Chemical modification of proteins during peroxidation of phospholipids

Chemical modification of proteins by advanced glycation and lipoxidation end products is implicated in the pathogenesis of macrovascular disease in aging and diabetes. To identify biomarkers of the lipoxidative modification of protein, we studied the oxidation of phospholipids in the presence of the model protein RNase A and compared protein-bound products formed in these reactions with those formed during oxidation of plasma proteins. Metal-catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylcholine or 1-palmitoyl-2-linoleoyl-phosphatidylcholine in the presence of RNase led to the loss of amino groups in RNase and the incorporation of phosphate, hexanoate, pentanedioate, nonanedioate, and palmitate into protein. Protein-bound palmitate and phosphate correlated strongly with one another, and protein-bound pentanedioate and nonanedioate, derived from arachidonate and linoleate, respectively, accounted for ∼20% of the cross-linking of lipid phosphorus to protein. Similar results were obtained on oxidation of total plasma or isolated LDL.We conclude that alkanedioic acids are quantitatively important linkers of oxidized phospholipids to proteins and that measurement of protein-bound phosphate and long-chain fatty acids may be useful for assessing long-term lipid peroxidative damage to proteins in vivo. Analyses of plasma proteins from control and diabetic patients indicated significant increases in lipoxidative modification of protein in diabetic compared with control subjects. Chemical modification of proteins by advanced glycation and lipoxidation end products is implicated in the pathogenesis of macrovascular disease in aging and diabetes. To identify biomarkers of the lipoxidative modification of protein, we studied the oxidation of phospholipids in the presence of the model protein RNase A and compared protein-bound products formed in these reactions with those formed during oxidation of plasma proteins. Metal-catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylcholine or 1-palmitoyl-2-linoleoyl-phosphatidylcholine in the presence of RNase led to the loss of amino groups in RNase and the incorporation of phosphate, hexanoate, pentanedioate, nonanedioate, and palmitate into protein. Protein-bound palmitate and phosphate correlated strongly with one another, and protein-bound pentanedioate and nonanedioate, derived from arachidonate and linoleate, respectively, accounted for ∼20% of the cross-linking of lipid phosphorus to protein. Similar results were obtained on oxidation of total plasma or isolated LDL. We conclude that alkanedioic acids are quantitatively important linkers of oxidized phospholipids to proteins and that measurement of protein-bound phosphate and long-chain fatty acids may be useful for assessing long-term lipid peroxidative damage to proteins in vivo. Analyses of plasma proteins from control and diabetic patients indicated significant increases in lipoxidative modification of protein in diabetic compared with control subjects. Oxidation of LDL is considered an early event in the conversion of LDL to a proatherogenic form, setting the stage for atherogenesis (1Witztum J.L. Steinberg D. The oxidative modification hypothesis of atherosclerosis: does it hold for humans?.Trends Cardiovasc. Med. 2001; 11: 93-102Google Scholar). In support of this hypothesis, products of lipid peroxidation, such as malondialdehyde (MDA) and hydroxynonenal (HNE) adducts to protein, have been detected in plasma of patients with macrovascular disease and in atherosclerotic plaque by both chemical and immunochemical methods (2Tsimikas S. Witztum J.L. Measuring circulating oxidized low-density lipoprotein to evaluate coronary risk.Circulation. 2001; 103: 1930-1932Google Scholar, 3Tsimikas S. Noninvasive imaging of oxidized low-density lipoprotein in atherosclerotic plaques with tagged oxidation-specific antibodies.Am. J. Cardiol. 