Formation of pentosidine during nonenzymatic browning of proteins by glucose. Identification of glucose and other carbohydrates as possible precursors of pentosidine in vivo

A fluorescent compound has been detected in proteins browned during Maillard reactions with glucose in vitro and shown to be identical to pentosidine, a pentose-derived fluorescent cross-link formed between arginine and lysine residues in collagen (Sell, D. R., and Monnier, V. M. (1989) J. Biol. Chem. 264, 21597-21602). Pentosidine was the major fluorophore formed during nonenzymatic browning of ribonuclease and lysozyme by glucose, but accounted for less than 1% of non-disulfide cross-links in protein dimers formed during the reaction. Pentosidine was formed in greatest yields in reactions of pentoses with lysine and arginine in model systems but was also formed from glucose, fructose, ascorbate, Amadori compounds, 3-deoxyglucosone, and other sugars. Pentosidine was not formed from peroxidized polyunsaturated fatty acids or malondialdehyde. Its formation from carbohydrates was inhibited under nitrogen or anaerobic conditions and by aminoguanidine, an inhibitor of advanced glycation and browning reactions. Pentosidine was detected in human lens proteins, where its concentration increased gradually with age, but it did not exceed trace concentrations (less than or equal to 5 mumol/mol lysine), even in the 80-year-old lens. Although its precise carbohydrate source in vivo is uncertain and it is present in only trace concentrations in tissue proteins, pentosidine appears to be a useful biomarker for assessing cumulative damage to proteins by nonenzymatic browning reactions with carbohydrates.     

Site-specific synthesis of Amadori-modified peptides on solid phase.

Glycation of peptides and proteins is a slow chemical reaction of reducing sugars modifying the amino groups. The first intermediates of this nonenzymatic glycosylation are the Amadori products that can undergo further chemical reactions, finally leading to advanced glycation end products (AGEs). The formation of AGEs was not only linked to aging of tissues and organs in general but also to several diseases such as diabetes mellitus and Alzheimer's disease. Because of the importance of these modifications and their potential use as diagnostic markers, a global postsynthetic approach on solid phase was developed. The peptides were synthesized by Fmoc/(t)Bu-chemistry, with the lysine residue to be modified being protected with the very acid-labile methyltrityl group. Incubation of the peptides with D-glucose in DMF at elevated temperatures resulted in product yields of 35%. Neighboring residues with bulky protecting groups reduced the yields only slightly. The major by-products were the unmodified peptide and an oxidation product. Whereas the unmodified peptide eluted before the glycated peptide, all other by-products eluted later in RP-HPLC, allowing simple purification.     

Glucose autoxidation induces functional damage to proteins via modification of critical arginine residues

Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)β(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.     

Scavenging of Active Oxygen Species by Glycated Proteins

Scavenging of active oxygen species by glycated proteins was investigated. Glycated proteins were prepared from bovine serum albumin (BSA), insulin, and lysozyme incubated with glucose. Glycated BSA at concentration of 0.5% scavenged 34% of hydroxyl radicals by ESR experiments using DMPO as a spin-trapping reagent. The ability to scavenge hydroxyl radicals by glycated BSA was higher than that by BSA. Hydrogen peroxides also were largely scavenged with an increase in the concentration of glycated proteins. However, the ability to scavenge superoxides by glycated BSA was lower than that by BSA because glycated proteins produced superoxides. Experiments using model compounds such as Amadori compound and caproyl pyrraline suggested that the scavenging ability of glycated proteins against hydroxyl radicals depends on Maillard reaction products in the advanced stage, while the ability against hydrogen peroxides is dependent upon Maillard reaction products in the early stage and brown pigments.     

Formation of the molten globule-like state during prolonged glycation of human serum albumin

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reaction. We studied the impact of incubation of human serum albumin (HSA) with glucose, at various concentrations and incubation times, on the extent of HSA glycation and structural changes using circular dichroism (CD), fluorescence, and microviscometer techniques. The number of moles of glucose bound per mole of HSA (r), the number of reacted lysine and arginine residues, and the Amadori product formation during glycation were determined using 3-(dansylamino) phenyl boronic acid, fluorescamine, 9, 10 phenanthrenequinone, and p-nitroblue tetrazoliumchloride, respectively. The formation of advanced glycation end products (AGE) was detected using the autofluorescence characteristic of samples. We identified three stages of Maillard reaction for HSA upon incubation with the physiological level of glucose (0-630 mg/dl): the early, intermediate and late stages, which occurred after 7-14, 21, and >28 days of incubation, respectively. Structural information, Stokes radius, and 1-anilinonaphthalene-8-sulfonate (ANS) binding data indicated the formation of a molten globule-like state of HSA after 21 days of incubation with 35 mM (630 mg/dl) glucose. Thus, the extent of the Maillard reaction was influenced by the concentration of glucose and incubation time, such that longer exposure of HSA to glucose may have a more deleterious effect on its structure and especially on its half-life and turnover in the circulation. Our results suggest that in acute diabetes mellitus patients, HSA, after 21 days of glycation, passes through a molten globule-like state and may contribute to the pathogenesis of diabetes, and perhaps other diseases.     

