The botanical extracts of Achyrocline satureoides and Ilex paraguariensis prevent methylglyoxal-induced inhibition of plasminogen and antithrombin III

Endogenously produced dicarbonyls, such as methylglyoxal (MG), are involved in advanced glycation end-product formation and thus linked to the pathophysiology of diabetic chronic complications. While the search for synthetic new antiglycation agents continues, little attention has been paid to putative antiglycation agents in natural compounds. Given the link between glycation and oxidation, in this work, we study the effects of methylglyoxal on two model systems; plasminogen and antithrombin III (AT III), then we set out to unravel a possible antiglycation effect for extracts of the flavonoid-rich common herbal species Achyrocline satureoides (AS) and Ilex paraguariensis (IP). Using SAR-PRO-ARG-pNA as a specific thrombin substrate, we show that incubation of plasma with MG decreases heparin activation of AT III by up to a 70%, in a dose-dependent manner. A parallel dose-dependent decrease in plasminogen activity reaching more than 50% was shown using D-BUT-CHT-lys-pNA as a plasmin-specific substrate. Extracts of AS and IP display a dose dependent inhibition of the action of the dicarbonyl, already significant at a 1/100 dilution of the herbal infusions. The inhibition was comparable to that obtained by using millimolar concentrations of known AGE inhibitors such as aminoguanidine and carnosine as well as micromolar concentrations of the antioxidant ascorbic acid. We believe our system of whole plasma glycation over 16 h with micromolar concentrations of MG, coupled with the measurement of activities of plasminogen and AT III by specific substrates provides a straightforward, practical method for monitoring the action of putative antiglycation agents. If predictably milder glycated forms of AT III and plasminogen were to be secreted in vivo, the loss of activities shown here could act synergistically to generate hyperthrombicity.     

Free alanine,aspartic acid,or glutamic acid reduce the glycation of human lens proteins

The amino acids lysine and glycine are reported to react with glucose at physiological pH and temperature and undergo non-enzymic glycation. Three other amino acids present in relatively larger amounts in the lens i.e. alanine, aspartic acid and glutamic acid were also found to undergo non-enzymic glycation as found by incorporation of uniformly labelled (U-[¹⁴C]) glucose into the amino acids. The glucose incorporation was 1.6 to 2.5% for alanine, 35 to 50% for aspartic acid and 2.3 to 3.3% for glutamic acid. Each amino acid of varying concentrations lowered the extent ofin vitro glycation of lens proteins significantly in glucose-treated homogenates of normal lens from humans. The decrease in glycation for alanine was between 32 and 69%, that for aspartate was between 18 and 74%, and for glutamate was between 52 to 74%. Decreased glycation was greater for higher concentrations of glucose. Scavenging of intracellular glucose and decreasing the extent of glycation of lens proteins could be the mechanism of action by which the amino acids alanine, aspartic acid and glutamic acid could exercise a beneficial effect on cataract and diabetic retinopathy.     

Inhibitory effect of some acetyl esters and acetamides on glycation of the histone H1

Non-enzymatic glycosylation (glycation) is a spontaneous set of reactions between reducing sugars and free amino groups in proteins or other biomolecules leading to the formation of fluorescent and coloured compounds known as advanced glycation end products (AGEs). AGEs cause structural changes of key proteins in humans, and therefore they are related with a number of physiological processes and diseases such as aging, atherosclerosis, cataract, arthritis, Alzheimer's disease. Two main strategies have been employed to prevent the formation of AGEs: a) low carbohydrate diet and b) pharmacological intervention. The latter includes treatment with reactive compounds which might be either sugar competitors (type A), carbonyl traps (type B) or free radical trapping antioxidants (type C). Acetylsalicylic acid (ASA, aspirin) is a good example of sugar competitor capable of inhibiting glycation by acetylating epsilon-amino groups of lysine residues in proteins. Taking into consideration the inhibiting effect of ASA on glycation we designed to study the antiglycation activity of other acetyl group-containing compounds (acetamides and acetyl esters) using the lysine-rich protein histone H1 as a model. The glycation of the histone H1 was carried out by either fructose or a complex mixture of glycating agents obtained from E. coli and monitored by fluorescent spectroscopy, SDS-PAGE and measurement of the content of reactive carbonyl groups in the target protein. Our results showed that the inhibitory effect of phenyl acetate, acetanilide, 4-acetamidophenylacetic acid and isopropenyl acetate was comparable to that of ASA. Based on the obtained results we conclude that these compounds act as free radical scavengers protecting proteins from the damaging effect of reactive oxygen species produced during the formation of AGEs.     

Glycation by ascorbic acid causes loss of activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and its increased susceptibility to proteases

Glycation is a process whereby sugar molecules form a covalent adduct with protein amino groups. In this study, we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein in chloroplast tissues, and examined effects of glycation on the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with AsA and Rubisco in vitro at physiological conditions (10 mM AsA, pH 7.5, 25 degrees C in the presence of atmospheric oxygen). At the early stage of glycation (phase 1), the amount of AsA attaching to Rubisco increased at an almost linear rate (0.5-0.7 mol AsA incorporated (mol Rubisco)(-1) d(-1)). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, N( epsilon )-(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation, and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation of high molecular weight aggregates cross-linked between subunits. Glycation led to a decrease in Rubisco activity (half-life about 7-8 d). Furthermore, glycated Rubisco of phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of phase 1 compared to that of native Rubisco. Results obtained here suggest that AsA is possibly an important factor in the loss of activity and turnover of Rubisco.     

