Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.     

Human lens coloration and aging. Evidence for crystallin modification by the major ultraviolet filter, 3-hydroxy-kynurenine O-beta-D-glucoside.

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.     

Identification of the new UV filter compound cysteine-L-3-hydroxykynurenine O-beta-d-glucoside in human lenses

UV filters protect the human lens and retina from UV light-induced damage. Here, we report the identification of a new UV filter, cysteine-l-3-hydroxykynurenine O-beta-d-glucoside, which is present in older normal human lenses. Its structure was confirmed by independent synthesis. It is likely this novel UV filter is formed in the lens by nucleophilic attack of cysteine on the unsaturated ketone derived from deamination of 3-hydroxykynurenine O-beta-d-glucoside. Quantitation studies revealed considerable variation in normal lens levels that may be traced to the marked instability of the cysteine adduct. The novel UV filter was not detected in advanced nuclear cataract lenses.     

Identifying sites of attachment of UV filters to proteins in older human lenses

Recent results indicate that covalent modification of proteins by tryptophan-derived UV filters may explain the age-dependent coloration of human lenses, and play a role in age-related cataract. The sites of attachment of the UV filters to the lens crystallins, however, have not been determined. This study utilized a database of predicted masses of UV filter-modified tryptic peptides to target sites of UV filter attachment. Proteins were isolated from old normal lenses and digested with trypsin at pH 6, in order to preserve the integrity of the sites of modification. Peptides were separated by high-performance liquid chromatography and characterized by mass spectrometry. Major colored and fluorescent peaks in the digest were found to correspond to cysteine-containing peptides in which the sulfur atom of the sidechain was linked to the major UV filter compound, 3-hydroxykynurenine glucoside. Three of the peptides originated from gammaS-crystallin and one from betaB1-crystallin. These results show that a predicted mass database can be used to facilitate the identification of sites of UV filter modification in human lens crystallins. Furthermore, this work represents the first evidence that UV filters bind to specific residues on lens proteins in vivo, and suggests that sulfhydryl groups may be important sites for the attachment of UV filters.     

A general mechanism of polypeptide cross-linking by 3-hydroxykynurenine

Abstract

The human lens contains a group of fluorescent compounds, derived from tryptophan, which act to absorb UV light in the 300–400 nm region of the spectrum.¹ The major component is the glucoside of 3-hydroxykynurenine (3HK), 3-hydroxykynurenine glucoside (3HKG).²In the lens, 3HKG represents a unique pathway of tryptophan metabolism. Smaller amounts of kynurenine and 3HK have been detected in human lens extracts.^3,4 . More recently, a new UV-filter compound derived from tryptophan, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside (AHBG), was identified, and constitutes the second most abundant UV-filter in the human lens.⁵     

Novel protein modification by kynurenine in human lenses.

It is known that human lenses increase in color and fluorescence with age, but the molecular basis for this is not well understood. We demonstrate here that proteins isolated from human lenses contain significant levels of the UV filter kynurenine covalently bound to histidine and lysine residues. Identification was confirmed by synthesis of the kynurenine amino acid adducts and comparison of the chromatographic retention times and mass spectra of these authentic standards with those of corresponding adducts isolated from human lenses following acid hydrolysis. Using calf lens proteins as a model, covalent binding of kynurenine to lens proteins has been shown to proceed via side chain deamination in a manner analogous to that observed for the related UV filter, 3-hydroxykynurenine O-beta-D-glucoside. Levels of histidylkynurenine and lysylkynurenine were low in human lenses in subjects younger than 30, but thereafter increased in concentration with the age of the individual. Post-translational modification of lens proteins by tryptophan metabolites therefore appears to be responsible, at least in part, for the age-dependent increase in coloration and fluorescence of the human lens, and this process may also be important in other tissues in which up-regulation of tryptophan catabolism occurs.     

