Abstract
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.
Highlights
Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation
The objectives of this study were as follows: 1) to investigate whether ␣-crystallin– client protein complexes are stable in human lenses; 2) to test whether ␣-crystallin– client protein complexes are more prone to intermolecular nondisulfide cross-linking by advanced glycation end products (AGEs); and 3) to determine whether the accu
␣AC and ␣BC constitute ϳ40% of total proteins in human lenses [27]. They prevent aggregation of client proteins in the lens, thereby keeping the lens proteins soluble and maintaining lens transparency. ␣-Crystallin binds to aggregation-prone proteins, possibly through a “holdase” mechanism that does not require ATP hydrolysis and prevents further denaturation of the protein [28]
Summary
Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. 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 ␣-crystallin– client complexes could contribute to lens aging and presbyopia. The eye lens is an avascular tissue It has to remain transparent during aging to focus light onto the retina. Glutathione (GSH) levels are reduced [12], which could lead to increased ascorbate oxidation and to the formation of glycation precursors. The loss of GSH could decrease MGO metabolism and allow MGO to form AGEs in lens proteins
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