September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Mini-chaperone peptide binding sites in mutant αA-G98R protein and its efficacy to stabilize mutant protein in HLE-B3 cells
Author Affiliations & Notes
  • Murugesan Raju
    Ophthalmology, University of Missouri, Mason Eye Institute, Columbia, Missouri, United States
  • Puttur Santhoshkumar
    Ophthalmology, University of Missouri, Mason Eye Institute, Columbia, Missouri, United States
  • Ashutosh Phadte
    Biochemistry, University of Missouri, Columbia, Missouri, United States
  • K Krishna Sharma
    Ophthalmology, University of Missouri, Mason Eye Institute, Columbia, Missouri, United States
    Biochemistry, University of Missouri, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Murugesan Raju, None; Puttur Santhoshkumar, None; Ashutosh Phadte, None; K Krishna Sharma, None
  • Footnotes
    Support  NIH Grant: EY19878 and EY023219
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 732. doi:
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      Murugesan Raju, Puttur Santhoshkumar, Ashutosh Phadte, K Krishna Sharma; Mini-chaperone peptide binding sites in mutant αA-G98R protein and its efficacy to stabilize mutant protein in HLE-B3 cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):732.

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

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Abstract

Purpose : The hallmark of αA-G98R mutation in humans is reduced stability and altered chaperone function leading to cataract. In a previous study, we have demonstrated that mini-chaperone peptide [70DFVIFLDVKHFSPEDLTVK88] interacts with the mutant αA-G98R protein, stabilizes the aggregation prone protein and rescues the lost chaperone function. The present study was undertaken to identify the mini-chaperone interacting site(s) during the stabilization of the mutant αA-G98R protein.

Methods : We substituted one of the three phenylalanine residues in mini-chaperone with benzoylphenylalanine (Bpa) to obtain Bpa labeled mini-chaperone (70DFVIFLDVKH [Bpa] SPEDLTVK88). The Bpa-mini-chaperone-peptide was incubated with αA-G98R mutant protein (1:1 ratio) at 37oC for 1hr. After removing the free peptides by 10kDa cut of filter, the mixture was exposed to UV light for photolysis at 4oC. Covalently cross linked mini-chaperone-αAG98R complex was excised from SDS-PAGE and subjected to in gel-digestion with trypsin. Tryptic peptides were analyzed by LC-MS+MS/MS. Furthermore, the aggregation preventing efficacy of mini-chaperone peptide against αA-G98R was analyzed ex vivo using HLE-B3 cells.

Results : Mass spectrometric analysis of tryptic peptides obtained from mini-chaperone-αAG98R complex showed three Bpa-mini-chaperone cross-linked fragments. 1) ion mass 404.804 5+ corresponding to Mini-chaperone [10-19] H [BPA] SPEDLTVK with αA-G98R [113EFHRR117], 2) ion mass 579.04 4+ assigned to mini-chaperone [10-19] H [BPA] SPEDLTVK with αA-G98R [13TLGPFYPSR21], and 3) ion mass 536.26 5+ corresponding to mini-chaperone [10-19] H [BPA] SPEDLTVK with αA-G98R [1EMDVTIQHPWFK11]. The MS/MS data suggests that the mini-chaperone peptide interacts with mutant protein at N-terminal and α-crystallin domain. The transduced mutant αA-G98R protein forms larger aggregates in HLE-B3 cells compared to the αA-Wt. However, the mutant protein aggregation was significantly reduced when mini-chaperone peptide was co-transduced suggesting that the mini-chaperone is preventing the aggregation of mutant protein.

Conclusions : Mini-chaperone rescues the mutant αA-G98R protein from its aggregation propensity by binding at the N-terminal and crystallin domain region. The present data suggests that the mini-chaperone could serve as potential therapeutic agent for diseases associated with protein conformational disorders

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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