Isolation of a mutant of Salmonella typhimurium strain TA1535 with decreased levels of glutathione (GSH-). Primary characterization and chemical mutagenesis studies

A mutant of Salmonella typhimurium strain TA1535 with decreased glutathione (GSH) levels was isolated after treatment with UV and selection for N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) resistance; this GSH- mutant also exhibited increased resistance to MNNG, the methyl analog of ENNG. Estimation of the cellular GSH content showed that the GSH- derivative contained about 20% of the GSH levels found in TA1535. In mutagenicity tests (hisG46 leads to His+), the GSH- strain required the presence of GSH or L-cysteine in the medium for an optimal phenotypic expression and/or growth of spontaneous and induced His+ revertants, and may, therefore, be allelic to cys mutants of Salmonella described earlier. The mutagenic activity of MNNG, ENNG and 1,2-dibromoethane (DBE), but not that of N-ethylnitrosourea (ENU), was strongly reduced in TA1535/GSH-; pretreatment of the strain with GSH restored the mutagenicity of the first 3 chemicals to levels normally found in TA1535. The results support the current view that MNNG, ENNG and DBE, but not ENU, can be activated via reaction with GSH to species of higher reactivity and mutagenicity. It is concluded that the present GSH- strain can be used to study more systematically the role of GSH in the bioactivation and -deactivation of xenobiotics to mutagenic factors.     

Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation

GSH was readily depleted by a flavonoid, H(2)O(2), and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H(2)O(2) and peroxidase co-oxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H](+) and m/z 592 [M + H](+) in the positive-ion mode, respectively. (1)H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.     

In vitro attachment of bilins to apophycocyanin. III. Properties of the phycoerythrobilin adduct

Addition of phycoerythrobilin (PEB) to apophycocyanin at pH 7.0 resulted in covalent adduct formation. The adduct showed absorbance maxima at 575 and 605 nm and fluorescence emission maxima at 582 and 619 nm. Analysis of bilin peptides obtained upon tryptic digestion of the adduct showed residues alpha-Cys-84 and beta-Cys-82 to be the sites of bilin addition. The product of PEB addition at the alpha-Cys-84 site was shown by 1H NMR analysis to be a dihydrobiliviolinoid peptide-linked pigment differing in structure from that of the naturally occurring PEB-adduct by the presence of a double bond in between C2 and C3 of ring A. At the beta-Cys-82 site both a dihydrobiliviolinoid and a PEB adduct were obtained. Biliverdin also formed a covalent adduct with apophycocyanin with a lambda max of 669 nm. These results show that the spontaneous in vitro addition of bilins to apophycocyanin does not exhibit the site selectivity of bilin addition observed in vivo. This offers the opportunity to form novel semisynthetic phycobiliproteins.     

一品红苞片花色素的分离及初步鉴定

用紫外-可见光分光光度计、高效液相色谱（HPLC）和质谱（MS）技术对一品红（Euphorbia pulcherrima）红色苞片中的花色素提取液进行了初步鉴定.一品红花色素的甲醇溶液分别在270、340和520 nm处有3个吸收峰;在440 nm吸光度与可见光最大吸收波长520 nm吸光度的比值为0.29;花色素的甲醇溶液中加入AlCl3后发生红移,再加入HCl后发生蓝移;色素溶液在紫外光下无荧光;色素样品经液相色谱分离后在270 nm检测有5个比较明显的吸收峰;质谱中得到595、611、381、571和589等对应的分子离子峰;花色素酸解液高效液相色谱图谱和鼠李糖、葡萄糖的出峰时间一致.由这些结果可推断一品红花色素样品中主要含有5种组分：矢车菊花色素芸香苷、飞燕草花色素芸香苷、飞燕草花色素苯甲酰基葡糖苷、矢车菊花色素苯甲酰基葡糖苷和一种未知成分.     

Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde.

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.     

