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I. Nemet, X. Fan, V. M. Monnier; Novel Nonenzymatic Modifications of Lens Crystallins by Vitamin C Target Arginine Residues. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4618.
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Lens crystallins are prone to chemical modifications that affect chaperone function and favor toward aggregation and cataractogenesis. Vitamin C degradation products participate significantly in these chemical pathways in the lens. However, the products described so far can be formed from sugars other than Vitamin C and therefore do not reflect the specific role of Vitamin C in the modification. Here we characterized the structure of novel crystallin ascorbylation adducts and crosslinks with target amino acids and lens crystallins in vitro and from mice expressing the human vitamin C transporter 2 (hSCVT2) in the lens.
Boc protected amino acids, lysine and arginine were incubated with dehydroascorbic acid (DHA) under metal free conditions. Major incubation products were isolated by RP-HPLC and identified by NMR and mass spectrometry. These products were measured by LC-MSMS in bovine lens crystallins incubated with ascorbic acid or DHA (5 and 10 mM) for 7 days or crystallins from hSCVT2-mouse.
From the Lys-Arg-DHA incubation two previously described crosslinks were isolated Lys-Arg-3-deoxyerythrosone (1) and Lys-Arg-erythrosone (2). In the arginine-DHA model systems three novel hydroimidazolone products derived from xylosone (3), erythrosone (4) and 3-deoxyerythrosone (5) were identified.In crystallins incubated in vitro with 5 mM ascorbic acid or DHA crosslink 1 reached the levels of 2.0 and 41.6 pmol/mg of protein, while crosslink 2 reached the levels of 66 and 664 pmol/mg of protein, respectively. Level of hydroimidazolone 3 was 10.9 pmol/mg of protein in crystallins incubated with 10 mM DHA. Surprisingly, only the hydroimidazolone 3 was identified in levels of 226 pmol/mg protein from 3 months old hSVCT2 mouse lens, but crosslinks 1 and 2 were not detected.
Novel products of protein modifications by Vitamin C which target arginine in crystallins were characterized and identified in vitro and in vivo systems. They could destabilize arginine residues and contribute toward impaired chaperone function during aging.
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