2002; 90: 22L-27LGoogle Scholar, 4Sattler W. Malle E. Kostner G.M. Methodological approaches for assessing lipid and protein oxidation and modification in plasma and isolated lipoproteins.Methods Mol. Biol. 1998; 110: 167-191Google Scholar, 5Kovacs I.B. Jahangiri M. Rees G.M. Gorog P. Elevated plasma lipid hydroperoxides in patients with coronary artery disease.Am. Heart J. 1997; 134: 572-576Google Scholar). Oxidized low density lipoprotein (OxLDL) is also recognized by scavenger receptors on macrophages, the cell type in which lipids accumulate during the early stages of atherogenesis (6Platt N. Haworth R. Darley L. Gordon S. The many roles of the class A macrophage scavenger receptor.Int. Rev. Cytol. 2002; 212: 1-40Google Scholar, 7Boullier A. Bird D.A. Chang M.K. Dennis E.A. Friedman P. Gillotre-Taylor K. Horkko S. Palinski W. Quehenberger O. Shaw P. et al.Scavenger receptors, oxidized LDL, and atherosclerosis.Ann. N. Y. Acad. Sci. 2001; 947 (discussion 222–223): 214-222Google Scholar). Zhang, Yang, and Steinbrecher (8Zhang H. Yang Y. Steinbrecher U.P. Structural requirements for the binding of modified proteins to the scavenger receptor of macrophages.J. Biol. Chem. 1993; 268: 5535-5542Google Scholar) demonstrated the loss of lysine amino groups during the oxidation of lipoproteins, and Friedman et al. (9Friedman P. Horkko S. Steinberg D. Witztum J.L. Dennis E.A. Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol concentration.J. Biol. Chem. 2002; 277: 7010-7020Google Scholar) have proposed that the dominant epitope recognized by the major macrophage scavenger receptor, CD36, is a phosphorycholine adduct on oxidized phospholipid linked to lysine residues on LDL as a Schiff base adduct. The linker, pentanoic acid semialdehyde (oxovalerate), was identified as a product of the oxidation of arachidonic acid at the sn-2 position of the phospholipid. Podrez et al. (10Podrez E.A. Poliakov E. Shen Z. Zhang R. Deng Y. Sun M. Finton P.J. Shan L. Gugiu B. Fox P.L. et al.Identification of a novel family of oxidized phospholipids that serve as ligands for the macrophage scavenger receptor CD36.J. Biol. Chem. 2002; 277: 38503-38516Google Scholar, 11Podrez E.A. Poliakov E. Shen Z. Zhang R. Deng Y. Sun M. Finton P.J. Shan L. Febbraio M. Hajjar D.P. et al.A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions.J. Biol. Chem. 2002; 277: 38517-38523Google Scholar) have identified a more complex array of compounds, α,β-unsaturated, γ-hydroxy, or oxo acids or aldehydes derived from arachidonate or linoleate, which may also cross-link phospholipids to protein by Schiff base (imine), Michael reactions, and amide linkages. Finally, using immunohistochemical techniques, Osawa and colleagues detected Nε-(hexanoyl)lysine (12Kato Y. Mori Y. Makino Y. Morimitsu Y. Hiroi S. Ishikawa T. Osawa T. Formation of Nε-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.J. Biol. Chem. 1999; 274: 20406-20414Google Scholar) adducts and nonanedioic (azelaic) acid (NDA) cross-links (13Kawai Y. Fujii H. Kato Y. Kodama M. Naito M. Uchida K. Osawa T. Esterified lipid hydroperoxide-derived modification of protein: formation of a carboxyalkylamide-type lysine adduct in human atherosclerotic lesions.Biochem. Biophys. Res. Commun. 2004; 313: 271-276Google Scholar) to lysine residues in oxidized lipoproteins. In none of these studies, however, has a dominant lipid adduct to protein been identified, nor have the cross-link structures been quantified in lipoxidized proteins. In the present study, we have focused on the measurement of two of the characteristic linkages of oxidized phospholipids to protein, those mediated by the dicarboxylic acids pentanedioic (glutaric) acid (PDA) and NDA, derived from arachidonate and linoleate, respectively (Fig. 1). We present preliminary studies on the modification of a model protein, bovine pancreatic RNase A (RNase), by peroxidizing phospholipids containing arachidonate and linoleate and compare the products formed in these model reactions with those formed during Cu2+-catalyzed oxidation of plasma proteins and purified LDLs. Our data indicate that protein-bound phosphate and fatty acids are present in lipoxidized proteins at much higher concentrations than MDA or HNE and that the dicarboxylic acids PDA and NDA may constitute a quantitatively important class of cross-links between oxidized phospholipids and proteins. We conclude that the measurement of protein-bound palmitate and stearate, which are components