May 2004
Volume 45, Issue 13
ARVO Annual Meeting Abstract  |   May 2004
Decreased stability of beta B2–crystallin by interacting oxidized lens crystallin peptide
Author Affiliations & Notes
  • P.E. G. Udupa
    University Missouri, Columbia, MO
  • K.K. Sharma
    Ophthalmology and Biochemistry,
    University Missouri, Columbia, MO
  • Footnotes
    Commercial Relationships  P.E.G. Udupa, None; K.K. Sharma, None.
  • Footnotes
    Support  NIH grants EY 09855, EY 14795, RPB
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3978. doi:
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      P.E. G. Udupa, K.K. Sharma; Decreased stability of beta B2–crystallin by interacting oxidized lens crystallin peptide . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3978.

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

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Abstract: : Purpose: It has been hypothesized that crystallin fragments play a role in lens protein aggregation and cataractogenesis. Earlier we reported that oxidized beta B3–crystallin peptide has the ability to modulate aggregation of denaturing proteins [J. Biol.Chem. (2002) 277, 39136]. The purpose of this study was to investigate the interaction of oxidized beta B3–crystallin fragment [residues 152–166] with bovine beta–L–crystallins. Methods: Aggregation assay of beta–L–crystallin was performed at 55°C and 37° C, and the light scattering was measured at 360 nm. Assays were performed in the presence of oxidized and unoxidized beta B3–crystallin peptide and in the presence or absence of alpha crystallin. Peptide–induced change in hydrophobicity was determined by bis–ANS binding studies. Beta B3–crystallin peptide binding sites were identified by sulfo–SBED labeling, cross–linking and mass spectrometric analysis. Results:Oxidation of the beta B3–crystallin peptide [152AINGTWVGYEFPGYR166] resulted in the formation of dityrosine, as well as oxidation of Tyr and Trp residues. Enhanced aggregation of beta–L–crystallin (>80%) was observed in the presence of oxidized beta B3–crystallin peptide compared to beta–L–crystallin with out oxidized beta B3–peptide. The increased aggregation was dependent on the concentration of the oxidized peptide. Peptides by themselves did not show aggregation under experimental conditions. Increased aggregation of beta–L–crystallin–oxidized peptide mixture was observed despite the presence of alpha crystallin in the assay. Increased bis–ANS binding was observed in the oxidized beta B3–peptide treated beta–L–crystallin. Reverse phase HPLC analysis of the oxidized peptide interacted beta–L–crystallin precipitate showed preferential precipitation of beta B2–crystallin compared to other members of beta–L–fraction. Sulfo–SBED labeling and mass spec analysis further confirmed a specific interaction between beta B2–crystallin and the oxidized beta B3–peptide. Conclusions: Our data suggest that oxidized beta B3–crystallin peptide interacts with denaturing beta B2–crystallin and enhances its aggregation. Bis–ANS binding study suggests a peptide induced conformational change and enhanced hydrophobicity in the target protein which, in turn, may be responsible for the enhanced aggregation.

Keywords: crystallins • oxidation/oxidative or free radical damage • protein structure/function 

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