Human Lens Coloration and Aging

Abstract

The human lens becomes increasingly yellow with age and thereby reduces our perception of blue light. This coloration is associated with lens proteins (crystallins), but its molecular basis was unknown. Here we show that the coloration occurs because of the interaction of crystallins with a UV filter compound, 3-hydroxykynurenine glucoside (3-OHKG). Crystallin modification results from deamination of the 3-OHKG amino acid side chain, yielding an unsaturated ketone that is susceptible to nucleophilic attack by cysteine, histidine, and lysine residues. This novel protein modification contributes to age-related lens coloration and may play a role in human nuclear cataractogenesis.As part of the normal process of aging, the human lens becomes progressively more yellow and fluorescent (1-3), leading to a concomitant increase in light absorption in the 300 -500 nm range (1) and thus diminishes our perception of violet and blue light. The age-related increase in lens coloration and fluorescence is associated with the major proteins of the lens, the crystallins, and is particularly prominent in the lens nucleus (2, 3). Because there is little or no protein turnover in the lens nucleus (4), the proteins are as old as the individual. The post-translational modifications that result in crystallin coloration, therefore, accumulate throughout life and may eventually contribute to age-related nuclear cataract. The latter condition is characterized by a brown coloration of the lens nucleus and extensive protein oxidation (5, 6). The human lens also contains a family of Trp-derived UV filter compounds, of which 3-OHKG 1 is present at the highest concentration (Ϸ500 M) (7, 8). These UV filters are thought to play a protective role by preventing potentially damaging UV light from reaching the retina. Several investigators have considered the possibility that the UV filters could covalently modify lens crystallins with deleterious consequences, including alteration of protein conformation and increased sensitivity to UV light (9 -11). However, the mechanism leading to crystallin modification by 3-OHKG was not determined nor was there any evidence that 3-OHKG induces protein modification in vivo.We have recently elucidated a novel pathway that leads to the formation of a glutathione (GSH) adduct of 3-OHKG in the human lens (12). The GSH-3-OHKG adduct was formed via deamination of the 3-OHKG amino acid side chain to form an ␣, ␤-unsaturated carbonyl that was highly susceptible to nucleophilic attack by the cysteine of GSH (12). The aim of the present studies is to assess the relevance of analagous reactions in the modification of crystallins and also to probe for evidence of crystallin-3-OHKG adducts in human lenses. EXPERIMENTAL PROCEDURESMaterials-All organic solvents were HPLC grade (Ajax, Unichrom, Auburn, NSW, Australia). Poly-L-lysine was from Sigma, trifluoroacetic acid (Ͼ99% pure) was from Aldrich, and acetic acid (Ͼ99.8% pure) from BDH (Poole, UK). 3-hydroxykynurenine O-␤-D-glucoside (3-OHKG) and 2-amino-3-hydroxyacetopheno...

Novel Protein Modification by Kynurenine in Human Lenses

“…For example, the concentrations of the His-Kyn and Lys-Kyn adducts in a 71-year-old lens were 0.70 and 0.12 nmol/mg protein, respectively. These levels, and the degree of scatter noted as a function of age, are comparable with those of 3-OHKG bound to human lens protein (36). The concentrations of Kyn-modified amino acids were observed to increase in concentration with the age of the individual.…”

“…By contrast, free Kyn is barely fluorescent. In recent work (13,36), we have identified a novel UV filter compound, the 3-OHKG adduct of GSH, which is also formed by a Michael-type addition, and have shown that 3-OHKG is also bound to lens crystallins. Thus it can be inferred that 3-OHKyn, which is also present at significant levels in human lenses, will react in a similar manner to 3-OHKG and Kyn.…”

3-Hydroxykynurenine-mediated Modification of Human Lens Proteins

“…Michael addition at C-9 on oxidized tryptophan has been well documented, and several additive products of lysine, histidine, and cysteine have been isolated and detected in the human lens (12,19,22). Our antibody reacted nearly as well with several 3OHKYN modifications, including N ␣ -acetyl lysine, N ␣ -acetyl histidine, N ␣ -acetyl arginine, and N ␣ -acetyl cysteine, although the N ␣ -acetyl histidine modifications reacted more strongly than products of the other amino acids.…”

“…Kynurenines react with nucleophilic amino acids, such as cysteine, histidine, and lysine in lens proteins (18 -20) and cysteinyl residue in GSH through Michael addition to form covalent adducts. One of the products of the reaction of 3OHKYNG with lens GSH is glutathionyl-3-hydroxykynurenine glucoside (21), which accumulates during lens aging and accumulates to rel-atively high levels in cataractous lenses (19). Vazquez et al (22,23) demonstrated modification of histidine and lysine residues of lens proteins by KYN and their accumulation in aging lenses, but these authors also noted that the modified products decrease in senile cataracts.…”

Human α-1-Microglobulin Is Covalently Bound to Kynurenine-derived Chromophores

“…It is known that deamination of the amino acid side chain of Kyn yields an unsaturated ketone that is susceptible to attack by nucleophilic amino acids, such as His, Lys, and Cys and that the process is favored when the pH is increased to 9.5 (25). The amniotic fluid ␣-1-m was treated with Kyn in carbonate buffer at pH 9.5 (15) and, after gel filtration at pH 7.0, the modification was verified following the optical properties of the adduct.…”