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Takumi Takata, Rintaro Inoue, Ken Morishima, Nobuhiro Sato, Masaaki Sugiyama, Noriko Fujii; The Site-specific Impacts of Modifications of Aspartate Residues on Lens αA-Crystallin. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3042.
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© ARVO (1962-2015); The Authors (2016-present)
α-crystallin (α-Crys) interacts with β- and γ-crystallin families and forms high molecular weight (HMW) aggregates in lens. One of α-crystallin subunit, αA-crystallin (αA-Crys) is involved in HMW as molecular chaperone, or aggregate substrate. While recent proteomics research has shown that many aspartate residues (Asp) in αA-Crys inverted to isomers in aged lens, the contributions of those for αA-Crys have been unknown. Therefore, the purpose of this study is to investigate distribution and contribution of Asp isomers for the αA-Crys in HMW fractions.
Lenses of many different ages were homogenized, centrifuged, and the soluble fraction was independently applied to size-exclusion chromatography (SEC). The HMW fractions were isolated, and then digested by trypsin. Each tryptic peptide was applied to mass spectrometry equipped with liquid chromatography to extract each peak of αA-Crys-derived peptides containing Asp isomers from elution profile. Next, to estimate the contribution of site-specific isomerization, a few of Asp residues (Asp 58 or Asp 151) of αA-Crys were replaced by other amino acid using site directed mutagenesis and recombinantly expressed by E.coli. Those properties were compared to the recombinantly expressed wild type αA-Crys.
The isomerization of Asp 58 was increased age-dependent, while that of Asp 151 was increased shortly after birth in lens. The substitution of Asp 58 or Asp 151 into other amino acid residues decreased heat stability and chaperone function of αA-Crys. The mutants, which induced hydrophobicity at Asp 58, increased the size of αA-Crys. Those were not observed in Asp 151 mutants. The heat-inducing isomerization assay demonstrated that modifications at Asp 151 in αA-Crys accelerated the isomerization of Asp 58 in αA-Crys.
The modifications of Asp residues alter the properties of αA-Crys depends on the site of residue and kind of amino acid. Since the predominant modification of Asp residues in lens crystallin is isomerization, each site isomerization would site-specifically impact on the property of αA-Crys. One of isomerization (Asp 151) is occurred shortly after birth, thus that contribution for αA-Crys would be over a long term during life. This includes the effect for other post-translational modifications to contribute the formation of senile cataract.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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