Novel Protein Modification by Kynurenine in Human Lenses

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 were to was used in the of all lenses were from the Eye or from the S. with from the of calf lenses were from and and Kyn were all from was from phase high pressure liquid chromatography was on a with a and a model For a was used with the following phase for by a of by a of and in the phase for The was were the same conditions as those for the that a was used with a of ionization mass spectra were on a mass were in acid and the mass by a at a of was used as the and at and The was and the from to The was to of the mass was were in at mass were to For tandem mass spectrometry the conditions used were the same as those that the of the first was to a to increase of the The used was at a pressure of and the laboratory was and to achieve the of with than of the were were electrospray mass spectrometry was on a The were the electrospray ionization with at a of molecular were a mass from a of a of in with was an and UV at nm with the mass adducts were on an at a of an a of and the at All spectra were in with spectra by and and spectra were on a For the following were and All were in and to are in spectra were on a fluorescence in were nm for excitation and nm and the was spectra were a was used as the in all lenses were on and the nucleus was a and from the The nucleus was in for at the was for at The liquid was and the was in and The was and the was The of Kyn was in The amino or were in The was to with and the was with in and at for the to and with the was by or the The high mass and for adduct are based on for of based on for on based on for The and adducts were by incubation at in the adduct was or of of protein or Kyn-modified amino were with for at in an the was and in of and of The was by of was in The of Kyn was and the of the was to with The was with in and at for The was on a in The protein was with to that all Kyn was model Kyn and calf lens proteins shown that Kyn was of binding to the proteins and that Lys, His, and Cys may be the of covalent (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). the first in our to Kyn adducts were in human the synthesis of authentic standards of Kyn adducts of these amino we that 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 and Cys were at amino were used in the of Lys and to reaction of the Cys was as the was the of reaction with Kyn J.A. Truscott R.J.W. Biochem. Biophys. Res. Commun. 2000; 276: 216-223Crossref PubMed Scopus (36) Google Scholar). The reaction were by and the adducts were by of isolated or by The major were found to be the amino and the acid adduct with Kyn and kynurenine the from of Kyn T. S. Y. T. J. Chem. Scholar, A. of and Scholar). The Kyn adducts of and Cys were by The were and therefore were not of the adducts was a of fluorescence tandem mass and and spectra were for of the three Kyn adducts to the of covalent The chemical for of the adducts are shown in The spectra revealed for and with chemical and with an Kyn The side chain of Kyn was observed in the spectra of with the for the The chemical for and were of covalent of amino at of the Kyn side chain. The of these modifications was confirmed by The observed in these spectra are shown in In were observed of Kyn and the of the amino Lys, His, and These modifications were confirmed by not the corresponding to those in were In the of the the and were also The via were with those from a mechanism nucleophilic attack by the or amino of the amino on the unsaturated side chain of for of the of of of of for of the of in a tandem mass spectrometry of the cysteinylkynurenine and the adducts lysylkynurenine and histidylkynurenine was also The of and a significant molecular for and of of the of the major for the and Kyn adducts of Lys and were the thus the of the Kyn adducts in the acid For of the Kyn the of the amino acid was confirmed by the of a for the amino acid and for Lys, His, and Cys, The spectra of all of the Kyn adducts of the Kyn This a at for the of Kyn. The UV and fluorescence spectra of following of the are shown in of the not the The UV of an at The adduct was also a fluorescence at nm excitation and emission at and nm and UV and fluorescence spectra UV fluorescence UV fluorescence Kyn at nm but is fluorescent at Because we to the of these Kyn in proteins, we first the of the adducts under the conditions used for of Kyn-modified amino acid adduct was to acid were and the adducts by a of the of the adducts was in high For was for and for Kyn-modified amino be from proteins that been to was first with Kyn at to promote the reaction of Kyn with the Kyn-modified was yellow in It was and as under The was by the for the The major colored were by the UV at nm and the for the molecular of at at and at and Furthermore, the spectra of of these molecular were to the spectra of the authentic standards not a Kyn was to to hydrolysis. corresponding to Kyn adducts were observed in these the possibility of formation of these the of the acid human lens proteins were from the of human with subjects in age from to were to acid hydrolysis. a of human lens protein in which the of major amino acid adducts corresponding to and are It should be that this which the of the adducts and is from the used for shown in The of these was by the with the standards to the retention In the individual were by and tandem mass spectrometry following a step to and to by In to the model system, in which levels of the adducts were the Kyn adducts were not by of the human lens was observed at the retention time for however, the of the not be confirmed by of the high amino acid We considered the possibility that the Cys adduct may be and thus oxidative of the acid of the human lens protein was also in the of an S. R. M. Truscott R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). This however, in an increased of the and it was that the observed of the levels in the for the low concentration of in lens protein is that it is at the of the human This was confirmed by on at was observed at not All human lenses contained levels of and The of of the Kyn adducts were found to increase as a of age, as shown in The concentration of the adduct was more than times than that of the age a of in the Kyn adduct levels from the of was by the low levels particularly in younger and by the of another that For this the levels