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Deamidation of Human ?S-Crystallin Increases Attractive Protein Interactions: Implications for Cataract.

ABSTRACT: Deamidation of proteins is one of the most prevalent post-translational modifications found upon aging, and in age-onset diseases. Specific asparagine and glutamine residues are often selectively deamidated during this process. In the human lens, deamidation has been shown to occur in many crystallins, but it is not clear how these deamidated proteins lead to lens opacity or cataract. Here we have modeled in vitro the effect of deamidation of specific asparagine and glutamine residues in human recombinant ?S-crystallin (HGS) on the solution properties of the protein. The residues selected for deamidation in vitro are those that are found to be deamidated in aged and cataractous lenses in vivo. Two derivatives were prepared, one with Asn76 and Asn143 deamidated (2N-HGS) and the other with two additional Gln residues (92 and 120) deamidated (2N2Q-HGS). Isoelectric focusing measurements showed the expected lowering of the pI from 6.9 in HGS to ?6.5 in 2N-HGS and to ?6.1 in 2N2Q-HGS. However, spectroscopic studies showed no significant change in the secondary and tertiary structures of the deamidated proteins relative to the wild type. The stability of 2N-HGS and 2N2Q-HGS, as measured by guanidinium hydrochloride unfolding, also remained comparable to that of HGS. The main difference was the altered protein-protein interaction among the three proteins. The net repulsive interactions that are characteristic of HGS are diminished in the deamidated derivatives as evidenced by static light scattering measurements of the second virial coefficient, B2 (B2 values for HGS, 2N-HGS, and 2N2Q-HGS of 8.90 × 10(-4), 7.10 × 10(-4), and 6.65 × 10(-4) mL mol g(-2), respectively). Further substantiation is provided by estimates of the excess binding energy of protein-protein interactions in the condensed phase, obtained from measurements of the PEG-induced liquid-liquid phase separation profiles for the three proteins. The data suggest that enhanced attractive protein-protein interactions, arising from the deamidation of HGS, promote protein aggregation, thereby leading to increased light scattering and opacity over time.

SUBMITTER: Pande A

PROVIDER: S-EPMC5760270 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Deamidation of Human γS-Crystallin Increases Attractive Protein Interactions: Implications for Cataract.

Pande Ajay A Mokhor Natalya N Pande Jayanti J

Biochemistry 20150729 31

Deamidation of proteins is one of the most prevalent post-translational modifications found upon aging, and in age-onset diseases. Specific asparagine and glutamine residues are often selectively deamidated during this process. In the human lens, deamidation has been shown to occur in many crystallins, but it is not clear how these deamidated proteins lead to lens opacity or cataract. Here we have modeled in vitro the effect of deamidation of specific asparagine and glutamine residues in human r ...[more]

PMID: 26158710

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Project description:Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (μs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

Dataset Information

Deamidation of Human ?S-Crystallin Increases Attractive Protein Interactions: Implications for Cataract.

Publications

Deamidation of Human γS-Crystallin Increases Attractive Protein Interactions: Implications for Cataract.

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