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• W2040792371 abstract "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-hydroxykynurenineO-β-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. 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-hydroxykynurenineO-β-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. The lens of the eye plays a crucial role in vision. Its chemical composition is unusual in that proteins represent ∼38% of the wet mass. The high concentration of protein is needed to achieve the refractive index necessary for focusing (1Sen A.C. Ueno N. Chakrabarti B. Photochem. Photobiol. 1992; 55: 753-764Crossref PubMed Scopus (41) Google Scholar). Crystallins constitute more than 90% of the lens protein and comprise three main classes, α, β, and γ, based on their aggregation behavior and sequence homology (2Bloemendal H. Science. 1977; 197: 127-138Crossref PubMed Scopus (346) Google Scholar). The tightly packed and ordered distribution of the crystallins is essential for maintaining lens transparency and therefore vision (3). The lenses of humans and other primates contain low molecular weight compounds that act as intraocular filters by absorbing UV light in the 300–400 nm region (4van Heyningen R. Ciba Found. Symp. 1973; 19: 151-168Google Scholar, 5Cooper G.F. Robson J.G. J. Physiol. 1969; 203: 411-417Crossref PubMed Scopus (92) Google Scholar), thus preventing UV-induced photodamage to the retina (6Zigman S. Paxhia T. Exp. Eye Res. 1988; 47: 819-824Crossref PubMed Scopus (43) Google Scholar). These filters are produced through the catabolism of tryptophan. The first step in this process involves the oxidative cleavage of the pyrrole ring of tryptophan toN-formyl-l-kynurenine, catalyzed by indoleamine 2,3-dioxygenase (7Yamazaki F. Kuroiwa T. Takikawa O. Kido R. Biochem. J. 1985; 230: 635-638Crossref PubMed Scopus (210) Google Scholar, 8Takikawa O. Littlejohn T.K. Truscott R.J.W. Exp. Eye Res. 2001; 72: 271-277Crossref PubMed Scopus (37) Google Scholar). The major UV filters in primate lenses in decreasing order of abundance are 3-hydroxykynurenineO-β-d-glucoside (3-OHKG), 1The abbreviations used are:3-OHKG3-hydroxykynurenine O-β-d-glucosideKynkynurenine3-OHKyn3-hydroxykynurenineMSmass spectrometryHis-KynhistidylkynurenineLys-KynlysylkynurenineCys-KyncysteinylkynurenineRP-HPLCreversed phase high pressure liquid chromatographyESI-MS/MSelectrospray ionization tandem mass spectrometryCLPcalf lens proteinLC/MSliquid chromatography/mass spectrometryGSHglutathionet-Boctert-butoxycarbonylAHBG4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acidO-β-d-glucosideRtretention timeExexcitationEmemissionHIVhuman immunodeficiency virus 1The abbreviations used are:3-OHKG3-hydroxykynurenine O-β-d-glucosideKynkynurenine3-OHKyn3-hydroxykynurenineMSmass spectrometryHis-KynhistidylkynurenineLys-KynlysylkynurenineCys-KyncysteinylkynurenineRP-HPLCreversed phase high pressure liquid chromatographyESI-MS/MSelectrospray ionization tandem mass spectrometryCLPcalf lens proteinLC/MSliquid chromatography/mass spectrometryGSHglutathionet-Boctert-butoxycarbonylAHBG4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acidO-β-d-glucosideRtretention timeExexcitationEmemissionHIVhuman immunodeficiency virus4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acidO-β-d-glucoside (AHBG), kynurenine (Kyn), and 3-hydroxykynurenine (3-OHKyn) (4van Heyningen R. Ciba Found. Symp. 1973; 19: 151-168Google Scholar, 9Truscott R.J.W. Wood A.M. Carver J.A. Sheil M.M. Stutchbury G.M. Zhu J. Kilby G.W. FEBS Lett. 1994; 348: 173-176Crossref PubMed Scopus (57) Google Scholar, 10van Heyningen R. Nature. 