of the oxidized phospholipids bound to plasma proteins, provides a sensitive technique for assessing lipoxidative damage to proteins and demonstrate that these chemical modifications are increased in total plasma proteins of diabetic compared with control subjects. Unless indicated otherwise, all reagents were purchased from Sigma-Aldrich (St. Louis, MO), including 1-palmitoyl-2-arachidonoyl-phosphatidylcholine (PAPC), 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC), and RNase (RNase type XII-A). Heavy labeled [2H11]hexanoic acid was purchased from Cambridge Isotope Laboratories (Andover, MA), and [2H4]palmitic acid and [2H8]lysine were from C/D/N Isotopes (Pointe-Claire, Quebec, Canada). All solvents were of the highest purity available from Acros Chemicals (Atlanta, GA). RNase (0.3 mM, 4.4 mg/ml = 3 mM lysine) was incubated with varying amounts of PAPC or PLPC in the presence of 75 μM CuSO4 for 24 h at 37°C in 0.2 M sodium phosphate buffer, pH 7.4. Aliquots (1 ml) were removed before the addition of CuSO4 and at 24 h after the addition of CuSO4; oxidation was quenched by the addition of diethylenetriaminepentaacetic acid (DTPA) (final concentration = 4 mM). Samples were then dialyzed in 3500 MWCO 4 of containing mM for 24 h at with of the were in and in of To PAPC or PLPC and lipid were with to the J. M. A for the and of total from Biol. Chem. and the was The and protein at the were in and in of Aliquots were removed for the measurement of amino groups using the acid W. A acid for amino acids and in Scholar) and protein concentration by the measurement with the Biol. Chem. Scholar). of protein-bound fatty of of [2H11]hexanoic acid and of acid in was to the of which was then with an of and for h at of the was for phosphate using the as modified by et al. J. M. E. The of methods to the incorporation of into the of Biophys. Scholar). were from were using by the of the of plasma was by at for at was oxidized using a concentration of mM CuSO4 a mM CuSO4 by at Aliquots (1 ml) were at and quenched by the addition of (final concentration = and at incubated in the of as a The of the oxidation was by of U.P. Witztum J.L. S. Steinberg D. in amino groups during oxidation or cell modification of LDL. Correlation with in Scholar) using MDA as a of protein-bound bound lipids were by the addition of of by at for at and the was this was two more that this of protein and of lipid The protein was in of for the measurement of protein and of protein, containing of was in of as The phosphate of the protein was after also as for lipoprotein were from LDL was isolated by from plasma = as by et al. S. M. D. R. The of LDL for the measurement of oxidized LDL by Scholar). The LDL was dialyzed M containing μM pH and a for and of then in the at (final protein concentration using a LDL was oxidized in pH at 37°C for to 24 h concentration = CuSO4 concentration = S. M. D. R. The of LDL for the measurement of oxidized LDL by Scholar). LDL incubated in the of as a To the of of both LDL to a protein concentration of were for of formation by at The oxidation was by the addition of to a concentration of Samples were then dialyzed in MWCO 4 of containing mM for 24 h at with of the were in and in of To bound were with 4 of as and of protein-bound fatty acids and phosphate was as for acids by acid of proteins were from the using an of the was a of MA), were by the addition of of M in by at for The were into of and was by were on a model to an using a and The was at and the at The was as of hold for then to to to and hold for 4 was on using from of a of labeled and amounts of fatty The formed after the loss of were to fatty = = = = = NDA, = = = and = In an to identify and the cross-link in oxidized (9Friedman P. Horkko S. Steinberg D. Witztum J.L. Dennis E.A. Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol concentration.J. Biol. Chem. 2002; 277: 7010-7020Google we the product of of the Schiff base cross-link between and protein. was from semialdehyde and semialdehyde was by of acid mM in M phosphate buffer, pH using of containing as by and M. Y. K. with in Biophys. 1999; Scholar). The was for 4 h at and the of was by of the adduct of semialdehyde M. Y. K. with in Biophys. 