2-Ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) Is a Newly Identified Advanced Glycation End Product in Cataractous and Aged Human Lenses

Abstract

Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the nonproteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nα-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nα-t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties. Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the nonproteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nα-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nα-t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties. 2-Ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses. Vol. 279 (2004) 6487–6495Journal of Biological ChemistryVol. 280Issue 19PreviewPage 6488, “Experimental Procedures”: Under the subheading “Synthesis of OP-lysine,” the beginning of the second paragraph was incomplete. The sentence should read: Briefly, Boc-Lys (4.92 g, 20 mmol) was dissolved in 50 ml of water, pH of the solution was brought to 9.5 with 4 m sodium hydroxide, sodium nitroprusside (9.43 g, 32 mmol) was added within 30 min, and the temperature was maintained at 60 °C for 6 h. Full-Text PDF Open Access Glycation is a natural process observed in living systems, consisting in post-translational modifications of the side chains of amino acids in proteins by reactive carbonyl components. Glycation is a multistage and multidirection process. Initially, so-called “early glycation products” are formed. At this stage the modifications of amino acids are reversible. Further reactions lead to the formation of advanced glycation end products (AGEs), 1The abbreviations and trivial names used are: AGEs, advanced glycation end products; Asc, l-ascorbic acid; Boc-Lys, Nα-tert-butoxycarbonyllysine; CL, cataractous lenses; DHA, l-dehydroascorbic acid; ESIMS, electrospray ionization mass-spectrometry; FCL, fetal calf lenses; HFBA, heptafluorobutyric acid; IE, ion-exchange; NAL, normal aged lenses; ODS, octadecylsulfate; OP-lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate; PDA, photo diode array; RP, reverse-phase; HPLC, high performance liquid chromatography; WI, water-insoluble; WS, water-soluble; DTPA, diethylenetriaminepentaacetic acid; HOP-lysine, 4-hydroxymethyl derivative of OP-lysine. which are relatively stable and tend to accumulate in biological or model systems with time. AGEs are important from a medical point of view because their concentration increases during aging (1Frye E.B. Degenhardt T.P. Thorpe S.R. Baynes J.W. J. Biol. Chem. 1998; 273: 18714-18719Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar) or in different medical complications such as cataract formation (2Cheng R. Lin B. Lee K.W. Ortwerth B.J. Biochim. Biophys. Acta. 2001; 1537: 14-26Crossref PubMed Scopus (74) Google Scholar), retinopathy (3Sugimoto H. Shikata K. Wada J. Horiuchi S. Makino H. Diabetologia. 1999; 42: 878-886Crossref PubMed Scopus (149) Google Scholar), and nephropathy (4Chibber R. Molinatti P.A. Rosatto N. Lambourne B. Kohner E.M. Diabetologia. 1997; 40: 156-164Crossref PubMed Scopus (125) Google Scholar). A natural protective mechanism against glycation and other harmful post-translational modifications is protein turnover. The vast majority of protein molecules has a limited life span and is periodically degraded and rebuilt. A remarkable exception are the proteins in lens fiber cells, especially those in the lens nucleus, being as old as the individual to whom they belong. Thus, the lens is a very appropriate tissue to study the accumulation of AGEs in vivo. The characterization and quantification of AGEs are based on chemical, spectral, and immunological techniques. Two major types of experiments are used for this purpose: either the properties of the biological sample are compared with those of in vitro glycated protein standard(s) or the tissue specimens are characterized/quantified with respect to a specific AGE with known structure. The first approach is popular probably because the preparation of in vitro glycated standards is rapid, inexpensive, and requires little labor. A serious drawback of such an approach, however, is that the conditions for preparation of in vitro glycated proteins are very different from in vivo glycated proteins. Usage of enormous amounts of glycating agent and/or elevated reaction temperatures is more the rule than the exception. There is no guarantee that the structures, and the relative amounts of products built in vitro, match those of AGEs formed in vivo. In the contrary, model glycation reaction mixtures can contain major glycation products, which do not exist in vivo (5Tessier F.J. Monnier V.M. Sayre L.M. Kornfield J.A. Biochem. J. 2003; 369: 705-719Crossref PubMed Scopus (85) Google Scholar). Another problem is that the first approach refers to an abstract or imaginary