Carnosine prevents the glycation-induced changes in electrophoretic mobility of aspartate aminotransferase

Carbohydrate-derived aldehydes cause irreversible loss of protein function via glycation. We previously observed that glyceraldehyde 3-phosphate (Glyc3P) abolishes the enzyme activity of cardiac aspartate aminotransferase (cAAT). We also examined the protective effects of carnosine against Glyc3P-induced loss of enzyme activity. The present study looked at carnosine's prevention of Glyc3P-induced change in protein structure. Purified cAAT (2 mg protein/mL) was incubated with various concentrations of carnosine (1-20 mM) in the presence of Glyc3P (500 microM) for 4 days at 37 degrees C. Following incubation, samples were analyzed by SDS-polyacrylamide gel electrophoresis. Carnosine showed prevention of protein modification at carnosine-to-Glyc3P ratios of 10:1 or greater. There was a progressive loss of the unmodified cAAT protein band as Glyc3P concentration was increased. Additionally, the gel position of the Glyc3P-modified cAAT protein varied over time. The apparent molecular weight (MWapp) of the Glyc3P-modified cAAT protein that formed after 1 day at 37 degrees C (500 microM) was greater than its MWapp after 2 days, suggesting that a chemical rearrangement of the initial adduct occurs. These observations support the hypothesis that carnosine is an antiglycation agent and that its mechanism of action involves prevention of protein modification.     

Novel biomarkers of the metabolism of caffeic acid derivatives in vivo

The purpose of this study was to investigate biomarkers of the bioavailability and metabolism of hydroxycinnamate derivatives through the determination of the pharmacokinetics of their urinary elimination and identification of the metabolites excreted. Coffee was used as a rich source of caffeic acid derivatives and human supplementation was undertaken. The results show a highly significant increase in the excretion of ferulic, isoferulic, dihydroferulic acid (3-(4-hydroxy-3-methoxyphenyl)-propionic acid), and vanillic acid postsupplementation relative to the levels presupplementation. Thus, ferulic, isoferulic, and dihydroferulic acids are specific biomarkers for the bioavailability and metabolism of dietary caffeic acid esters. Isoferulic acid is a unique biomarker as it is not a dietary component, however, dihydroferulic acid may well derive from other flavonoids with a structurally related B-ring. 3-Hydroxyhippuric acid has also been identified as an indicator for bioavailability and metabolism of phenolic compounds, and shows a highly significant excretion increase postsupplementation. The results reveal isoferulic acid (and possibly dihydroferulic acid) as novel markers of caffeoyl quinic acid metabolism.     

Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging

Until now, the glycation reaction was considered to be a nonspecific reaction between reducing sugars and amino groups of random proteins. We were able to identify the intermediate filament vimentin as the major target for the AGE modification N(epsilon)-(carboxymethyl)lysine (CML) in primary human fibroblasts. This glycation of vimentin is neither based on a slow turnover of this protein nor on an extremely high intracellular expression level, but remarkably it is based on structural properties of this protein. Glycation of vimentin was predominantly detected at lysine residues located at the linker regions using nanoLC-ESI-MS/MS. This modification results in a rigorous redistribution of vimentin into a perinuclear aggregate, which is accompanied by the loss of contractile capacity of human skin fibroblasts. CML-induced rearrangement of vimentin was identified as an aggresome. This is the first evidence that CML-vimentin represents a damaged protein inside the aggresome, linking the glycation reaction directly to aggresome formation. Strikingly, we were able to prove that the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in human tissues such as skin into strong relationship with loss of organ contractile functions.     

The polyamines spermine and spermidine protect proteins from structural and functional damage by AGE precursors: a new role for old molecules?

Due to the importance of glycation in the genesis of diabetic complications, an intense search for synthetic new antiglycation agents is ongoing. However, a somewhat neglected avenue is the search for endogenous compounds that may inhibit the process and be a source of protodrugs. Based on their ubiquity, their polycationic nature, their essential role in growth, their relatively high concentrations in tissues, and their high concentrations in sperm, we hypothesized that polyamines inhibit glycation and that might be one of their so far elusive functions. In this study we demonstrate a potent antiglycation effect of physiological concentrations of the polyamines spermine and spermidine. We employed two approaches: in the first, we monitored structural changes on histones and ubiquitin in which polyamines inhibit glycation-induced dimer and polymer formation. In the second we monitored functional impairment of catalytic activity of antithrombin III and plasminogen. Protection is afforded against glycation by hexoses, trioses and dicarbonyls AGE precursors and is comparable to those of aminoguanidine and carnosine.     