1999; Scholar). A from the adduct of was for and indicated that the from the was 4 the was removed using and the was a was to the to a concentration of mM, and the was then incubated for h at 37°C to Schiff base was mM and the was for 4 h at The was in and then in for 3 h at Aliquots of the were and then to the as a of oxidative in Mol. Biol. 2002; Scholar) for by as The of the synthetic on the at and major at and of to the amino and oxidized and control were with mM in M sodium buffer, pH or with mM in M sodium phosphate buffer, pH for 4 h at was then with an of acid and for at at and the was lipids were with of as of protein-bound of of to et al. of and aging on in human and of was to the which was as the were in 3 of containing acid and to 3 to and before for The were and then for measurement of and lysine as by as a of oxidative in Mol. Biol. 2002; Scholar). The was at and the at The was as of hold for 4 then to to to and hold for 4 was on using from of a of labeled and amounts of and The were = = = and = was to the lysine of the was as the labeled for the measurement of = of disease and were on than the and and type diabetic = were from the at The was by the and A was and were and at of with or and of macrovascular disease of diabetic and control for diabetic control are for diabetic control subjects. in a are were after an before including was by and a was and and lipids were by the and results are in and were by and at using methods Unless indicated otherwise, data are as of or more were using the To model the of protein with products of lipid peroxidation, RNase was incubated with phospholipids or in phosphate at pH for 24 We the of modification of the protein by phospholipids of protein-bound palmitate fatty and phosphate to the however, we also for the dicarboxylic acid PDA and NDA (Fig. 1). (Fig. the presence of protein-bound PDA from linoleate in and NDA from arachidonate in in the of modification of lysine residues in RNase by both PAPC and PLPC increased with the of phospholipid to protein. the amino groups of RNase lysine residues the amino were modified PAPC or PLPC was present at a lysine The of protein-bound phosphate with the of acid during that the of phospholipids was linked to protein a structure derived from the at the sn-2 position in the phospholipid. the PAPC the of phospholipid phosphate and palmitate bound to protein with the of amino however, protein-bound phospholipid and palmitate accounted for of lysine that in this products than phospholipid adducts also to the loss of lysine amino RNase modified by PAPC or the palmitate and phosphate were by (1 M in for h at with the structures proposed in of amino groups and in palmitate and phosphate in RNase incubated with peroxidizing are of two in a are of two with palmitate and phosphate, the concentrations of PDA and NDA also increased with the of phospholipid to protein (Fig. on data in 3 and PDA accounted for ∼20% and of lysine modification and palmitate in the reactions of PAPC and PLPC with The PDA or NDA of palmitate lysine was of the of PAPC or PLPC to lysine that phospholipid in this model was in the lipid at the of protein and that oxidation products with protein. on these dicarboxylic acids may of the cross-linking of phospholipid to of protein-bound phospholipid and dicarboxylic acids in RNase incubated with peroxidizing nonanedioic (azelaic) pentanedioic (glutaric) are from two in a NDA, nonanedioic (azelaic) pentanedioic (glutaric) are from two 3 also for both PAPC (Fig. and PLPC (Fig. the of bound increased with the of phospholipid to The of modification of the protein by from PLPC was that with that to be a more sensitive of the modification of the protein by arachidonate compared with The in and PDA or NDA of the protein that the and dicarboxylic acids may be formed by the To evaluate the of PDA and NDA in the of oxidized phospholipids to plasma proteins, plasma was exposed to oxidation and protein modifications were (Fig. concentrations of were to the of and The of the in MDA and protein-bound phosphate (Fig. were and both a after h of The of a characteristic product of lipid H. H. and of and Biol. Med. 11: was that of phosphate bound to protein. The fatty acids derived from