value, “total AGEs,” whereas it is clear that at least the known AGEs belong to completely different structural classes having little in common. In contrast, the approach based on characterization and quantification of known AGEs gives specific information about the progress and extent of glycation in vivo. The only limitation of this approach is that it could be applied only to AGEs whose structure has already been identified. In this article for the first that the amino acid 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate is a of the proteins in aged and cataractous lenses. a derivative of lysine, the trivial OP-lysine for compound present a reaction for its formation as a of post-translational of lysine on proteins. In experiments OP-lysine in of cataractous lens acid hydrolysates as of the major at and the at at The spectral and properties of this compound were very to those of glycation In a study Lin B. Ortwerth B.J. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar) that 4-hydroxymethyl derivative of can be formed from and in vitro with The to as was as a of proteins in human based on R. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to about the structure of the lens protein with at and at at a the presence of OP-lysine in cataractous and aged lenses was compound is because the structure of OP-lysine is for other glycation Lin B. Ortwerth B.J. Biochim. Biophys. Acta. 2003; PubMed Scopus Google R. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), structure Biochem. 2003; PubMed Scopus Google Scholar), structure the Monnier V.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) to as structure and the post-translational Biochem. Biophys. PubMed Scopus Google Scholar), structure heptafluorobutyric acid and the were from and were from was from and were from were from The were with and with acid in to of in were using from to the calf lenses were from human lenses were from the Cataractous lenses were by S. and were lenses were at °C to performance liquid chromatography were on a consisting of a and The was by a The were using the reverse-phase were using a (3Sugimoto H. Shikata K. Wada J. Horiuchi S. Makino H. Diabetologia. 1999; 42: 878-886Crossref PubMed Scopus (149) Google Scholar) from The used are as The used are as min, min, min, at a of and were using a (3Sugimoto H. Shikata K. Wada J. Horiuchi S. Makino H. Diabetologia. 1999; 42: 878-886Crossref PubMed Scopus (149) Google Scholar) The used are as in The used are as min, min, min, at a of ion-exchange was using a A from The used are as pH pH The used are as min, min, at a of min, at a of was on 60 from on were using for chromatography from of was to a by 1999; Scopus Google Scholar). The reaction is in Briefly, Boc-Lys (4.92 g, 20 mmol) was dissolved in 50 ml of pH of the solution was brought to 9.5 with 4 m sodium hydroxide, and the temperature was maintained at 60 °C for 6 h. pH of the reaction mixture was to 9.5 periodically during The sample was and The was with sodium pH was brought to and the solution was with The were with ml of and sodium The sample was under The was of the compound of this product was with ml of which of the compound. 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The solution was and by liquid chromatography of in for the of carbonyl DHA, and glycolaldehyde, were with mixtures carbonyl 50 Boc-Lys, DTPA, and pH were at °C for under The mixtures were by The were for in the of and the at at of the reaction mixtures showed a with spectral and to those of the synthetic OP-lysine the reaction mixture Boc-Lys The from this reaction was under and using of acid at °C for acid was by a of and the was by ion-exchange chromatography in the and presence of the synthetic OP-lysine using ion-exchange chromatography and of OP-lysine from Boc-Lys and of reaction mixture 50 glycolaldehyde, 50 50 Boc-Lys, DTPA, and pH was at °C under for The reaction mixture was on an g, 60 using in of ml were and by The a to the derivative of OP-lysine were under The was with of acid for at acid was in a of and the product was by Two major having at and at at were The were and by The to OP-lysine were under and by and A of was based on the of OP-lysine in the The product was to experiments with a synthetic of OP-lysine using and reaction mixture 50 of 50 was by of OP-lysine with UVA in the of were using a and A solution OP-lysine, DTPA, and 50 pH was with for The sample was for 60 at °C in a with and with The of the mixture was in the of The was in a at and the was as a solution the components ascorbate was and under the and liquid were on a consisting of and with and performance The were with the The of the and the in the and liquid experiments were within a on the of The used for experiments was 20 study of OP-lysine of amounts of the compound from cataractous lenses, of its structure based on and and characterization