Molecular Damage to Lens Crystallins by Vitamin C Products in Age-Related Cataracts

Hom, Grant

Vitamin C (ascorbic acid) is an essential antioxidant and ultraviolet (UV) filter in the eye lens. However, with age, Vitamin C is slowly oxidized to dehydroascorbate, which forms reactive sugars that can chemically react with nucleophilic amino acid residues in proteins by the so-called Maillard/glycation reaction. The goals of this research are to investigate the hypothesis that the Maillard reaction by Vitamin C may explain lens crystallin browning, precipitation and crosslinking that occurs in human cataracts, and to identify the sites of the protein damage by ascorbylation. To test this hypothesis, calf lens crystallin homogenate was separated into α-, βH-,βL- and γ-crystallin fractions by gel filtration chromatography, and fractions were incubated with 20 mM ascorbic acid until precipitation occurred. Proteomic analysis with mass spectrometry was used to reveal and identify protein modification sites that are selectively present in precipitated vs. soluble proteins. The results show that glycation of selective lysine (K) and arginine (R) residues, and oxidation of embedded tryptophan (W) residues occurred in precipitated but not soluble fractions of CRYBB1, CRYBB2, CRYBA4 and CRYBA1 crystallins. Molecular modeling studies are in progress to find out if ascorbylation favors protein unfolding and exposure of the protected tryptophan residues. In summary, this study is the first to provide molecular insights into how long-time exposure of lens proteins to Vitamin C might contribute to lens protein aging and cataract formation via destabilization and precipitation of selected crystallins, in particular beta crystallins.