1971; 230: 393-394Crossref PubMed Scopus (141) Google Scholar, 11Wood A.M. Truscott R.J. Exp. Eye Res. 1993; 56: 317-325Crossref PubMed Scopus (125) Google Scholar, 12van Heyningen R. Exp. Eye Res. 1973; 15: 121-126Crossref PubMed Scopus (44) Google Scholar). Recent work in our laboratory has led to the discovery of glutathionyl-3-hydroxykynurenineO-β-d-glucoside, a novel fluorescent UV filter that was found to increase in concentration with the age of the individual (13Garner B. Vazquez S. Griffith R. Lindner R.A. Carver J.A. Truscott R.J. J. Biol. Chem. 1999; 274: 20847-20854Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 3-hydroxykynurenine O-β-d-glucoside kynurenine 3-hydroxykynurenine mass spectrometry histidylkynurenine lysylkynurenine cysteinylkynurenine reversed phase high pressure liquid chromatography electrospray ionization tandem mass spectrometry calf lens protein liquid chromatography/mass spectrometry glutathione tert-butoxycarbonyl 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acidO-β-d-glucoside retention time excitation emission human immunodeficiency virus 3-hydroxykynurenine O-β-d-glucoside kynurenine 3-hydroxykynurenine mass spectrometry histidylkynurenine lysylkynurenine cysteinylkynurenine reversed phase high pressure liquid chromatography electrospray ionization tandem mass spectrometry calf lens protein liquid chromatography/mass spectrometry glutathione tert-butoxycarbonyl 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acidO-β-d-glucoside retention time excitation emission human immunodeficiency virus The young human lens is pale yellow in color; however, with age, an increase in lens coloration and fluorescence is observed (14Weale R.A. J. Physiol. 1988; 395: 577-587Crossref PubMed Scopus (227) Google Scholar, 15Lerman S. Borkman R. Ophthalmic Res. 1976; 8: 335-353Crossref Scopus (182) Google Scholar, 16Yu N.T. Barron B.C. Kuck J.F., Jr. Exp. Eye Res. 1989; 49: 189-194Crossref PubMed Scopus (36) Google Scholar). This increase is particularly prominent in the lens nucleus and is associated with changes to the crystallins (15Lerman S. Borkman R. Ophthalmic Res. 1976; 8: 335-353Crossref Scopus (182) Google Scholar, 17Bessems G.J.H. Keizer E. Wollensak J. Hoenders H.J. Investig. Ophthalmol. Vis. Sci. 1987; 28: 1157-1163PubMed Google Scholar). Because lens proteins, once produced, show little or no turnover (18Wannemacher C.F. Spector A. Exp. Eye Res. 1968; 7: 623-625Crossref PubMed Scopus (65) Google Scholar), any post-translational modifications accumulate with age and may eventually contribute to age-related cataract (19Bando M. Nakajima A. Satoh K. Exp. Eye Res. 1975; 20: 489-492Crossref PubMed Scopus (23) Google Scholar). Several investigators have considered the possibility that UV filters may covalently modify lens crystallins (4van Heyningen R. Ciba Found. Symp. 1973; 19: 151-168Google Scholar, 20Stutchbury G.M. Truscott R.J.W. Exp. Eye Res. 1993; 57: 149-155Crossref PubMed Scopus (58) Google Scholar, 21Bando M. Mikuni I. Obazawa H. Exp. Eye Res. 1985; 40: 813-818Crossref PubMed Scopus (7) Google Scholar, 22Dillon J. Skonieczna M. Mandal K. Paik D. Photochem. Photobiol. 1999; 69: 248-253Crossref PubMed Scopus (32) Google Scholar), and most have proposed a role for UV light in this modification (23Dillon J. Doc. Ophthalmol. 