation and Insolubilisation: Role of A-Crystallin-Dervied Peptide. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5805.

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

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Purpose: : Earlier we reported identification of αA-crystallin-derived fragment - αA66-80 accumulating in aged and cataract human lenses. (J. Biol. Chem. Vol 283, 8477-8485, 2008). This study was undertaken to determine the role of this crystallin fragment in protein aggregation in lenses.

Methods: : Synthetic αA66-80 peptide (SDRDKFVIFLDVKHF, Genscript Corp) was incubated with α-crystallin and the resulting complex was isolated. Water-insoluble sonicated supernatant (WISS) fraction from human lenses was prepared as described earlier. Recombinant human αBT162C and βL- and γ-crystallins isolated from bovine lens were labeled with Alexa Flour 488. The Alexa Fluor-labeled proteins were added to α-crystallin-peptide or WISS-peptide complexes and incubated at 37oC from 0 to 24 hrs. The reaction mixtures were examined under fluorescence microscope. As control Pro-substitutes αA66-80 peptide was used in the study. The structure of the peptides used in the study was investigated by CD spectroscopy.

Results: : The CD spectroscopy showed that the αA66-80 peptide has β-sheet conformation whereas Pro substitution completely abolished this feature. Co-incubation of αA66-80 peptide with α-crystallin resulted in aggregation and precipitation whereas Pro-substituted αA66-80 and α-crystallin did not aggregate or precipitate. Alexa Fluor labeled αB-, βL- and γ-crystallins interacted with α-crystallin-αA66-80 and WISS-αA66-80 complexes whereas as the presence of Pro-substituted -αA66-80 peptide in incubations with α- or WISS fraction did not cause the binding of labeled proteins. Further, the binding of labeled αB-crystallin to α-crystallin-αA66-80 took place in a shorter incubation period compared to the binding of labeled βL- or γ-crystallins.

Conclusions: : Our study shows that αA-crystallin-derived αA66-80 peptide interaction with α-crystallin leads to the formation of initial aggregates that attract α-, β-and γ-crystallins. Additionally, the WIS fraction from human lens becomes an aggregation prone nucleus after αA66-80 peptide binding. The absence of crystallin aggregation in presence of αA66-80-Pro peptide suggests that peptide conformation has the greater effect on its activity than the charge property. Collectively, these data show that crystallin-derived αA66-80 peptide present in aged and cataract human lenses may be responsible, at least in part, to the early events in age-related cataractogenesis.

Keywords: crystallins • cataract • protein structure/function 

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