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  • Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of α-crystallin: Implications for lens aging and presbyopia.

Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of α-crystallin: Implications for lens aging and presbyopia.

The Journal of biological chemistry (2020-03-19)
Sandip K Nandi, Rooban B Nahomi, Johanna Rankenberg, Marcus A Glomb, Ram H Nagaraj
ANOTACE

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein-cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin-γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens-epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone-client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone-client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin-client complexes could contribute to lens aging and presbyopia.

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Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
Sigma-Aldrich
Anti-alpha B-crystallin Antibody, from rabbit, purified by affinity chromatography
Sigma-Aldrich
Citrate Synthase from porcine heart, ammonium sulfate suspension, ≥100 units/mg protein
Sigma-Aldrich
Alcohol Dehydrogenase from Saccharomyces cerevisiae, ≥300 units/mg protein, lyophilized powder (contains buffer salts), Mw 141-151 kDa