of amounts of OP-lysine, experiments that the the biological and the quantification of OP-lysine in acid hydrolysates of lens proteins and liquid experiments that OP-lysine in lens Our was by the of the and spectral properties this specific and of lenses and known The of the study was that OP-lysine is of the known AGEs in cataractous and aged lenses. and of was using the synthetic in The preparation is a of a by 1999; Scopus Google Scholar). The major was the of methyl of the of Boc-Lys of The of compound 6 with was and the only major side product was which to chromatographic In the of methyl of for the of the and amino of product the spectral of compound by the by 1999; Scopus Google Scholar), The important was and its spectral are as of OP-lysine a at which was in with the The showed major at and A of OP-lysine is in of under OP-lysine was by using of in in the presence of or found that the of results in a of OP-lysine compared with chromatographic to was for other AGEs L.M. 2001; PubMed Scopus Google Scholar). more appropriate for of OP-lysine from mixtures or its chromatographic is than and with of and from OP-lysine. the other the concentration of OP-lysine in acid systems in the of the acid of compound acid as 1999; Scopus Google Scholar). used for preparation of a for quantification of OP-lysine by and for preparation of for OP-lysine based the at and at at The of OP-lysine on pH as can be in to the of different At pH which is for OP-lysine showed at and At pH which to the the were at and match the for pH and and pH and J. Chem. Scopus Google Scholar). The of OP-lysine was pH at pH at and of OP-lysine in from cataractous and aged human lenses as as fetal calf lenses were to water-soluble and water-insoluble The water-insoluble of calf lenses was The proteins were and in to of the components of the experiments with synthetic OP-lysine that it is not under conditions and to the of the At the of the by was on the of the The on was used as a first in of OP-lysine from hydrolyzed proteins for and as A of hydrolyzed water-insoluble to cataractous lenses, was on 60 ml of and by The to the spectral and chromatographic properties of OP-lysine was at a was and by of the of the product isolated from the lenses is in The and the structure of the of the of this sample of the synthetic experiments using and the the sample isolated from hydrolyzed cataractous lenses and the synthetic of OP-lysine. 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Lin B. Lee K.W. Ortwerth B.J. Biochim. Biophys. Acta. 2001; 1537: 14-26Crossref PubMed Scopus (74) Google Scholar) of the sample of cataractous lens proteins by The of of OP-lysine at appropriate is that this post-translational of lysine in cataractous lenses. of OP-lysine in Boc-Lys and the of potential carbonyl to produce OP-lysine with Boc-Lys under conditions that do not in of and anaerobic DHA, glyceraldehyde and are reactive carbonyl known to be to the amino of lysine in lens proteins Lin B. Ortwerth B.J. Biochim. Biophys. Acta. 2003; PubMed Scopus Google B.J. PubMed Scopus Google Monnier V.M. Full Text PDF PubMed Scopus Google PubMed Scopus Google K.W. Ortwerth B. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Ortwerth B.J. Biochim. Biophys. Acta. PubMed Scopus Google J. Biochem. PubMed Scopus Google J. Google Scholar). The showed that of the carbonyl was to produce amounts of OP-lysine with Boc-Lys at pH for based on and HPLC of the reaction In the of was a having a and to those of OP-lysine. The product was using and with of the was by and The in the presence of only in the having at and as as the of the were completely different compared with those of the synthetic sample of OP-lysine. It is that the product isolated is compound which has for the at and to Biochem. 2003; PubMed Scopus Google The of the product from the model mixture Boc-Lys glyceraldehyde and the synthetic OP-lysine showed different with and min, that the were not identical. and argue that the product formed from glyceraldehyde and Boc-Lys was not OP-lysine. that of the model systems can produce amounts of OP-lysine, a reaction mixture glycolaldehyde, and The for study of such a was that OP-lysine to the amino of the lysine and the of and glyceraldehyde can The analysis of an of the reaction mixture showed a having an at and at at as as and to of the derivative of OP-lysine. The reaction mixture was using with and The product to that of the synthetic OP-lysine as as and experiments using and the the reaction mixture Boc-Lys, glycolaldehyde, and of however, were not to with spectral properties and to those of OP-lysine. of OP-lysine by UVA in the or of study B.J. PubMed Scopus Google Scholar) from this showed that the irradiation of aged human lenses with UVA in the presence of of their in the and It was to OP-lysine this The irradiation of solution of OP-lysine in presence of under in a of the at A irradiation in the of in of the at The of OP-lysine at not in the presence of irradiation The results in argue that is an in the photochemical bleaching of by The major of this article is to for the of OP-lysine in vivo as a newly identified AGE of lysine on proteins. to other known AGEs, the of OP-lysine in the biological was not for the of its which is of the of the structure of an were however, to for the to the relatively high of this AGE in cataractous human lenses. a structure of the AGE based on its and spectral as as its with which isolated from a model reaction Lin B. Ortwerth B.J. