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1From the Save Sight Institute, University of Sydney, Sydney, NSW, Australia; the 2Department of Chemistry, University of Wollongong, Wollongong, NSW, Australia; the 3Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia; and the 4Heart Research Institute, Camperdown, Sydney, NSW, Australia.
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Abstract |
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METHODS. The t-Boc–protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC.
RESULTS. At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in ß-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys.
CONCLUSIONS. These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.
In the normal human lens, levels of protein-bound UV filters increase in an age-dependent manner, with a significant increase in the levels of modification occurring after middle age.4 5 6 A corresponding decrease with age in the levels of free UV filters in lenses is also observed, with 3-OHKG, 3-OHKyn, and Kyn all declining by approximately 12% per decade.2
UV filters are intrinsically unstable under physiological conditions and undergo side chain deamination to yield 
,ß-unsaturated ketones.2 7 These can react with nucleophilic species in the lens through Michael addition. Reaction with protein side chains is particularly favored because the lens has the highest protein concentration of any human tissue.8 Approximately 90% of the proteins are crystallins,8 making these major targets for modification.
Around middle age in humans, a barrier forms in the lens9 10 that hinders the diffusion of low molecular–weight species, such as the antioxidant glutathione, from the cortex into the nucleus and traps small molecules, including UV filters, in the lens nucleus. In proteins in the nuclei of older lenses, this results in greater susceptibility to modification by compounds such as deaminated UV filters.
Protein turnover in the human lens is negligible11 ; hence, modifications accumulate over a person’s lifetime12 that may affect protein tertiary structure and predispose the lens to age-related nuclear cataract. For example, model studies show that proteins to which Kyn is attached become oxidized after exposure to wavelengths of light that penetrate the cornea.13
The present study investigated the rate of formation of Kyn-amino acids and the stability of these adducts under physiological conditions in vitro. These data point to an important role for protein cysteine (Cys) residues in the initial phase of modification by UV filters and suggest that glutathione may play a key role in preventing posttranslational modification of lens proteins.
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Materials and Methods |
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-t-Boc-L-His, N--t-Boc-L-Lys, N--t-Boc-L-Cys, and Cys), DL-kynurenine and DL-kynurenine (Kyn) sulfate salt, HCl (6 M, sequencing grade), glutathione, guanidine HCl, phenol, thioglycolic acid, trifluoroacetic acid (TFA), formic acid, and sodium hydroxide were all purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Protein concentrators (MWt cut-off, 10,000 Da) were purchased (Vivaspin; Vivascience Sartorius AG, Göttingen, Germany). All organic solvents were HPLC grade (Ajax, Auburn, NSW, Australia).
Rate of Formation of Kyn-Amino Acids
N--t-Boc (tert-butyloxycarbonyl) amino acids were used to confine chemical reactions to the amino acid side chains and therefore to serve as a model for the amino acid residues in proteins. DL-Kyn (3 mM) and either N--t-Boc-L-His, N--t-Boc-L-Lys, N--t-Boc-L-Cys, or L-Cys (75 mM) were dissolved in 200 mM phosphate buffer (pH 7.2). For these and subsequent incubations, each vial was bubbled with argon, sealed, wrapped in foil, and incubated at 37°C. Triplicate samples (70 µL) were taken at the indicated times and diluted with aqueous 0.05% (vol/vol) TFA (70 µL).
HPLC Measurement of Kyn-Amino Acid Formation
Reverse-phase (RP)-HPLC was performed on an HPLC system with a diode array detector (SPD-M10AVP; Shimadzu, Kyoto, Japan). For analytical scale separation, an analytical C18 column (100 Å, 5 µm, 4.6 mm x 250 mm; Microsorb-MV; Varian, Palo Alto, CA) was used under the following mobile-phase conditions: solvent A (0.05% vol/vol TFA) for 5 minutes, followed by a linear gradient of 0% to 40% solvent B (80% acetonitrile/H2O, 0.05% vol/vol TFA) over 50 minutes, then a linear gradient of 40% to 0% solvent B over 5 minutes at a flow rate of 1 mL/min.
Stability of Kyn-Amino Acid Adducts
The t-Boc–protected Kyn-amino acid adducts Kyn-t-Boc-His and Kyn-t-Boc-Lys and the sulfoxide of Kyn-t-Boc-Cys were prepared as previously described,5 dissolved (at 1.0–2.5 mM) in 200 mM phosphate buffer, pH 7.2, and incubated. Triplicate samples were taken every 20 hours for RP-HPLC.