the position of plasma phospholipids and increased with The total of long-chain fatty acid bound to oxidized plasma proteins (Fig. was to the phosphate of the proteins (Fig. that than or in were the of chemical modifications in the oxidized of the formation of malondialdehyde protein-bound phosphate and long-chain fatty acids during oxidation of was oxidized with mM CuSO4 for h at and were quenched at the indicated with of the formation of by the of the of protein-bound phosphate, by the of the formation of protein-bound fatty acids after acid of by acid protein at the of from control oxidized and NDA concentrations also increased in with the in the phosphate of the protein (Fig. with the protein of PDA and NDA accounted for cross-linking of ∼20% of the lipid to proteins in oxidized The of PDA to NDA was than during the 4 h of oxidation with that in was oxidized more than the concentration of protein-bound NDA was higher than that of the higher of linoleate arachidonate in plasma phospholipids P. The and fatty acid of plasma phospholipids in and with acid Chem. Med. 2002; Scholar). To evaluate the of in the modification of amino acid residues in proteins, we the concentration of protein-bound of the to or we were to amounts of in protein from oxidation of plasma or in of RNase incubated with PAPC of that we were to and of of of protein, which is of the of PDA concentration in plasma To evaluate the of PDA and NDA in the modification of oxidized lipoproteins, we the formation of these during the Cu2+-catalyzed oxidation of LDL. The of the in at in a after h of oxidation in the fatty acids and increased in with the phosphate of the in with the results of plasma oxidation The of phosphate, and LDL in the and oxidized LDL, respectively, were the as those in studies to the of the cross-link (9Friedman P. Horkko S. Steinberg D. Witztum J.L. Dennis E.A. Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol concentration.J. Biol. Chem. 2002; 277: 7010-7020Google Scholar, Horkko S. Witztum J.L. Steinberg D. Oxidized linked to of oxidized LDL, are ligands for macrophage scavenger Res. Scholar). both PDA and NDA increased during LDL oxidation and with the of The of PDA and NDA accounted for the cross-linking of of the lipid to the proteins, with the results of on plasma oxidation of protein-bound phosphate and acid of LDL and oxidized 24 are from two in a are from two the results of the of protein-bound fatty acids and phosphate in total plasma proteins from a of control and type diabetic Protein-bound fatty acids and phosphate were detected in these and were increased = in plasma proteins of diabetic in patients with lipid and in the model studies the total concentration of protein-bound fatty acids with the phosphate of the plasma protein. an of for plasma proteins, are of protein-bound phosphate of protein in we have at this it that the phospholipid adducts be in the lipoprotein of plasma and acid adducts in total plasma proteins of control and type diabetic for type diabetic control for type diabetic control for type diabetic control are and are as of plasma for type diabetic control subjects. in a are and are as of plasma protein. We have that during the oxidation of PAPC or PLPC in the presence of the modification of lysine residues is by increases in the bound phosphate and palmitate of the protein. data the binding of phospholipids to protein during lipoxidation reactions (9Friedman P. Horkko S. Steinberg D. Witztum J.L. Dennis E.A. Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol concentration.J. Biol. Chem. 2002; 277: 7010-7020Google Scholar, E.A. Poliakov E. Shen Z. Zhang R. Deng Y. Sun M. Finton P.J. Shan L. Gugiu B. Fox P.L. et al.Identification of a novel family of oxidized phospholipids that serve as ligands for the macrophage scavenger receptor CD36.J. Biol. Chem. 2002; 277: 38503-38516Google Scholar, 11Podrez E.A. Poliakov E. Shen Z. Zhang R. Deng Y. Sun M. Finton P.J. Shan L. Febbraio M. Hajjar D.P. et al.A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions.J. Biol. Chem. 2002; 277: 38517-38523Google Scholar). We have also quantified hexanoate, and NDA bound to lipoxidized RNase and oxidized plasma proteins in plasma proteins and LDL, and NDA of the cross-linking of phosphate to protein. Our results support the of et al. (13Kawai Y. Fujii H. Kato Y. Kodama M. Naito M. Uchida K. Osawa T. Esterified lipid hydroperoxide-derived modification of protein: formation of a carboxyalkylamide-type lysine adduct in human atherosclerotic lesions.Biochem. Biophys. Res. Commun. 