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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. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
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.
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.
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.
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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