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T. Takata, K. Lampi; Deamidations in βA3-crystallin Are Highly Correlated With Insolubulization During Aging. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3801.
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Recently, βA3-crystallin was reported to be the most deamidated crystallin in the insoluble proteins in aged and cataractous lenses (Wilmarth et al, 2006). Many of the deamidation sites were detected on the surface of βA3 in vivo. The purpose of this study was to determine the effects of reported deamidations on the surface and in the connecting linker of βA3 in vitro.
Deamidations were mimicked on the surface of the N-terminal domain of βA3 at Q42 and N54, on the C-terminal domain at N133 and N155, and in the linker peptide at N120 by replacing the amides with the respective acidic residues by site-directed mutagenesis. Far and near UV-circular dichroism, multi-angle light scattering (MALS), quasi-elastic light scattering, size exclusion chromatography, and analytical ultra-centrifugation were performed to characterize the structure and formation of the dimer. The stability of proteins was investigated by measuring the turbidity during heating and the fluorescence during unfolding/refolding in urea. Formation of hetero-oligomers with βB1-crystallin was determined by size exclusion chromatography with MALS and native-electrophoresis at 37 °C.
All proteins folded with similar predicted secondary structure. Differences were detected in tertiary structure particularly for N120D, N133D, and N155D. All of the βA3- expressed proteins were predicted by analytical sedimentation and light scattering to have very elongated elliptical structures without dimer dissociation. Deamidation at Q42, N120, N133, and N155 decreased stability, with deamidation at N120 and at N155 decreasing stability the most. βA3 formed a hetero-oligomer with βB1 at a one-to-one ratio. The deamidated mutants, Q42E, N120D, and N133D formed similar size hetero-oligomers. However, N54D and N155D formed hetero-oligomers with a lower molecular weight, suggesting disruption of the oligomer formation.
These results suggest that the effect of deamidation on the structure, stability, and interactions with β-subunit, βB1, are dependent on the specific location of the deamidation. These in vitro experiments strongly suggest that specific deamidations on the surface of βA3 alter subunit interactions and higher oligomer state, and may be contribute to the insolubilization reported in aged and cataractous lenses in vivo.
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