Changes in Lysozyme due to Interaction with Vaporized Hexanal

In order to elucidate the mechanism of the alteration of proteins induced by vaporized aldehydes, unmodified and chemically-modified lysozymes were exposed in the solid state to vaporized hexanal at 50°C and 5.8 or 75% relative humidity (RH). On exposure at 75%RH, the unmodified lysozyme exhibited polymerization, browning, loss of solubility, fluorescence production and impairment of lysine, tryptophan and methionine residues. Methionine residues seemed to be oxidized to methionine sulfoxide residues. The polymerization did not proceed at 5.8RH. All the above alterations were almost completely prevented by the removal of oxygen from the reaction cells. Acetylation of lysozyme retarded these alterations fairly well except that the impairment of tryptophan residues was unaffected.

On the basis of all the results it is suggested that at the first step the concerned reaction essentially requires hexanal derivatives such as peroxyhexanoic acid and/or related radicals induced through the reaction with oxygen. The second step seems to consist at least of two routes which are independent of each other and require water. One route is assumed to be an amino-carbonyl reaction involving lysine residues. The other route seems responsible for the attack on tryptophan and methionine residues through oxidation involving the radicals.     

Mechanism of formation of the Maillard protein cross-link pentosidine. Glucose, fructose, and ascorbate as pentosidine precursors

Pentosidine is a recently discovered fluorescent protein cross-link from human extracellular matrix that involves lysyl and arginyl residues in an imidazo (4, 5b) pyridinium ring. Pentosidine could be synthesized in vitro by the reaction of ribose, lysine, and arginine. The potential biological significance of the molecule prompted us to investigate its mechanism of formation from D-ribose and key Maillard intermediates, as well as from other potential precursor sugars. The yield of pentosidine from N alpha-t-Boc-lysine, N alpha-t-Boc-arginine, and D-ribose was highest at pH 9.0 and 65 degrees C, but was unaffected by reactant ratios at alkaline pH suggesting an important role for base catalysis. Ribated Boc-lysine on incubation with N alpha-t-Boc-arginine afforded a fluorescent compound with UV, fluorescence, 1H NMR, and MS properties identical with those from native or synthetic pentosidine. 3-Deoxypentosone, however, was not a major pentosidine precursor. Pentosidine became slowly detectable in bovine serum albumin incubated with 0.25 M and 1.0 M glucose and reached, at 30 days, 13.2 and 17 pmol/mg bovine serum albumin, respectively. Spectroscopical properties of glucose-derived pentosidine were identical with those from ribose-derived pentosidine. Pentosidine formed from glucated Boc-lysine with N alpha-t-Boc-arginine in higher yields than from glucose under standard conditions. Fructose, and unexpectedly ascorbate, also formed pentosidine in similar yields as glucose. The discovery that pentosidine can form not only from pentoses but also from hexoses and ascorbate raises major new questions concerning biochemical pathways of the Maillard reaction in vivo.     

Arginine-specific modification of proteins with polyethylene glycol

In this study, the residue-selective modification of proteins with polymers at arginine residues is reported. The difficulty in modifying arginine residues lies in the fact that they are less reactive than lysine residues. Consequently, typical chemo-selective reactions which employ "kinetic" selectivity (active esters, Michael addition, etc.) cannot be used to target these residues. The chemistry exploited herein relies on "thermodynamic" selectivity to achieve selective modification of arginine residues. ω-Methoxy poly(ethylene glycol) bearing an α-oxo-aldehyde group was synthesized and used to demonstrate the selective modification of lysozyme at arginine residues. In addition, the optimization of reaction conditions for coupling as well as the stability of the formed adduct toward dilution, toward a nucleophilic buffer, and toward acidification are reported. It was concluded that this approach is a convenient, mild, selective, and catalyst-free method for protein modification.     

Production and characterization of antibodies to advanced glycation products on proteins

Antibodies directed against advanced glycation products formed during Maillard reaction have been generated and characterized. These antibodies reacted specifically with advanced glycation products in common among proteins incubated with glucose, but not early-stage compounds such as a Schiff base adduct and Amadori rearrangement products. Incubation of bovine serum albumin with glucose caused a time-related increase in immunoreactivity and a concomitant increase in fluorescence intensity. These antibodies may serve as a useful tool to elucidate pathophysiological roles of advanced Maillard reaction in diabetic complications and aging processes.     