1994; 88: 339-344Crossref PubMed Scopus (28) Google Scholar, 24Finley E.L. Dillon J. Crouch R.K. Schey K.L. Protein Sci. 1998; 7: 2391-2397Crossref PubMed Scopus (182) Google Scholar, 25Ellozy A.R. Wang R.H. Dillon J. Photochem. Photobiol. 1994; 59: 479-484Crossref PubMed Scopus (26) Google Scholar, 26Tomoda A. Yoneyama Y. Yamaguchi T. Shirao E. Kawasaki K. Ophthalmic Res. 1990; 22: 152-159Crossref PubMed Scopus (27) Google Scholar). Our approach, however, has focused on the binding of these compounds to lens proteins without the involvement of UV light. Support for this proposal came from the mechanism of formation of novel human UV filters. For example, the glutathione adduct of 3-hydroxykynurenine glucoside, GSH-3-OHKG (13Garner B. Vazquez S. Griffith R. Lindner R.A. Carver J.A. Truscott R.J. J. Biol. Chem. 1999; 274: 20847-20854Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), is formed via deamination of the 3-OHKG amino acid side chain, yielding an α,β-unsaturated ketone that is highly susceptible to nucleophilic attack by the Cys residue of glutathione (GSH) (13Garner B. Vazquez S. Griffith R. Lindner R.A. Carver J.A. Truscott R.J. J. Biol. Chem. 1999; 274: 20847-20854Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Reduction of the unsaturated side chain in the lens yields another UV filter, AHBG (27Bova L.M. Wood A.M. Jamie J.F. Truscott R.J. Investig. Ophthalmol. Vis. Sci. 1999; 40: 3237-3244PubMed Google Scholar). Because Kyn has the same amino acid side chain as 3-OHKG, it should also undergo deamination. Indeed, in a model system, reaction of Kyn with calf lens protein (CLP) under nonoxidative conditions has been found to generate colored, fluorescent protein. Peptide mapping of Kyn-modified crystallins has revealed that all of the colored peptides contained His, Cys, or Lys residues (28Garner B. Shaw D.C. Lindner R.A. Carver J.A. Truscott R.J.W. Biochim. Biophys. Acta. 2000; 1476: 265-278Crossref PubMed Scopus (71) Google Scholar, 29Aquilina J.A. Truscott R.J.W. Biochem. Biophys. Res. Commun. 2001; 285: 1107-1113Crossref PubMed Scopus (20) Google Scholar, 30Aquilina J.A. Truscott R.J.W. Biochem. Biophys. Res. Commun. 2000; 276: 216-223Crossref PubMed Scopus (36) Google Scholar). In this paper we demonstrate that proteins from human lenses contain covalently bound Kyn. Furthermore, we show that the Kyn is attached primarily to His, Lys, and to a lesser extent, Cys residues and that this pattern of covalent modification can be reproduced by incubation of the lens proteins with Kyn under conditions that promote deamination of the amino acid side chain. All organic solvents and acids were HPLC grade (Ajax, Auburn, New South Wales, Australia). Milli-Q® water (purified to 18.2 milliohms/cm2) was used in the preparation of all solutions. Human lenses were obtained from the Sydney Eye Bank (New South Wales, Australia) or from the National Disease Research Interchange (U. S. A.) with ethical approval from the University of Wollongong Human Ethics Committee (HE99/001). Fresh calf lenses (<2 years old) were obtained from Parrish Meats (Yallah, New South Wales, Australia). Amino acids (N-α-t-Boc-l-histidine,N-α-t-Boc-l-lysine, and cysteine) and Kyn sulfate salt were all obtained from Sigma. Sequencing grade HCl (6 m) was purchased from Pierce. Reversed phase high pressure liquid chromatography (RP-HPLC) was performed on a Beckman System Gold® HPLC system equipped with a 127S solvent