一品红苞片花色素的分离及初步鉴定

用紫外-可见光分光光度计、高效液相色谱(HPLC)和质谱(MS)技术对一品红(Euphorbia pulcherrima)红色苞片中的花色素提取液进行了初步鉴定。一品红花色素的甲醇溶液分别在270、340 和520 nm 处有 3 个吸收峰; 在 440 nm 吸光度与可见光最大吸收波长 520 nm 吸光度的比值 为0.29; 花色素的甲醇溶液中加入AlCl3后发生红移, 再加入HCl后发生蓝移; 色素溶液在紫外光下无荧光; 色素样品经液相色谱分离后在 270 nm检测有 5 个比较明显的吸收峰; 质谱中得到 595、611、381、571 和 589 等对应的分子离子峰; 花色素酸解液高效液相色谱图谱和鼠李糖、葡萄糖的出峰时间一致。由这些结果可推断一品红花色素样品中主要含有 5 种组分: 矢车菊花色素芸香苷、飞燕草花色素芸香苷、飞燕草花色素苯甲酰基葡糖苷、矢车菊花色素苯甲酰基葡糖苷和一种未知成分。     

3-Hydroxykynurenine oxidizes alpha-crystallin: potential role in cataractogenesis

The alpha-, beta-, and gamma-crystallins are the major structural proteins of mammalian lenses. The human lens also contains tryptophan-derived UV filters, which are known to spontaneously deaminate at physiological pH and covalently attach to lens proteins. 3-Hydroxykynurenine (3OHKyn) is the third most abundant of the kynurenine UV filters in the lens, and previous studies have shown this compound to be unstable and to be oxidized under physiological conditions, producing H2O2. In this study, we show that methionine and tryptophan amino acid residues are oxidized when bovine alpha-crystallin is incubated with 3-hydroxykynurenine. We observed almost complete oxidation of methionines 1 and 138 in alphaA-crystallin and a similar extent of oxidation of methionines 1 and 68 in alphaB-crystallin after 48 h. Tryptophans 9 and 60 in alphaB-crystallin were oxidized to a lesser extent. AlphaA-crystallin was also found to have 3OHKyn bound to its single cysteine residue. Examination of normal aged human lenses revealed no evidence of oxidation of alpha-crystallin; however, oxidation was detected at methionine 1 in both alphaA- and alphaB-crystallin from human cataractous lenses. Age-related nuclear cataract is associated with coloration and insolubilization of lens proteins and extensive oxidation of cysteine and methionine residues. Our findings demonstrate that 3-hydroxykynurenine can readily catalyze the oxidation of methionine residues in both alphaB- and alphaA-crystallin, and it has been reported that alpha-crystallin modified in this way is a poorer chaperone. Thus, 3-hydroxykynurenine promotes the oxidation and modification of crystallins and may contribute to oxidative stress in the human lens.     

Isolation and identification of two isomeric forms of malonyl-coenzyme A in commercial malonyl-coenzyme A

Two isomers of malonyl-coenzyme A (malonyl-CoA) were detected in a commercial preparation of malonyl-CoA. These compounds were separated by preparative high-performance liquid chromatography (HPLC) and characterized by HPLC/ultraviolet (UV)/mass spectrometry. Both compounds had a UV absorbance maximum at 259-260 nm. Both compounds underwent negative electrospray ionization to produce a [M-H](-)quasi-molecular ion at m/z 852 and both compounds underwent collision-induced dissociation to produce a characteristic fragment at m/z 808, all consistent with the structure of malonyl-CoA. Nuclear magnetic resonance spectrometry showed that the two chromatographically distinguishable malonyl-CoAs are structural isomers: the major component is the naturally occurring malonyl-CoA and the contaminant is 3'-dephospho- 2'-phospho-coenzyme A.     

delta 5,7,9(11)-Cholestatrien-3 beta-ol: a fluorescent cholesterol analogue

Structural analysis, a purification scheme and stability information on a fluorescent cholesterol analogue, which has been used as a probe in several model and biological systems, are presented. The proposed structure for the fluorophore, cholestatrien-3 beta-ol, closely resembles that of cholesterol. However, problems of low yield during synthesis and rapid decomposition have impeded its use. This study concerns the synthesis and purification of cholestatrien-3 beta-ol by reverse phase high performance liquid chromatography (HPLC). Unlike cholestatrien-3 beta-ol recrystallized from solvents, the fluorescent sterol purified by HPLC was stable over several months at -70 degrees C either as a white, crystalline powder or in ethanolic solution. In model membranes the fluorescence of cholestatrien-3 beta-ol was stable to ultraviolet (UV) light. A simple spectroscopic assay for purity is presented. Included are detailed absorbance, fluorescence, mass, 1H-NMR, and 13C-NMR spectral analyses. The data confirm the structure of cholestatrien-3 beta-ol proposed, but not proven, over 50 years ago, delta 5,7,9(11)-cholestatrien-3 beta-ol.     

Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage

The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.     

Spectroscopic studies of brunescent cataractous lenses

The absorption spectra of brunescent cataractous lenses and their homogenates were analyzed under various conditions by using a double wavelength spectrophotometer. The absorption spectra of the samples were in good agreement with those of synthetic xanthommatin derived from 3-hydroxykynurenine. The results provided evidence that brown pigment in the brunescent cataractous lenses is mainly composed of xanthommatin.     

Assessment of in situ cellular glutathione labeling with naphthalene-2,3-dicarboxaldehyde using high-performance liquid chromatography

We have presently studied a dialdehydic reagent, i.e. naphthalene-2,3-dicarboxaldehyde (NDA), as a fluorogenic probe for the labeling of intracellular reduced glutathione (GSH), using a yeast strain Candida albicans as a cell model. Chemical reactivity of NDA with both amino and sulfhydryl groups of the GSH molecule leads to a highly selective detection. Moreover, fluorescence properties of the resulting adduct fit well with most of modern instruments adapted for in situ measurements, and equipped with an argon laser. After incubation of cells with 100 microM of NDA for 20 min, cells were harvested and corresponding lysates obtained after a freezing cycle, were suspended in 0.2M borate buffer pH 9.2 and analysed with HPLC (column: Spherisorb ODS-2 (125 mm x 4.6 mm i.d.) 5 microm; mobile phase: methanol-0.01 M phosphate buffer pH 6.5 (20:80, v/v) at a flow rate of 0.8 mL min(-1); spectrofluorimetric detection: lambda(exc)=430 nm and lambda(em)=530 nm). The GSH-NDA adduct was identified in the yeast strain extracts using the reported HPLC technique and quantified versus a calibration curve of NDA derivatized with an excess of GSH (linearity range: 9-230 nM). The cell loading step of the free probe NDA and the extraction efficiency of the resulting NDA-GSH adduct were optimized.     

Morphine metabolism revisited. II. Isolation and chemical characterization of a glutathionylmorphine adduct from rat liver microsomal preparations

Incubation of tritium-labeled morphine and cold glutathione (GSH) or cold morphine and tritiated GSH with liver microsomal preparations obtained from phenobarbital-treated rats led to the identification by high performance liquid chromatography (HPLC) of a glutathionylmorphine adduct. Liquid secondary ion mass spectral analysis established the molecular weight of the metabolite to be 590 which corresponds to the mass of a mono-GSH-morphine adduct. High resolution (360 and 500 MHz) 1H-NMR experiments have led to the tentative assignment of the structure of this metabolite as 10-alpha-S-glutathionylmorphine. Based on both in vivo and in vitro data, the formation of this product appears to be mediated by cytochrome P-450 and to involve a reactive intermediate that may be responsible for the observed covalent binding of radiolabeled morphine to proteins and, at least in part, for the morphine-induced depletion of GSH in the rat.     

Identification of astaxanthin isomers in Haematococcus lacustrisby HPLC-photodiode array detection

An HPLC method was developed for the separation and identification of the isomers of astaxanthin from the saponification products of the individual astaxanthin ester fractions and the total pigment extract from Haematococcus lacus-tris. Six astaxanthin ester fractions were initially separated and collected by HPLC. These astaxanthin ester fractions and the total pigment extract were subsequently respectively hydrolysed. Four isomers of astaxanthin in the saponified mixtures were separated and identified respectively as (3S, 3¢S)- trans-astaxanthin (478.8 nm), (3S, 3¢S)-9-cis-astaxanthin (470.4 nm), (3S, 3¢S)-13- cis-astaxanthin (371.8 and 468.0 nm) and (3R, 3¢R)- trans-astaxanthin (477.6 nm) according to their absorbance spectra and absorption maxima by photodiode array detection. The relative contents of these isomers were determined to be 72.8, 9.7, 8.9 and 8.6%, respectively, in Haematococcus lacustris.