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

Lens proteins become increasingly crosslinked through non-disulfide linkages during aging and cataract formation. One mechanism that has been implicated in this crosslinking is glycation through formation of advanced glycation endproducts (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein-crosslinking 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 crosslinking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of crosslinking 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 crosslinking and higher crosslinking AGE levels. These results uncover a protein crosslinking mechanism in the lens and suggest that AGE-mediated crosslinking of α-crystallin-client complexes could contribute to lens aging and presbyopia.