module and a model 166 UV-visible detector. For analytical scale separations, a Varian (Microsorb-MV™ C18, 100 Å, 5 μm, 4.6 × 250 mm) column was used with the following mobile phase conditions: solvent A (aqueous 4 mmammonium acetate, pH 6.5) for 5 min followed by a linear gradient of 0–50%; solvent B (80% acetonitrile/H2O, 4 mmammonium acetate) over 20 min followed by a linear gradient of 50–100% solvent B over 15 min and re-equilibration in the aqueous phase for 15 min. The flow rate was 1 ml/min. Semi-preparative separations were performed using the same conditions as those for the analytical separations except that a (Hypersil® BDS C18, 5 μm, 10 × 250 mm) column was used with a flow rate of 3 ml/min. Electrospray ionization mass spectra were acquired on a VG Quattro triple quadrupole mass spectrometer (VG Biotech Ltd., now Micromass, Altrincham, UK). Samples were dissolved in 50% aqueous acetonitrile containing 1% formic acid and introduced into the mass spectrometer by a Harvard Apparatus 22 syringe pump (South Natick, MA) at a rate of 10 μl/min. Nitrogen was used as both the bath and nebulizing gas, flowing at 350 and 10 liter/h, respectively. The capillary voltage was 3.2 kV, and the cone voltage ranged from 20 to 60 V. The source temperature was set to 85 °C. Calibration of the mass spectrometer was achieved using NaI. Spectra were acquired in positive ion mode at unit mass resolution using multichannel analysis. Typically 10–20 scans (scan rate 100m/z per s) were summed to obtain representative spectra. For tandem mass spectrometry the conditions used were the same as those described above except that the resolution of the first quadrupole was set to a minimum to increase transmission of the selected precursor ion. The collision gas used was argon at a pressure of 3.5 × 10−4 millibars, and the laboratory collision energy was varied between 25 and 45 eV to achieve the desired level of fragmentation. Only peaks with intensity greater than 20% of the base peak were reported unless additional data were pertinent. High resolution electrospray mass spectrometry was performed on a Micromass QTOF. The samples were infused into the electrospray ionization source with 50% aqueous acetonitrile at a flow rate of 10 μl/min. Protonated molecular ions were calibrated against a lock mass arising from a co-injection of a solution of polyethylene glycol in 50% aqueous acetonitrile with 1% ammonia added. LC/MS was carried out using an Applied Biosystems 140B solvent delivery system and 785A UV detector set at 360 nm with the VG Quattro mass spectrometer. Kynurenine adducts were separated on an Alltech Alltima C18 column (250 × 2.1 mm, 5 μm, 300 Å) at a flow rate of 200 μl/min using an 0–80% acetonitrile/H2O (each containing 1% formic acid) gradient over 40 min, a column oven temperature of 25 °C, and the source temperature maintained at 170 °C. All spectra were acquired in continuum mode with representative spectra obtained by summing 10–50 scans. One-dimensional and two-dimensional1H and 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer (1H, 400 MHz;13C, 100 MHz). For each compound, the following two-dimensional experiments were performed:1H-1H correlation spectroscopy (COSY),1H-1H rotating frame nuclear Overhauser effect spectroscopy (ROESY), and 1H-13C heteronuclear multiple bond correlation. All experiments were run in D2O and referenced to residual HDO. Coupling constants (J) are given in Hz. Fluorescence spectra were