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar). of OP-lysine which was a of a 1999; Scopus Google Scholar). to produce a of synthetic to characterization by and spectral The of known reactions during the the and spectral properties of the synthetic and the of and spectral by and those by 1999; Scopus Google Scholar) were that the synthetic of OP-lysine the structure in The relative high of OP-lysine acid to this for its from proteins. used the synthetic in to the of OP-lysine in vivo. compared the properties of the synthetic of OP-lysine with the substance isolated from hydrolyzed cataractous lenses in the The in 6 showed that the and the of in the of the substance isolated from hydrolyzed lenses were to those of the synthetic of OP-lysine. the and of the were not Two chromatographic experiments using and showed of the an the synthetic sample and those isolated from the lenses, which that OP-lysine can be found in hydrolyzed cataractous lenses. used to biological for the presence of OP-lysine. is a for of an especially in The of requires that a specific in experiments with should be using the and its should be for of The of the synthetic OP-lysine major at and which can be to its structure The of having the for of OP-lysine is a for of OP-lysine in the only in the of the were for the other of hydrolyzed human lens and aged lens proteins not important in this study is OP-lysine in vivo or its presence in hydrolysates is an to the acid The of the of of OP-lysine for hydrolyzed cataractous proteins and hydrolyzed calf lens proteins in that OP-lysine be during acid of proteins. the presence of for of OP-lysine in the of lens proteins the of this derivative of lysine in vivo. the of OP-lysine in of the water-insoluble and water-soluble from cataractous and aged human lenses. The in argue that amounts of OP-lysine can be to and of water-soluble to water-insoluble lens proteins. to the in the which are not completely in this are on of an quantification based on of of OP-lysine. in the study an the from and HPLC which that the in are In the for OP-lysine are than those for lysine J.A. Thorpe S.R. Baynes J.W. PubMed Scopus Google Scholar), J.A. Thorpe S.R. Baynes J.W. PubMed Scopus Google E.B. Degenhardt T.P. Thorpe S.R. Baynes J.W. Biochem. J. 1997; PubMed Scopus Google Scholar), and E.B. Degenhardt T.P. Thorpe S.R. Baynes J.W. Biochem. J. 1997; PubMed Scopus Google Scholar) and are of the as for K. 2001; 42: Google Scholar). the of model reaction systems based on Boc-Lys and and AGE to OP-lysine under anaerobic the and of the mixtures not amounts of this compound. At the was an for OP-lysine in the Boc-Lys glyceraldehyde The to OP-lysine, were and the product showed a to those of the synthetic and the substance isolated from hydrolyzed lenses The of the product of the model was as and were experiments with the synthetic using and The argue that the of the carbonyl is a for of OP-lysine from Boc-Lys than of the carbonyl in In model experiments used conditions that that other and acid could be OP-lysine under A reaction for formation of OP-lysine a lysine and as an and by of are no that products are in glycation in it is known that are reactive in reaction at °C J. 273: Scopus Google Scholar). Our results argue that of the AGEs in vivo could be products of reactive carbonyl and side chains of amino In the biological systems, lysine and side chains are in a of glycation and it should be not a that in the reaction of more than carbonyl compound. is in with results Monnier V.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) that the of to a reaction mixture of and results in a of the of lysine The formation of OP-lysine on protein molecules could their properties. with the lysine OP-lysine an because the is at pH could the and structure of proteins. the of a in to a of to results in of the structure of this protein S. PubMed Scopus Google Scholar), of its and has been as a for of cataract 1998; PubMed Scopus Google Scholar). that UVA can in the human lens J. 1999; PubMed Scopus Google Scholar) and that the high of ascorbate PubMed Scopus Google Scholar), can that OP-lysine could an important in photochemical in the lens, based on the for its in for the UVA N. for with analysis and