Incubation of Kyn-Cys in the Presence of Excess N--t-Boc-L-His
Kyn-Cys (1.0 mg) was dissolved in 2.0 mL of 200 mM phosphate buffer (pH 7.2), with a 20-fold molar excess of N--t-Boc-L-His. Aliquots (100 µL) were removed every 12 hours and analyzed by RP-HPLC.
Modification of Calf Lens Proteins with Kyn
Proteins were extracted from bovine lenses as described previously.14 Calf lens protein (CLP; 10 mg/mL) and DL-Kyn sulfate (2 mg/mL) were dissolved in 200 mM phosphate buffer, pH 7.2. Chloroform (100 µL) was added as an antibacterial agent. After 14 days of incubation, modified CLP was dialyzed against 1 mM phosphate buffer (pH 6) at 4°C and lyophilized.
Effect of Glutathione on CLP Modification
CLP (100 mg/mL) and DL-Kyn sulfate (2 mM) were dissolved in chelating resin (Chelex; Bio-Rad, Hercules, CA)–treated phosphate buffer (200 mM), in the absence and presence of glutathione (2 mM). Chloroform (10 µL) was added as an antibacterial agent. The solution was placed under argon, sealed, and incubated at 35°C for 4 days. Modified protein was isolated from the reaction mixture with the use of a centrifugal filter unit (Centricon, with YM-3 membrane; Millipore). Aliquots of the modified protein (200 µL) were loaded onto units (Centricon; Millipore) and washed with ultrapure water (1 mL; Milli-Q; Billerica), followed by 6 M guanidine HCl (1 mL). The centrifugal filter unit (Centricon; Millipore) was inverted and spun at 1000g for 3 minutes to remove the protein from the membrane. UV-Vis spectra of the modified proteins were obtained on a spectrophotometer (UV-1700; Shimadzu) to determine levels of modification using absorbance at 360 nm.
Preparation of Human Lens Proteins
Human tissue was handled in accordance with the tenets of the Declaration of Helsinki. Normal human lenses were obtained from the NSW Lions Eye Bank (Sydney, NSW Australia). Nuclei were obtained with a cork borer (5 mm), and the ends (1 mm) were removed. Nuclei were homogenized in 500 µL of 6 M guanidine HCl and dialyzed overnight against 100 mM sodium acetate/acetic acid buffer, pH 4, and lyophilized.
Acid Hydrolysis of Kyn-Modified Proteins and Human Lens Proteins
Kyn-modified lens proteins (4–8 mg) or human lens proteins (5 mg) were hydrolyzed with 6 M HCl for 24 hours at 110°C, then analyzed by HPLC as described previously.6
Rate of Formation of Kyn-Amino Acid Adducts on Proteins
CLP (10 mg/mL) was incubated with Kyn (2 mg/mL) in 200 mM phosphate buffer, pH 7.2, under argon at 37°C. Aliquots (10 mg protein) were removed every 2 days and modified protein filtered (6000g, 30 minutes, 10°C) in a concentrator (Vivaspin; Vivascience Sartorius AG) and washed three times with 1 mL water. Lyophilized protein (4–7 mg) was acid hydrolyzed and analyzed by RP-HPLC. The filtrate was also analyzed by RP-HPLC using conditions for measuring the formation of Kyn-amino acid adduct.
Stability of Protein-Bound Kyn
CLP (5 mg/mL, covalently modified with Kyn at pH 7.2) was incubated in 200 mM phosphate buffer, pH 7.2, at 37°C. Aliquots (7–10 mg protein) were filtered with a concentrator (Vivaspin; Vivascience Sartorius AG) and washed with water (1 mL), and the protein was lyophilized. Protein samples were acid hydrolyzed and analyzed by RP-HPLC.
Tryptic Digestion
CLP (1.0 mg) was modified with Kyn in 50 mM ammonium bicarbonate buffer, pH 9.0, for 48 hours and then digested with trypsin (1 mg/mL) in 0.1 M morpholino ethane sulfonic acid (MES) buffer, pH 6.0, for 48 hours at 37°C at a substrate/enzyme ratio of 50:1 (Cys-Kyn is stable at pH 6.0).
Incubation of Kyn-t-Boc-Cys with Excess Glutathione
Kyn-t-Boc-Cys (1 mM) was dissolved in 100 mM phosphate buffer, pH 7.2, containing glutathione (5, 20, or 40 mM). Chloroform (20 µL) was added. Aliquots were taken every 24 hours for RP-HPLC.