obtained on a Hitachi F-4500 fluorescence spectrometer (Tokyo, Japan) in three-dimensional scan mode. Slit widths were 5 nm for excitation and 5 nm emission, and the scan speed was 12 000 nm/min. UV-visible absorbance spectra were obtained using a Shimadzu UV-265 spectrophotometer (Kyoto, Japan). Milli-Q® water was used as the solvent in all experiments. Individual lenses were placed on dry ice, and the nucleus was cored (using a 6-mm cork borer) and separated from the cortex. The nucleus was then homogenized in absolute ethanol. After cooling for 1 h at −20 °C, the homogenate was centrifuged for 20 min at 14,000 rpm. The supernatant liquid was removed, and the pellet was re-extracted in 80% ethanol and centrifuged again. The supernatant was discarded, and the pellet was vacuum-dried. The sulfate salt of Kyn (50 mg) was dissolved in 50 mm Na2CO3, NaHCO3buffer, pH 9.5 (30 ml). The amino acids (N-α-t-Boc-l-histidine andN-α-t-Boc-l-lysine or cysteine) were added in 10-fold molar excess. The pH was readjusted to 9.5 with 0.1 m NaOH if required, and then the resulting solution was bubbled with argon, sealed, wrapped in foil, and incubated at 37 °C for 48 h. After adjusting the pH to between 4 and 5 with glacial acetic acid, the resulting mixture was separated by semi-preparative or analytical HPLC using the methods detailed above. The respective yields, high resolution exact mass measurements, NMR, and MS data for each adduct are given below. N-α-tert-Butoxycarbonyl-l-lysyl-d,l-kynurenine: 40 mg, 56% yield based on kynurenine. Found: MH+, 438.2239. Calculated for C21H32N3O7: MH+, 438.2240; δH 7.72 (1H, dd, J 8.2, 1.2, H-6), 7.31 (1H, ddd, J 8.4, 8.2, 1.2, H-4), 6.77 (1H, d,J 8.4, H-3), 6.70 (1H, dd, J 8.2, 8.2, H-5), 3.97 (1H, “broad t,” J ∼5, H-9), 3.82 (1H, m, H-15), 3.62 (2H, m, CH2-8), 3.04 (2H, t, J 7.4, CH2-11), 1.65 (4H, m, CH2-12, CH2-14), 1.55 (2H, m, CH2-13), 1.32 (9H, s, 3× CH3); δC 199.9 (CO-7), 178.1 (CO), 173.3 (CO), 150.7 (C-2), 136.1 (C-4), 131.7 (C-6), 118.4 (C-1), 117.2 (C-3), 116.2 (C-5), 81.4 (C-18), 58.2 (C-9), 55.7 (C-15), 47.4 (C-11), 38.6 (C-8), 31.4 (C-12), 28.0 (3× CH3), 25.5 (C-14), 22.6 (C-13); ESI-MS/MS of m/z 438 (MH+), 382 (26%), 338 (48%), 203 (100%), 192 (7%), 147 (11%), 136 (54%), 128 (37%). N-α-tert-Butoxycarbonyl-l-histidyl-d,l-kynurenine: 22 mg, 30% yield based on kynurenine. Found: MH+, 447.1870. Calculated for C21H27N4O7: MH+, 447.1880; δH 8.69 (1H, s, H-11), 7.71 (1H, dd,J 8.2, 1.2, H-6), 7.27 (1H, ddd, J 8.4, 8.2, 1.2, H-4), 7.27 (1H, H-13 superimposed on H-4), 6.72 (1H, d, J8.4, H-3), 6.67 (1H, dd, J 8.2, 8.2, H-5), 5.32 (1H, m, H-9), 4.10 (1H, m, H-15), 3.82 (2H, m, CH2-8), 3.12 (1H, br dd, J 15.4, ∼4, CH2-14), 2.85 (1H, dd,J 15.4, 9.8, CH2-14), 1.19 (∼4.5H, s, CH3s), 1.14 (∼4.5H, s, CH3s); δC 199.5 (CO-7), 199.4 (CO-7), 177.3 (CO-10), 173.8 (CO-16), 157.4 (CO-17), 150.6 (C-2), 135.9 (C-4), 135.3 (C-11), 135.3 (C-11), 131.6 (C-6), 130.6 (C-12), 119.8 (C-13), 118.4 (C-3), 117.9 (C-1), 117.2 (C-5), 81.4 (C-18), 60.6 (C-9), 55.0 (C-15), 54.9 (C-15), 42.1 (C-8), 28.2 (C-14), 28.1 (C-14), 27.9 (C-19), 27.8 (C-19); ESI-MS/MS ofm/z 447 (MH+), 391 (35%), 347 (100%), 192 (7%), 156 (13%). l-Cysteinyl-d,l-kynurenine: 25 mg, 49% yield based on kynurenine. Found: MH+, 313.0865. Calculated for C13H17N2O5S: MH+, 313.0858; δH 8.01 (1H, dd, J 8.1, 1.2, H-6), 7.56 (1H, ddd, J 8.1, 8.1, 1.2, H-4), 7.27 (1H, m, H-5), 7.19 (∼0.5H, d, J 8.1, H-3), 7.17 (∼0.5H, d,J 8.1, H-3), 4.17 (1H, m, H-12), 3.88 (1H, m, H-9), 