Rongzhu Cheng

Vitamin C mediates chemical aging of lens crystallins by the Maillard reaction in a humanized mouse model

Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. We hypothesized that the Maillard reaction, which leads browning and aroma development during the baking of foods, would occur between the lens proteins and the highly reactive oxidation products of vitamin C. To test this hypothesis, we engineered a mouse that selectively overexpresses the human vitamin C transporter SVCT2 in the lens. Consequently, lenticular levels of vitamin C and its oxidation products were 5- to 15-fold elevated, resulting in a highly compressed aging process and accelerated formation of several protein-bound advanced Maillard reaction products identical with those of aging human lens proteins. These data strongly implicate vitamin C in lens crystallin aging and may serve as a model for protein aging in other tissues particularly rich in vitamin C, such as the hippocampal neurons and the adrenal gland. The hSVCT2 mouse is expected to facilitate the search for drugs that inhibit damage by vitamin C oxidation products.

Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A(330) units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R(f) values for the long wavelength-absorbing fluorophores. Glycation with [U-(14)C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A(330)-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A(330)-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A(330)-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo.

Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue.

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A(330)-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A(330)-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60. Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins.

Separation of the yellow chromophores in individual brunescent cataracts

Quantitative changes in the 330 nm absorbing chromophores and 350/450 nm fluorophores of water-soluble (WS) and water-insoluble (WI) proteins of individual human cataract lenses were characterized and compared with aged normal human lens. Twenty-five brunescent cataract lenses from India were selected from five different stages (types I-V) based upon the color of the lens. The WS and WI proteins from each lens were collected and subjected to an extensive enzymatic digestion procedure under argon. The lens protein digests were separated by Bio-Gel P-2 size-exclusion chromatography and individual peaks were analyzed further by reversed-phase HPLC. The total WI proteins increased and the total WS protein decreased with the development of cataract, especially in the late stages of cataract (III-V). The total 330 nm absorbance and 350/450 nm fluorescence of the WI fraction also increased, however, the A(330) and fluorescence per mg lens protein were constant except for type V (black) lenses. Bio-Gel P-2 chromatography separated the chromophores and fluorophores into four fractions. The main fraction (designated as peak 2+3) from the cataract WI proteins was several times higher than that present in aged normal human lens WI proteins. A significant increase of this fraction was observed in WI proteins, but not in WS proteins with cataract development. Similarly, fractions 1 and 4 in the WI proteins also increased gradually but fraction 5 did not. Reversed-phase HPLC resolved fraction (2+3) of the water-insoluble sonicate supernatant proteins into four 330 nm absorbing peaks and eight fluorescent peaks. Among these peaks, a late-eluting peak (peak 8) increased 10 to 15-fold with the progress of cataract, and accounted for 80% of the total chromophores in type V lenses. This peak may represent limit digests of advanced glycation end-products (AGEs) derived protein cross-links. HPLC profiles of fraction 5 from both WS and WI proteins showed numerous new peaks which were not observed in either WS protein from cataract or WI proteins from aged normal human. The severe coloration and the higher levels of numerous novel chromophores and fluorophores in brunescent cataractous lenses reveal the possibility that a different chemistry occurs during cataract development.

K2P—A Novel Cross-Link from Human Lens Protein

Abstract: We report here the isolation of a novel acid‐labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by LC‐MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One‐ and two‐dimensional NMR analyses elucidated the structure as being 1‐(5‐amino‐5‐carboxypentyl)‐4‐(5‐amino‐5‐carboxypentyl‐amino)‐3‐hydroxy‐2, 3‐dihydropyridinium, a cross‐link between the ε‐amino groups of two lysine residues and a five‐carbon atom ring. We assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water‐soluble and water‐insoluble protein digests revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 ± 362 pmol/mg of water‐insoluble sonicate supernatant (WISS) protein or 85 ± 51 pmol/mg of water‐soluble [WS] protein) when compared with aged normal human lens proteins (261 ± 93 pmol/mg of WISS protein or 23 ± 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross‐link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.