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Results |
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,ß-Unsaturated Ketone Derived from Kyn,ß-unsaturated ketone that reacts with amino acid nucleophiles.7 The aim of this study was to investigate the rate of reaction of the N--t-Boc (tert-butyloxycarbonyl) nucleophilic amino acids N--t-Boc-L-His, N--t-Boc-L-Lys, and N--t-Boc-L-Cys with Kyn under physiological conditions (pH 7.2, 37°C). N--t-Boc derivatives were chosen to limit the reaction of Kyn to the amino acid side chains. Incubation of Kyn with these amino acid derivatives, at pH 7.2, yielded all three Kyn-amino acid adducts. The data obtained (Fig. 1) indicate that N--t-Boc-L-Cys was the best nucleophile, followed by N--t-Boc-L-His and then by N--t-Boc-L-Lys (Fig. 1) , with the apparent rates of reaction 6.0 (±0.8) x 10–6, 2.0 (±1) x 10–6, and 1.6 (±0.93) x 10–6 µmol/min, respectively. These figures do not take into account any possible decomposition under the reaction conditions.
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Incubation of Kyn-Cys under analogous conditions resulted in a 70% decrease in the levels of the adduct over 80 hours (Fig. 2) . Therefore, the t-Boc adduct was twofold to threefold more stable than Kyn-Cys under these conditions. These data show that protection of the -amino moiety with a t-Boc group significantly increased the stability of the Kyn-Cys adduct at pH 7.2, suggesting that the -amino group of Cys might have assisted the deprotonation necessary for the formation of deaminated Kyn7 or that the bulky t-Boc group might have obstructed the access of basic species (e.g., hydroxyl ion) to the Kyn side chain.
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-t-Boc-L-His-t-Boc-L-His, at pH 7.2. As shown in Figure 5 , Kyn-Cys rapidly decreased in concentration, with an 82% loss after 24 hours and a corresponding formation of deaminated Kyn (data not shown). Deaminated Kyn then decreased in concentration as it reacted with t-Boc-His. Most deaminated Kyn reacted with N--t-Boc-L-His; after 48 hours, the level of Kyn-t-Boc-His (confirmed by ESI-MS/MS) corresponded to 75% of the original level of Kyn-Cys.
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Ultrafiltrate from the incubation of Kyn with CLP was analyzed by RP-HPLC to determine the major Kyn-derived species formed in the presence of protein. A doublet peak, eluting at 41 to 42 minutes (HPLC profile not shown), had an elution time similar to that of an authentic standard of Kyn dimer.7 Mass spectrometry confirmed this identification, with a molecular ion at m/z 400 and characteristic fragment ions at m/z 383 (loss of NH3), 265 (loss of NH2PhCOCH3), and 192 (loss of Kyn).7 Deaminated Kyn was also identified in the filtrate. Concentrations of both species increased steadily with incubation time (Fig. 7B) . A similar trend was observed previously in which the incubation of 3 mM Kyn at pH 7, in the absence of protein, led to the formation of 1.25 mM Kyn dimer and approximately 0.3 mM deaminated Kyn after 7 days.7
Sites of Modification
Major sites of Kyn modification on CLP were identified by tryptic digestion at pH 6 (Kyn-Cys is stable at pH 6), HPLC separation of the peptides, and MS/MS analysis. Data are summarized in Table 1 . Peptides derived from ß-crystallins were found to predominate.
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-t-Boc-L-His provided evidence of transfer of the deaminated Kyn moiety from one amino acid to another (Fig. 5) . It was hypothesized that prolonged incubation of Kyn-modified lens proteins, which contain predominantly Kyn-Cys residues, may yield Kyn-His as a dominant modification. To test this hypothesis, CLPs were modified with Kyn (pH 7.2) for a period of 14 days and then were purified from unbound Kyn by exhaustive dialysis against 1 mM phosphate buffer, pH 6.0. The solution was readjusted to pH 7.2, and then the solution was incubated at 37°C. Aliquots of protein were removed at intervals, purified, and hydrolyzed in the presence of antioxidants. During the first 6 days of incubation, the concentration of Kyn-Cys decreased by 54% and then plateaued (Fig. 8) . In agreement with the proposed transfer of the deaminated Kyn moiety, the levels of Kyn-His increased by 38% after 14 days of incubation. Despite this, Kyn-Cys remained the dominant modification. Therefore, although the initial time points obtained from incubation of Kyn-modified CLP mimicked those observed from the amino acid studies, later ones did not. Thus, significant Kyn-Cys levels were observed in the CLP even after extended incubation, and Kyn-His increased only slowly, despite deaminated Kyn in the solution. These findings suggest an important role for protein structure and solvent accessibility in determining the final outcome of the modification. In agreement with this, significant levels of Kyn-Cys were found in proteins isolated from older normal human lenses (22 pmol/mg protein, 76 years; 26 pmol/mg protein, 73 years).