Maillard reactions by alpha-oxoaldehydes: detection of glyoxal-modified proteins

Proteins can be chemically modified by sugars by glycation, or the Maillard reaction. The Maillard reaction produces irreversible adducts on proteins that are collectively known as advanced glycation end products, or AGEs. Recent studies indicate that several alpha-dicarbonyl compounds, including glyoxal (GXL), are precursors of AGEs in vivo. We developed antibodies against a GXL-modified protein (GXL-AGE) and purified a mixture of GXL-AGE-specific antibodies by chromatography on GXL-modified bovine serum albumin (BSA-GXL) coupled to EAH-Sepharose. This preparation was then processed on a human serum albumin-carboxymethyllysine (HSA-CML)-NHS-Sepharose to remove CML-specific antibodies. We used the resulting purified antibody in a competitive ELISA to probe GXL-AGEs in vitro and in vivo. We found increasingly greater antibody binding with increasing concentrations of GXL-modified BSA, but the antibody failed to react with either free CML or protein-bound CML. Incubation experiments with BSA revealed that glyceraldehyde, ribose and threose could be precursors of GXL-AGEs as well. Experiments in which GXL was incubated with N-alpha-acetyl amino acids showed that the antibody reacts mostly with lysine modifications. The GXL-derived lysine-lysine crosslinking structure, GOLD was found to be one of the antigenic epitopes for the antibody. Analysis of human plasma proteins revealed significantly higher levels of GXL-AGE antigens in type II diabetic subjects compared with normal controls (P<0.0001). We also found GXL-AGEs in human lens proteins. Bovine aortic endothelial cells cultured for 7 days with 30 mM glucose did not accumulate intracellular GXL-AGEs. These studies underscore the importance of GXL for extracellular AGE formation (except in lens where it is likely to be formed intracellularly) and suggest that changes associated with age and diabetes might be prevented by alteration of GXL-AGE formation.     

Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg39 and Arg85, which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg10, while Arg33 appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg39 and Arg85 are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.     

2′-N-Acetylation of Butirosin A by Mutants of Streptomyces fradiae

To reveal the mechanism of reducing sugar-induced polymerization of proteins, monomeric lysozymes were isolated at various stages of storage from whole lysozyme (WL) being kept with glucose at 75% relative humidity and 50°C for up to 30 days, and their chemical properties were investigated and compared with the corresponding WL. The impairment of Lys, Arg, and Trp residues was observed in all the isolated monomeric lysozymes (IM) as well as in the WL.

When the IM were stored for another 10 days without glucose, they polymerized and had an additional impairment of Arg and Lys but not Trp residues. All IM exhibited almost the same polymerization rate, but the sum of additional losses of Lys and Arg residues varied. The IM was also found to cross-link untreated lysozymes even in the absence of glucose.

On basis of the results obtained hitherto, it is suggested that the glucose-induced polymerization of lysozymes proceeds through the following paths. At the first step, some bifunctional agents (BF), probably α-dicarbonyl compounds, generated from the reaction between ?-amino groups of lysine residues and glucose, attach to Arg, Lys, and Trp residues through one of their two functional sites. At the second step, some of those proteins with BF attached polymerize by binding of the other unoccupied functional site with the remaining Lys and Arg (not Trp) residues of the other protein molecules. The other of the proteins with BF attached polymerize through the combination between the other unoccupied functional sites themselves with no loss of amino acid residues.     

Modification of arginine and lysine in proteins with 2,4-pentanedione.

Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.     

In vitro and in vivo reactions of nucleic acids with reducing sugars

Reducing sugars such as glucose and glucose 6-phosphate have been shown to nonenzymatically react with the amino groups of proteins. The modification of proteins by reducing sugars can alter both physical characteristics and biological functions. Analogous to the reaction observed with proteins, the amino groups of DNA bases are also able to react nonenzymatically with reducing sugars. The modifications of DNA by reducing sugars result in the time- and sugar-concentration-dependent changes in biological properties. In this communication we review data describing in vitro and in vivo models we have used to investigate the biological consequences of the nonenzymatic glycosylation of DNA.     

Protein modifications during antiviral heat bioprocessing and subsequent storage

Antiviral heat treatment is routinely used in the bioprocessing of therapeutic proteins as a means of reducing viral load. However, in protein formulations containing sucrose this form of bioprocessing can lead to protein modifications. Using a model protein, hen egg white lysozyme, we investigated the effects of antiviral heat treatments in the presence of sucrose on protein integrity during subsequent long-term protein storage. Although heat treatment alone resulted in protein modification, subsequent medium- to long-term storage of both lyophilized and liquid samples at room temperature or above led to further protein modifications. The majority of these modifications were due to the formation of glycation and advanced glycation end products via the reaction of reducing sugars and their autoxidation products (derived from hydrolyzed sucrose) with function groups on the protein surface. These findings have implications for the improvement of therapeutic protein bioprocessing to ensure protein product quality.     

