September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Age-related Asp Isomerizations in Dissociated Alpha-crystallin from Aged Lens
Author Affiliations & Notes
  • Takumi Takata
    Dept of Biochemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan
  • Takashi Sato
    Dept of Biochemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan
  • Noriko Fujii
    Dept of Radiation Biochemistry and Biological Function, Research Reactor Institute, Kyoto University, Kumatori, Osaka, Japan
  • Footnotes
    Commercial Relationships   Takumi Takata, None; Takashi Sato, None; Noriko Fujii, None
  • Footnotes
    Support  Grant-in-Aid for Young Scientists (B)
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Takumi Takata, Takashi Sato, Noriko Fujii; Age-related Asp Isomerizations in Dissociated Alpha-crystallin from Aged Lens. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.

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

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Purpose : The passive chaperone lens α-crystallin (α-Crys), a small heat shock protein, is composed of two subunits (~ 20 kDa) αA- and αB-crystallin (αA-Crys and αB-Crys), which form a hetero-oligomeric and polydisperse complex with molecular mass of ~ 800 kDa in the vertebrate lenses. Recent studies by LC/MS/MS analysis have shown that many aspartyl residues (Asp) in αA-Crys inverted to isomers (Lβ-Asp, Dα-Asp, Dβ-Asp) with age. However, it is not well understood whether Asp isomers in native polymeric αA-Crys are different from those in dissociated αA-Crys. In the present study, we examined to clarify the isomerized Asp in dissociated αA-Crys, which may contribute to abnormal lens protein subunit-subunit interactions in aged lens.

Methods : Lens of four different ages (42, 54, 69 and 83 years old) were homogenized, centrifuged, and the soluble fraction was applied to size-exclusion chromatography (SEC) and non-reducing SDS-PAGE. The polymeric and monomeric αA-Crys were independently obtained, and then digested by trypsin. Each tryptic peptide was applied to mass spectrometry equipped with nano-scale liquid chromatography to extract each of αA-Crys-derived peptides containing Asp isomers. Peptides with Asp isomers were also obtained by in-gel digestion with a gel of non-reducing SDS-PAGE. The ratio of Asp isomers was determined by the comparison of peak area from four Asp isomer containing peptide.

Results : αA-Crys was identified as a polymeric and monomeric state in the soluble fraction of aged lens. The Asp 58, Asp 84 and Asp 151 of αA-Crys were highly isomerized in the monomeric fraction, but were not isomerized to the same level in the native polymeric fraction.

Conclusions : These results showed that the distribution of Asp isomers is different between the dissociated and aggregated states of α-Crys in aged lens. Furthermore, age-dependent Asp isomerization in α-Crys is likely to contribute to the solubility of lens protein in aged lens. The isomerization of Asp as well as many other modifications would reduce the normal subunit-subunit interaction of α-Crys with aging, resulting in senile cataract formation.

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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