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Discussion |
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In the lens, the role of glutathione appears to be crucial. Glutathione can inhibit the reaction of proteins with UV filters in three ways: by intercepting the initial reactive product formed by deamination of the kynurenine7 ; by promoting decomposition of protein Kyn-Cys adducts once formed (Fig. 8) and by covalently linking to the deaminated UV filters released from the modified protein so that they can diffuse from the lens.
It is therefore possible to rationalize the observation that young human lenses have little coloration, despite free UV filters being present in higher concentrations than in older lenses.2 Glutathione levels in young lenses are high. When deamination of a UV filter takes place, the ,ß-unsaturated ketone formed will predominantly react with glutathione before it can bind to proteins.2 17 To test whether glutathione at physiological concentrations could prevent covalent modification by UV filters, CLP was incubated with Kyn in phosphate buffer, pH 7.2, in the presence and absence of 2 mM glutathione. In the absence of glutathione, a linear increase in absorbance of the protein at 360 nm was observed over 4 days, in agreement with previous model studies.14 Inclusion of glutathione at the start of the incubation prevented this linear increase (data not shown).
The concentration of protein thiol groups, even in young lenses, is higher than that of glutathione, but it is unknown what percentage of these Cys residues are solvent exposed. If some UV filter decomposition molecules do bind to crystallins, the binding will most likely be to Cys residues. The Kyn-Cys adducts will decompose at pH 7, but the rate is increased considerably by high glutathione. In addition, because the barrier to diffusion between the cortex and the nucleus has not yet formed,9 10 18 the average residence time for UV filters in the lens center, and therefore the time available for deamination, is greatly reduced. After middle age, when glutathione levels decrease2 19 and the barrier forms, net covalent modification of proteins in the nucleus is observed.20
The present investigation aimed to explore some aspects of the kinetics of Kyn-amino acid formation and the relative stabilities of Kyn-amino acid adducts under physiological conditions. Studies using t-Boc–protected amino acids, as a model for His, Lys, and Cys residues in proteins, revealed that Cys reacts with Kyn threefold to fourfold faster than His or Lys (Fig. 1) . Cys was also more rapidly modified in CLP (Fig. 7) . These results are consistent with those of a previous study that examined the reactivity of -crystallin with Kyn in which the single Cys of A-crystallin was found to be the initial site of modification.21 Thiol compounds are known to be good nucleophiles.22 The same relative order of nucleophilicity of Cys, His, and Lys residues at physiological pH has been shown in other systems. For example, 4-hydroxynonenal, an oxidation product of lipids, reacts with proteins at nucleophilic sites through Michael addition.23
The environment of Kyn appeared to have a marked effect on its stability. Thus, Kyn-t-Boc-Cys was more stable than Kyn-Cys, with the t-Boc on the -amino group enhancing adduct stability by approximately threefold. The increased stability of Kyn-t-Boc-Cys might have resulted from the t-Boc group providing steric hindrance, the free -amino group of Cys participating in base-catalyzed hydrolysis of Kyn-Cys, or both. Incubation in the presence of t-Boc-His also increased the rate of breakdown of Kyn-Cys (Fig. 5 ; see Fig. 2 ).
Decomposition of Kyn-Cys was accompanied by the release of deaminated Kyn. This unsaturated ketone appears to be involved in three further reaction pathways: intramolecular condensation of the aromatic amino group of deaminated Kyn with the unsaturated side chain, leading to the formation of Kyn yellow16 ; reaction with the thiol group of Cys to re-form the Kyn-Cys adduct; or reaction with another molecule of deaminated Kyn, through intermolecular nucleophilic addition of the aromatic amino group to form a dimer of deaminated Kyn (Fig. 3) . In the human lens, whose absorbance in the 360- to 370-nm region increases with age,24 25 formation of a dimer of deaminated Kyn is likely to be a minor reaction compared with the reaction of deaminated Kyn with lens proteins because His, Cys, and Lys side chains are present in higher concentrations than Kyn.