2004; 313: 271-276Google NDA cross-links between oxidized phospholipids and proteins using immunochemical and this by the of the PDA cross-link and by the of both NDA and the we were to the cross-link in RNase oxidized in the presence of or in oxidized plasma protein or LDL, or in human plasma for this were to those for the and of MDA and HNE adducts to lysine residues in protein of malondialdehyde and adducts to lysine residues in and oxidized human low density J. 1997; Scholar). We conclude that the PDA and NDA have been may for to of the lipid modification of protein and may be quantitatively significant cross-links between oxidized phospholipids and protein. Oxidation of in is to a complex array of both protein-bound and in including W. K. J. adducts of a product of the in oxidized low density Biol. Chem. 1999; 274: Scholar, O. of the adducts formed from via the 1999; Scholar) and N. O. Formation of as products of the Biol. Chem. 2001; Scholar, O. Yang T. D. of proteins by oxidized Biol. Chem. 2004; Scholar) and and fatty acids E.A. Poliakov E. Shen Z. Zhang R. Deng Y. Sun M. Finton P.J. Shan L. Febbraio M. Hajjar D.P. et al.A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions.J. Biol. Chem. 2002; 277: 38517-38523Google Scholar). A of more and are also into and with protein to Schiff Michael and and that to the and cross-linking of oxidized lipoproteins. The of these is by the that a of products is many in low and that many of these products are to acid such as MDA or may to to products and cross-links an of advanced glycation reactions, also advanced lipoxidation Biol. Chem. Scholar). In protein-bound phosphate and long-chain fatty acids are end products that be in after acid in the of protein-bound phosphate and fatty acids in oxidized plasma proteins that of a of lipid peroxidation, by in the present of HNE and are to that of MDA in lipoxidized RNase and in oxidized LDL an of advanced glycation reactions, also advanced lipoxidation Biol. Chem. the of protein-bound phosphate and fatty acids provides for the of a protein to lipoxidative The of the present is to present a for phosphate and fatty acids to oxidized to demonstrate that measurement of these adducts may be useful for lipoxidative damage to protein. measurement of protein-bound fatty acids by provides and for lipoxidation reactions, are and is that these may be to more or for of the is the presence of of phosphate and fatty acids on proteins. this to be for plasma proteins, it may the of the for the of lipoxidation products on proteins, protein and are of proteins, it may be to on protein-bound or phosphate by be and such as be by of the of the of the phospholipid adducts and the of plasma proteins, of bound phosphate and long-chain fatty acids on plasma protein may be useful for oxidative damage to proteins a of to on the of the major proteins in the an may the measurement of for the of control and damage to protein in an of damage to protein. may also useful for assessing lipoxidative damage to plasma proteins as a of the of in lipid In preliminary studies we detected phosphate and long-chain fatty acids on total plasma proteins from human plasma and higher concentrations of these adducts in the plasma proteins of type diabetic The patients for this were for lipid than in the modification of proteins. studies are or in to these adducts in total plasma proteins and isolated plasma from both type and type diabetic as a of the and of the presence of and in with in with and in from atherosclerotic plaque from both diabetic and subjects. In these the phospholipid adducts may be of or the presence it is also that the fatty acids of phospholipids on the of lipoxidized proteins may serve as lipid binding these proteins to cell and oxidative and and the of The of for the LDL in this was by from the of and and

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.