3.75–3.65 (1H, m, H-8), 3.59 (∼0.5H, dd, J 18.3, 4.9, H-8), 3.54 (∼0.5H, dd, J 18.4, 5.1, H-8), 3.37 (∼0.5H, dd, J 14.8, 4.2, H-11), 3.31 (∼0.5H, dd, J14.8, 4.5, H-11), 3.23 (∼0.5H, dd, J 14.8, 7.2, H-11), 3.13 (∼0.5H, dd, J 14.8, 8.1, H-11); δC 201.1 (CO-7), 201.0 (CO-7), 176.4 (CO-10), 176.1 (CO-10), 171.4 (CO-13), 171.3 (CO-13), 135.9 (C-4), 132.2 (C-6), 126.0 (C-5), 125.8 (C-5), 123.3 (C-3), 123.2 (C-3), 118.2 (C-1), 53.4 (C-12), 53.1 (C-12), 42.9 (C-9), 42.1 (C-8), 41.7 (C-8), 32.5 (C-11), 31.6 (C-11); ESI-MS/MS ofm/z 313 (MH+), 202 (88%), 192 (100%), 174 (33%), 122 (34%). The N-α-t-Boc-l-histidyl-Kyn and N-α-t-Boc-l-lysyl-Kyn adducts were deprotected by incubation at 37 °C in 6 m HCl overnight. Following lyophilization the adduct was purified using semi-preparative or analytical HPLC.l-Lysyl-d,l-kynurenine: ESI-MS/MS of m/z 338 (MH+), 203 (93%), 192 (5%), 147 (14%), 128 (48%).l-Histidyl-d,l-kynurenine: ESI-MS/MS of m/z 347 (MH+), 301 (30%), 192 (28%), 174 (22%), 156 (20%), 146 (40%), 120 (60%), 109 (78%). Lens protein (∼10 mg) or Kyn-modified amino acids (N-α-t-Boc-l-histidine andN-α-t-Boc-l-lysine orl-cysteine) (2 mg) were hydrolyzed with 6m HCl (1 ml) for 24 h at 110 °C in an evacuated hydrolysis tube. After hydrolysis, the sample was lyophilized overnight and then dissolved in 400 μl of 0.1 mNaH2PO4 and 200 μl of 1 mNa2HPO4 (pH ∼ 5). The solution was then examined by RP- HPLC. 50 mg of CLP was dissolved in 50 mmNa2CO3, NaHCO3 buffer, pH 9.5 (10 ml). The sulfate salt of Kyn (10 mg) was added, and the pH of the resulting solution was readjusted to 9.5 with 0.1 m NaOH if required. The tube was bubbled with argon, sealed, wrapped in foil, and incubated at 37 °C for 4 days. The resulting mixture was separated on a Sephadex G25 PD-10 column (Amersham Biosciences Inc.) in Milli Q® water. The resulting protein fraction was extracted twice with ethanol to ensure that all unreacted Kyn was removed. Initial model studies involving Kyn and calf lens proteins had shown that Kyn was capable of binding to the proteins and suggested that Lys, His, and Cys may be the sites of covalent attachment (28Garner B. Shaw D.C. Lindner R.A. Carver J.A. Truscott R.J.W. Biochim. Biophys. Acta. 2000; 1476: 265-278Crossref PubMed Scopus (71) Google Scholar). Hence, the first stage in our investigation, to determine whether such Kyn adducts were present in human lenses, involved the synthesis of authentic standards of Kyn adducts of these amino acids. As we had demonstrated that basic conditions promote deamination of the Kyn side chain (27Bova L.M. Wood A.M. Jamie J.F. Truscott R.J. Investig. Ophthalmol. Vis. Sci. 1999; 40: 3237-3244PubMed Google Scholar), the Kyn amino acid adducts ofN-α-t-Boc-l-lysine (t-Boc-Lys),N-α-t-Boc-l-histidine (t-Boc-His), and Cys were prepared at pH 9.5.t-Boc-protected amino acids were used in the case of Lys and His to prevent reaction of the α-amino group. Cys was left unprotected, as the sulfhydryl group was the preferred site of reaction with Kyn (30Aquilina J.A. Truscott R.J.W. Biochem. Biophys. Res. Commun. 