of are not in The levels of in lenses to those observed for in an lens and in a lens the human lens protein and the produced Kyn-modified was that the levels of Kyn-modified amino were in the human lens protein. This is a of the and high Kyn concentration used in the model high yields of the Kyn In to the of it is that other as and with for the unsaturated ketone of Kyn it is formed the lens R. J. Ophthalmic Scholar). in the of human lens protein were shown in These which and were also observed at the same in the acid of and from acid of tryptophan T. T. Biochim. Biophys. Acta. 1976; PubMed Scopus Google Scholar). In the work here we have shown that Kyn is covalently to human lens proteins and that the bound with of human lens protein revealed that and were the major with levels of a are the order of of these amino Kyn at be to be by Cys a and Lys a In with this the levels of the adduct were found to be than the levels of the adduct in the human For example, the of the and adducts in a lens were and These and the of as a of age, are with those of 3-OHKG bound to human lens protein B. Truscott R.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The of Kyn-modified amino were observed to increase in concentration with the age of the individual. The concentration levels of and in the region of the human lens were found to increase from and protein in lenses younger than to a of and in an The than of that a of may a role in the concentration of the In to their a the to which residues are is to be by the of the protein and of residues for as well as the adduct at The low of observed may also the abundance of Cys residues in crystallins with and Lys M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the for the observed pattern to be the of the Kyn adducts at In of the involvement of Cys in the covalent binding of reaction with the Cys residue of the is observed is with Kyn at and Lys residues comprise of amino in α, β, and in α, in β, and in J.A. Truscott R.J.W. Biochem. Biophys. Res. Commun. 2000; 276: 216-223Crossref PubMed Scopus (36) Google Scholar). This may that Cys is an of binding in the lens, but of the of the Cys adducts at these adducts with of Cys and of which is to other adduct The of the adducts may therefore be primarily for the pattern of adducts we In on we found that at of the adduct incubation at for these and in the and Lys, the α,β-unsaturated ketone was to with the amino to which are at not a of a process in the lens a in the distribution of the Kyn adducts in of the formation of and and may thus the adduct is found at low levels in the The of covalent modifications of human lens proteins to have been found at low for F. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The levels of the Kyn adducts that we have here are with those of These are with those from model (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 in which the reaction Kyn and nucleophilic amino was The reaction via deamination of the Kyn amino acid side chain by a of the nucleophilic amino acid residue to the α,β-unsaturated The reaction mechanism is in the of Kyn adduct formation is on deamination of the Kyn amino acid side this has been found to be most at with a but at (27Bova L.M. Wood A.M. Jamie J.F. Truscott R.J. Investig. Ophthalmol. Vis. Sci. 1999; 40: 3237-3244PubMed Google Scholar). the of the amino which is related to their and to the of the M. J. Chem. Scopus Google Scholar, M. J. Chem. Scopus Google Scholar). revealed that in of the to a with the Kyn. This is in with the reaction of with Biol. 19: PubMed Scopus (27) Google Scholar). appears to reaction to the the from the side chain, for as Kyn. It is well known that human lenses more fluorescent with the fluorescence associated with has been as an excitation of nm and an emission of nm (15Lerman S. Borkman R. Ophthalmic Res. 1976; 8: 335-353Crossref Scopus (182) Google Scholar). binding of Kyn to lens proteins appears to be mechanism for this Kyn is fluorescent and nm Kyn is In work (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, B. Truscott R.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), we have a novel UV filter the 3-OHKG adduct of which is also formed by a and have shown that 3-OHKG is also bound to lens it can be that which is also at significant levels in human in a manner to 3-OHKG and Kyn. The of Kyn binding to lens protein are not understood. Kyn has the to the protein which we have confirmed in The of binding to human lens protein be more to protein through the amino acid side chain, as the is to undergo in may to formation of and S. B. Sheil M.M. Truscott R.J. Res. 2000; PubMed Scopus Google Scholar). of bound also protein J.A. Carver J.A. Truscott R.J.W. Exp. Eye Res. PubMed Scopus (71) Google and may also in protein and cataract as that are in the lens in high Biochem. J. PubMed Scopus Google Scholar, Jr. Exp. Eye Res. PubMed Scopus (41) Google Scholar, Exp. Eye Res. PubMed Scopus (67) Google may with the amino acid residues for the unsaturated ketone of Kyn and therefore crystallins from age, the concentration of in the region of the lens to Jr. Exp. Eye Res. PubMed Scopus (41) Google Scholar, L.M. Jamie J.F. Truscott R.J. Investig. Ophthalmol. Vis. Sci. 2001; Google Scholar), and this may therefore contribute to the nonoxidative of UV filters in this of the It has been proposed that the of an lens age may be the for these which to lens coloration and to age-related cataract R. J. Ophthalmic Scholar). In with this the levels of Kyn adducts in lens proteins were than those found in the proteins from the same lens not Kyn has been shown in this to be in the lens, tryptophan metabolites can also be increased in other For Kyn and with acid and are found in the system, and their increase in to and 1992; PubMed Scopus Google Scholar). The role of Kyn and in these conditions is not but their binding to proteins may a in the of these These modifications may also in other Kyn and Kyn metabolites are increased via the up-regulation of indoleamine In we have a novel post-translational modification of proteins by the tryptophan and shown that in the lens, Kyn is attached primarily to histidine to a lesser extent, lysine and residues. In the human lens, this process to the age-related increase in coloration and fluorescence and may a role in cataract Its in in which the levels of Kyn and can increase to be We for the high mass