Oxygen is not required for the browning and crosslinking of protein by pentoses: relevance to Maillard reactions in vivo

The chemical modification and crosslinking of proteins by the Maillard or browning reaction contributes to the aging of tissue proteins, and acceleration of this reaction during hyperglycemia is implicated in the pathogenesis of diabetic complications. Metal-catalyzed autoxidation reactions catalyze the browning of proteins by glucose, a process known as autoxidative glycosylation, but the effects of oxidative conditions on browning of proteins by smaller sugars has not been reported. In this work we studied the browning and crosslinking of the model protein, RNase A, by pentoses. Although antioxidative conditions inhibited the formation of glyoxal and the advanced glycation end-product, N epsilon-(carboxymethyl)lysine from arabinose, browning and crosslinking, and formation of the fluorescent crosslink pentosidine proceeded at comparable rates under oxidative and antioxidative conditions. These studies and other work on smaller dicarbonyl compounds indicate that Maillard reactions of simpler carbohydrates proceed efficiently in the absence of oxygen and suggest that antioxidant therapy for treatment of diabetic complications may have limited clinical efficacy.     

Independent selection by I-Ak molecules of two epitopes found in tandem in an extended polypeptide antigen

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.     

Propagation of protein glycation damage involves modification of tryptophan residues via reactive oxygen species: inhibition by pyridoxamine

Nonenzymatic modification of proteins is one of the key pathogenic factors in diabetic complications. Uncovering the mechanisms of protein damage caused by glucose is fundamental to understanding this pathogenesis and in the development of new therapies. We investigated whether the mechanism involving reactive oxygen species can propagate protein damage in glycation reactions beyond the classical modifications of lysine and arginine residues. We have demonstrated that glucose can cause specific oxidative modification of tryptophan residues in lysozyme and inhibit lysozyme activity. Furthermore, modification of tryptophan residues was also induced by purified albumin-Amadori, a ribose-derived model glycation intermediate. The AGE inhibitor pyridoxamine (PM) prevented the tryptophan modification, whereas another AGE inhibitor and strong carbonyl scavenger, aminoguanidine, was ineffective. PM specifically inhibited generation of hydroxyl radical from albumin-Amadori and protected tryptophan from oxidation by hydroxyl radical species. We conclude that oxidative degradation of either glucose or the protein-Amadori intermediate causes oxidative modification of protein tryptophan residues via hydroxyl radical and can affect protein function under physiologically relevant conditions. This oxidative stress-induced structural and functional protein damage can be ameliorated by PM via sequestration of catalytic metal ions and scavenging of hydroxyl radical, a mechanism that may contribute to the reported therapeutic effects of PM in the complications of diabetes.     

Carbohydrate binding specificity of monoclonal antibodies raised against lactose-protein Maillard adducts.

Three hybridoma antibodies (L101, L104, and L117) specific for lactose-protein amino carbonyl products (Maillard adducts) were obtained by immunizing mice with the lactose-ovalbumin Maillard adduct and by screening with the lactose-bovine serum albumin (BSA) adduct. They reacted with the Maillard adducts of lactose with several different proteins, but not with the adducts of several other reducing sugars. L101 reacted well with the lactose-BSA adducts formed by 2- to 16-day incubation, whereas L104 and L117 reacted with the advanced stage reaction products but not with the adducts of 2-day incubation. The competitive inhibition of the antibody binding by several mono- and disaccharides showed that lactulose (4-O-beta-D-galactopylanosyl-D-fructose) was the best inhibitor for all three antibodies, and that L104 and L117 were inhibited by methyl-beta-D-galactoside more effectively than L101. These results suggested that different components produced during the progress of the Maillard reaction could be antigenic determinants, and that the carbohydrate moiety including the terminal galactosyl residue played an important role in the antibody binding to the lactose-protein Maillard adducts.     

Testing biological activity of model Maillard reaction products: studies on gastric smooth muscle tissues

Water-soluble Maillard reaction products obtained from five different model systems were investigated for their effects upon the mechanical activity of rat gastric smooth muscle. Most of the total Maillard reaction products applied at concentration of 1.5 mg/ml evoked contractions; among them the product obtained from arginine and glucose (Arg-Glc) produced the most powerful contractions. The product obtained from glycine and ascorbic acid (Gly-AsA) was the only one that brought about relaxation response. The high molecular weight fractions (>3,500 Da) isolated from the reaction systems Arg-Glc and Gly-AsA demonstrated effects similar in type and amplitude to those evoked by non-fractioned reaction products. The results obtained suggest that moieties of molecules acting upon the muscle tonus originate mainly from lysine and arginine residues; that these structures are available in both low and high molecular pools in similar concentrations, and most likely these fragments act upon membrane-located cellular structures involved in calcium transport.