2000; 276: 216-223Crossref PubMed Scopus (36) Google Scholar). The reaction mixtures were analyzed by RP-HPLC, and the adducts were identified by ESI-MS of isolated peaks or by direct LC/MS. The major components were found to be the unreacted amino acid, Kyn, and the Kyn-amino acid adduct (∼1:1 diastereomeric mixture) along with deaminated Kyn and kynurenine yellow, the product resulting from intramolecular cyclization of deaminated Kyn (31Tokuyama T. Senoh S. Hirose Y. Sakan T. J. Chem. Soc. Jpn. 1958; 79: 752-761Google Scholar, 32Butenandt A. Schafer W. Gore T.S. Chemistry of Natural and Synthetic Colouring Matter. Academic Press, New York1962: 13-33Google Scholar). The Kyn adducts of t-Boc-Lys, t-Boc-His, and Cys were purified by semi-preparative RP-HPLC. The diastereomers were poorly resolved and therefore were not separated. Characterization of the adducts was achieved using a combination of UV-visible spectroscopy, three-dimensional fluorescence spectroscopy, tandem mass spectrometry, and NMR spectroscopy. One-dimensional and two-dimensional NMR spectra were acquired for each of the three Kyn adducts to confirm the site of covalent attachment. The one-dimensional 1H-NMR chemical shifts for each of the adducts are shown in TableI. The one-dimensional 1H-NMR spectra revealed four aromatic protons for each compound (H-3, H-4, H-5, and H-6) with chemical shifts and coupling patterns consistent with an unmodified Kyn aromatic ring. The side chain CH2-CH spin system of Kyn was observed in the COSY spectra of each adduct, with the diastereotopic methylene protons clearly discernible for the Cys-Kyn adduct. The downfield chemical shifts for CH-9 (δ 3.88-δ 5.32 ppm) and CH2-8 (δ 3.54–δ 3.82 ppm), were indicative of covalent attachment of amino acids at C-9 of the Kyn side chain. The site of these modifications was confirmed by ROESY experiments. The diagnostic cross-peaks observed in these spectra are shown in Fig. 1. In each case, cross-peaks were observed between H-9 of Kyn and the pertinent protons of the amino acids Lys, His, and Cys. These modifications were confirmed by heteronuclear multiple bond correlation experiments (data not shown), where the corresponding carbon-proton cross-peaks to those in Fig. 1 were observed. In the case of the t-Boc-His-Kyn adduct, cross-peaks between both the primary imidazole carbons and C-9 were also present. The structures determined via NMR spectroscopy were consistent with those predicted from a mechanism involving nucleophilic attack by the sulfhydryl or amino groups of the amino acids on the unsaturated side chain of deaminated Kyn.Table I1H NMR spectral assignmentsAdducts1H assignments (ppm)1-aSee Fig. 1 for numbering of the protons (1H).H-3H-4H-5H-6H-8H-9H-11H-12H-13H-14H-15H-19A6.777.316.707.723.623.973.041.651.551.653.821.32B6.727.276.677.713.825.328.697.273.12, 2.851-bDiastereotopic protons.4.101.19, 1.141-cResolved peaks of diastereomers.C7.19, 7.171-cResolved peaks of diastereomers.7.567.278.013.70, 3.59, 3.541-bDiastereotopic protons.1-cResolved peaks of diastereomers.3.883.37, 3.31, 3.23, 3.131-bDiastereotopic protons.1-cResolved peaks of diastereomers.4.17Adducts: A,N-α-t-boc-l-lysylkynurenine; B,N-α-t-boc-l-histidylkynurenine; C, cysteinylkynurenine.1-a See Fig. 1 for numbering of the protons (1H).1-b Diastereotopic protons.1-c Resolved peaks of diastereomers. Open table in a new tab Adducts: A,N-α-t-boc-l-lysylkynurenine; B,N-α-t-boc-l-histidylkynurenine; C, cysteinylkynurenine. ESI tandem mass spectrometry (ESI-MS/MS) of the adductst-Boc-Lys-Kyn, t-Boc-His-Kyn, cysteinylkynurenine (Cys-Kyn), and the t-Boc-deprotected adducts lysylkynurenine (Lys-Kyn) and histidylkynurenine (His-Kyn) was also investigated. The ESI-MS/MS of both t-Boc-Lys-Kyn and t-Boc-His-Kyn exhibited a significant protonated molecular ion for Lys-Kyn (m/z 338) and His-Kyn (m/z 347), respectively, because