April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
α-crystallin’S Structure And Function Are Altered By Acetylation
Author Affiliations & Notes
  • Ram H. Nagaraj
    Ophthalmology and Visual Sciences,
    Case Western Reserve University, Cleveland, Ohio
  • Shilpa Shanthakumar
    Ophthalmology and Visual Sciences,
    Case Western Reserve University, Cleveland, Ohio
  • Smitha Padmanabha
    Ophthalmology and Visual Sciences,
    Case Western Reserve University, Cleveland, Ohio
  • Mikhail D. Linetsky
    Ophthalmology and Visual Sciences,
    Case Western Reserve University, Cleveland, Ohio
  • Benlian Wang
    Center for Proteomics and Bioinformatics,
    Case Western Reserve University, Cleveland, Ohio
  • Alok Kumar Panda
    School of Basic Science, Indian Institute of Technology, Bhubaneswar, India
  • Ashis Biswas
    School of Basic Science, Indian Institute of Technology, Bhubaneswar, India
  • Footnotes
    Commercial Relationships  Ram H. Nagaraj, None; Shilpa Shanthakumar, None; Smitha Padmanabha, None; Mikhail D. Linetsky, None; Benlian Wang, None; Alok Kumar Panda, None; Ashis Biswas, None
  • Footnotes
    Support  R01EY-016219, R01EY-09912, P30EY-11373, RPB, OLERF
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4737. doi:
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      Ram H. Nagaraj, Shilpa Shanthakumar, Smitha Padmanabha, Mikhail D. Linetsky, Benlian Wang, Alok Kumar Panda, Ashis Biswas; α-crystallin’S Structure And Function Are Altered By Acetylation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4737.

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

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Abstract

Purpose: : α-crystallin’s lysine residues are acetylated in vivo. We have investigated the effect of acetylation on the structure and chaperone function of human αA-crystallin.

Methods: : Recombinant human αA-crystallin was acetylated with acetic anhydride (Ac2O). Lysine70 (K70) has been identified to be an acetylation site in αA-crystallin in vivo. To mimic acetylation at this site, lysine was replaced with glutamine (K70Q). The tertiary structure of the wild type and acetylated protein was determined by tryptophan fluorescence. Surface hydrophobicity was determined by TNS binding. The chaperone function of wild type and acetylated αA-crystallin was evaluated using several client proteins.

Results: : Western blotting using an antibody to acetyllysine indicated that α-crystallin (along with other crystallins) was acetylated in human lenses. Lysine residues were converted to acetyllysine by Ac2O treatment in a concentration-dependent manner and with a corresponding decrease in the lysine content. Western blotting with an antibody for acetyllysine and mass spectrometry confirmed acetylation in αA-crystallin. The acetylated αA-crystallin showed better chaperone function than the wild type protein in thermal aggregation assays using several client proteins (~2.5-fold with citrate synthase; ~6-fold with γ-crystallin and ~1.5-fold with βL-crystallin at a protein lysine: Ac2O molar ratio of 1:100). The TNS-fluorescence data indicated higher hydrophobicity of acetylated proteins relative to the unmodified protein. The intrinsic tryptophan fluorescence was increased in acetylated αA-crystallin suggesting perturbation in the tryptophan microenvironment. The acetylation mimic of αA-crystallin (K70Q) also showed increased chaperone function toward citrate synthase, lysozyme and βL-crystallin (1.5 to 2-fold) relative to the wild type protein.

Conclusions: : Acetylation of α-crystallin occurs in vivo and it increases the chaperone function of αA-crystallin by increasing its hydrophobicity. Acetylation could be an important chaperone-function protective post-translational modification of αA-crystallin in vivo.

Keywords: crystallins • chaperones • cataract 
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