Abstract
Purpose :
Lens proteins become increasingly crosslinked through non-disulfide linkages during aging and cataract formation. Glycation through formation of advanced glycation end products (AGEs) has been implicated for such crosslinking. α-Crystallin binds to other lens proteins to prevent their denaturation and aggregation during aging. Since the lens is largely metabolically inactive, we hypothesized that the α-crystallin-client complexes remain stable and become susceptible to crosslinking by AGEs.
Methods :
Human αA-, αB-, and γD-crystallins were cloned, expressed in E. coli and purified by ion-exchange and size exclusion chromatography. A fluorescence resonance energy transfer (FRET) assay was developed to determine stability of αA-crystallin-γD-crystallin complex.Thermal stress (25 to 43°C) was applied to α-crystallin-citrate synthase(CS) protein mixture and formation of chaperone-client protein complex was assessed by size exclusion chromatography. The complexes were glycated with erythrulose (250 μM) and methylglyoxal (200 μM) for 7 days at pH 7.4 and 37°C. Lens stiffness was measured in 13 to 75-year old human donor lenses. AGEs were measured by LC-MS/MS.
Results :
The FRET assay showed that αA-crystallin-γD-crystallin complex was stable under physiological conditions up to 12 days tested. Upon incubation with human lens epithelial cell lysate or lens homogenate (from young: 13 years or aged: 75 years), even when supplemented with 2 mM ATP, complexes remained stable. Upon glycation, α-crystallin-CS complex underwent greater levels of non-disulfide crosslinking than non-complexed protein mixtures. LC-MS/MS analysis showed that levels of crosslinking AGEs were significantly higher in glycated chaperone-client complexes than in glycated non-complexed or non-glycated protein mixtures. Mouse lenses subjected to thermal stress followed by glycation showed decreased resilience more than lenses subjected to glycation alone or control lenses. This was accompanied by increased protein crosslinking and crosslinking AGEs. Human lens stiffness increased with age and the increase positively correlated with the levels of protein crosslinking AGEs.
Conclusions :
Our study uncovers a novel biochemical mechanism for protein crosslinking and provides an explanation for stiffening and onset of presbyopia in aging lenses.
This is a 2020 ARVO Annual Meeting abstract.