of loss of the t-Boc group. Most of the major ions for the t-Boc and deprotected Kyn adducts of Lys and His were the same, thus facilitating the later identification of the Kyn adducts in the acid hydrolysates. For each of the Kyn adducts, the presence of the respective amino acid was confirmed by the observation of a product ion for the protonated amino acid (m/z 147, 156, and 122 for Lys, His, and Cys, respectively). The ESI-MS/MS spectra of all of the Kyn adducts showed product ions characteristic of the Kyn moiety. This included a diagnostic peak at m/z 192 for the deaminated form of Kyn. The UV and three-dimensional fluorescence spectra of His-Kyn following removal of the t-Boc-protecting group are shown in Fig.2. Removal of the t-Boc group did not alter appreciably the spectral characteristics. The UV spectrum of His-Kyn displayed an absorbance maximum at 361 nm. The His-Kyn adduct was also fluorescent, demonstrating a maximum fluorescence intensity at 410 nm excitation (Ex) and emission (Em) at 490 and 525 nm (Fig. 2, inset). Lys-Kyn and Cys-Kyn displayed similar UV and fluorescence spectra (Lys-Kyn UV maximum 361 nm, fluorescence Ex maximum 410, Em maximum 490, 527; Cys-Kyn UV maximum 357 nm, fluorescence Ex maximum 392, Em maximum 490, 527). By contrast, Kyn absorbs at 360 nm but is barely fluorescent at neutral pH. Because we wished to quantify the amounts of these Kyn derivatives in proteins, we first investigated the stability of the synthetic adducts under the conditions used for total hydrolysis of proteins. Therefore, each Kyn-modified amino acid adduct was subjected separately to acid hydrolysis (6 m HCl, 110 °C). Aliquots were removed every 6 h and the adducts analyzed by RP-HPLC. Although a small amount of decomposition occurred over the 24-h hydrolysis period, each of the adducts was recovered in high yield. For Cys-Kyn, recovery was 96 ± 2%, for Lys-Kyn 96 ± 2%, and for His-Kyn 99 ± 1%. To establish whether Kyn-modified amino acids could be recovered from proteins that had been exposed to Kyn, CLP was first incubated with Kyn at pH 9.5 to promote the reaction of Kyn with CLP. After 24 h, the Kyn-modified CLP was clearly yellow in color. It was purified and hydrolyzed as described under “Experimental Procedures.” The hydrolysate was then examined by LC/MS. Fig.3 shows the LC/MS data for the hydrolysate. The major colored peaks were identified by correlation between the UV absorbance at 360 nm (Fig. 3 A) and the ion currents for the protonated molecular ions of His-Kyn at m/z347, Rt = 16.4 min (Fig. 3 B), Lys-Kyn at m/z 338, Rt = 15.8 min (Fig.3 C), and Cys-Kyn at m/z 313, Rt = 18.4 and 18.7 min (isomers) (Fig.3 D). Furthermore, the ESI-MS/MS spectra of each of these protonated molecular ions were identical to the spectra of the authentic standards (data not shown). As a control experiment, Kyn was added to unmodified CLP immediately prior to hydrolysis. No ions corresponding to Kyn adducts were observed in these experiments, thereby eliminating the possibility of artifactual formation of these adducts. Having established the applicability of the acid hydrolysis method, human lens proteins were then examined. Proteins from the nuclei of 20 human lenses, with subjects ranging in age from 16" @default.
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• W2040792371 title "Novel Protein Modification by Kynurenine in Human Lenses" @default.
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