With t-Boc amino acids, it was demonstrated that the deaminated Kyn moiety can transfer from one amino acid to another. For example, incubation of Kyn-Cys with excess t-Boc-His at pH 7.2 resulted in a loss of Kyn-Cys and a corresponding increase in Kyn-t-Boc-His because of the release of deaminated Kyn from the breakdown of Kyn-Cys and its subsequent reaction with t-Boc-His (Fig. 5) .
Formation of Kyn-amino acid adducts on lens proteins was also examined. Bovine crystallins were used because of their considerable sequence homology with human proteins. Bovine lenses do not contain UV filter compounds26 ; therefore, the resultant CLPs are not modified by these compounds. Reaction of Kyn with CLP at pH 7.2 resulted in the formation of colored and fluorescent (Ex 365/Em 460–480 nm) protein. After 10 days of incubation, Kyn modification was observed at all three residues, with Kyn-Cys, Kyn-His, and Kyn-Lys present at 1.6, 0.1, and 0.07 mol/mol protein, respectively (Fig. 7) . Therefore, in proteins, Cys is the preferred site of Kyn modification, which is consistent with the amino acid data (Fig. 1) .
The stability of Kyn attached to Cys in proteins was comparable with that of the free Kyn-t-Boc-Cys adduct in the initial phase of the modified CLP incubation (Figs. 2 8) . After prolonged incubation, the levels of Kyn-His increased, primarily as the result of decreased Kyn-Cys. Kyn-Lys decreased to an even smaller extent. In contrast to the amino acid studies (Fig. 5) , a significant proportion of Kyn-Cys remained covalently bound to the protein, even after prolonged incubation. This suggests that some sites of attachment of Kyn to Cys may be buried within the tertiary structure of the proteins and may be unavailable for hydrolysis. This aspect will form the basis of a future study.
Tryptic digestion revealed that the ß-crystallins were major sites of modification by Kyn. ßB2-crystallin contains two solvent exposed sulfhydryls, Cys 37 and Cys 66,27 which are accessible to N-iodoacetyl-N’-(5-sulfo-1-naphthyl)ethylenediamine and which react with 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB).28 Our finding that these Cys residues were modified by Kyn is consistent with these observations. Cys 66 has been identified as a site of Kyn modification.29 The 
-crystallins are also rich in Cys residues,27 but surprisingly we detected only one Kyn-modified peptide, from S-crystallin.
At pH 7.0, Cys, Lys, and His were all modified, but Cys was by far the major site of modification. In bovine lens crystallins, Lys is the most abundant (, 7.8%; ß, 5.6%; , 7.3%) followed by His (, 4.6%; ß, 3.5%; , 3.1%) and then by Cys (, 0.3%; ß, 1.9%; , 3.4%).30 Therefore, the pattern of modification observed is not simply a function of amino acid abundance. Local environment and solvent accessibility of nucleophilic residues in the proteins are likely to be major determinants.
The present findings suggest that, in the lens, Cys residues are the most reactive, binding to deaminated Kyn at a faster rate than that of His or Lys. It is proposed that the Kyn moiety attaches preferentially to Cys residues in lens proteins. However, after the attachment of Kyn to Cys, a proportion of this protein-bound Kyn is lost because of the intrinsic instability of the Kyn-Cys adduct (Scheme 2 ). The extent of this decomposition may be influenced by the degree of solvent exposure and the environment of the Cys residues. This decomposition is accelerated by glutathione in a concentration-dependent manner (Fig. 6). These studies emphasize the importance of maintaining high glutathione levels in the lens if posttranslational modification by UV filters is to be minimized. It is unknown whether processes that involve reactions of thiols may facilitate the oxidation of Cys residues that accompany age-related nuclear cataract.
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Acknowledgements |
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Footnotes |
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Submitted for publication September 7, 2006; revised November 21, 2006, and March 21, 2007; accepted May 22, 2007.
Disclosure: N.R. Parker, None; A. Korlimbinis, None; J.F. Jamie, None; M.J. Davies, None; R.J.W. Truscott, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Roger J. W. Truscott, Save Sight Institute, GPO Box 4337, Sydney, NSW 2001, Australia; rjwt{at}eye.usyd.edu.au.
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References |
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-crystallin by kynurenine. Biochem Biophys Res Comm.. 2000;276:216–223.[CrossRef][Web of Science][Medline][Order article via Infotrieve]-crystallin: a model for lenticular aging. Photochem Photobiol.. 1999;69:248–253.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
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