Ferulic acid prevents methylglyoxal-induced protein glycation,DNA damage,and apoptosis in pancreatic β-cells

Methylglyoxal (MG) can react with amino acids of proteins to induce protein glycation and consequently the formation of advanced glycation end-products (AGEs). Previous studies reported that ferulic acid (FA) prevented glucose-, fructose-, and ribose-induced protein glycation. In this study, FA (0.1–1 mM) inhibited MG-induced protein glycation and oxidative protein damage in bovine serum albumin (BSA). Furthermore, FA (0.0125–0.2 mM) protected against lysine/MG-mediated oxidative DNA damage, thereby inhibiting superoxide anion and hydroxyl radical generation during lysine and MG reaction. In addition, FA did not have the ability to trap MG. Finally, FA (0.1 mM) pretreatment attenuated MG-induced decrease in cell viability and prevented MG-induced cell apoptosis in pancreatic β-cells. The results suggest that FA is capable of protecting β-cells from MG-induced cell damage during diabetes.     

Glycation changes the charge distribution of type I collagen fibrils

In aging and diabetes, glycation of collagen molecules leads to the formation of cross-links that could alter the surface charge on collagen fibrils, and hence affect the properties and correct functioning of a number of tissues. The electron-optical stain phosphotungstic acid (PTA) binds to positively charged amino acid side-chains and leads to the characteristic banding pattern of collagen seen in the electron microscope; any change in the charge on these side-chains brought about by glycation will affect the uptake of PTA. We found that, upon glycation, a decrease in stain uptake was observed at up to five regions along the collagen D-period; the greatest decrease in stain uptake was apparent at the c1 band. This reduction in PTA uptake indicates that the binding of fructose leads to an alteration in the surface charge at several sites along the D-period. Not all lysine and arginine residues are involved; there appear to be specific residues that suffer a loss of positive charge.     

Effects of several acetylated chitooligosaccharides on antioxidation,antiglycation and NO generation in erythrocyte

Three kinds of chitooligosaccharides (COS) with different degrees of deacetylation were prepared and named MD90, MD70 and MD50, respectively. Antioxidation, antiglycation and nitric oxide (NO) promotion in erythrocyte of these samples were investigated. The results showed that COS, especially MD90 had obviously inhibitory effects on oxidation and glycation. In addition, MD90 displayed stronger effect on increasing endogenous NO content than both MD70 and MD50, whose degrees of deacetylation were lower. The results indicated that amino group in COS has a certain effect on the activities of COS. As COS have a conformed activity to treat diabetes, the results of this study may be meaningful for further understanding the mechanism of the action.     

Preventive aspirin treatment of streptozotocin induced diabetes: blockage of oxidative status and revertion of heme enzymes inhibition

Some late complications of diabetes are associated with alterations in the structure and function of proteins due to glycation and free radicals generation. Aspirin inhibits protein glycation by acetylation of free amino groups. In the diabetic status, it was demonstrated that several enzymes of heme pathway were diminished. The aim of this work has been to investigate the in vivo effect of short and long term treatment with acetylsalicylic acid in streptozotocin induced diabetic mice. In both treatments, the acetylsalicylic acid prevented delta-aminolevulinic dehydratase and porphobilinogen deaminase inactivation in diabetic mice and blocked the accumulation of lipoperoxidative aldehydes. Catalase activity was significantly augmented in diabetic mice and the long term treatment with aspirin partially reverted it. We propose that oxidative stress might play an important role in streptozotocin induced diabetes. Our results suggest that aspirin can prevent some of the late complications of diabetes, lowering glucose concentration and probably inhibiting glycation by acetylation of protein amino groups.     

Chemical modification and amino terminal sequence of calotropin DI from Calotropis gigantea

The effect of chemical modification of histidine, lysine, arginine, tryptophan and methionine residues on the enzymatic activity of calotropin DI has been studied. 1,3-Dibromoacetone inhibited the enzyme completely, indicating that a single histidine residue and a cysteine residue are involved in its catalytic activity. Its second bistidine residue was modified with diethyl pyrocarbonate without loss of activity. Modification of seven of its 13 lysine residues with 2,4,6-trinitrobenzene sulphonic acid led to 90% loss of its activity, but no single lysine residue appears to be essential for its activity. Four of the 12 arginine residues by 1,2-cyclohexanedione can be modified with little loss of activity. Modification of a single tryptophan residue and two methionine residues did not inhibit enzymatic activity. The blocked amino-terminal amino acid residue of calotropin DI has been identified as pyroglutamic acid. Its amino-terminal amino acid sequence to residue 14 has been determined and compared with that of papain. They show an extensive homology in their amino-terminal amino acid sequences.     

Design and synthesis of amino acids-conjugated heterocycle derived ureas/thioureas as potent inhibitors of protein glycation

Protein glycation is believed to play an important role in the development of long-term disorders associated with diabetic complications. In view of the wide occurrence of advanced glycation end products (AGE’s) and the oxidative stress derived from them in a variety of diabetic complications, it would be of great interest to identify and develop AGE inhibitors. In this study, synthesis and in vitro antiglycation activity of a small library of forty urea/thiourea derivatives of Phe/Tyr/Glu/Lys-benzisoxazole hybrids are reported. Structures of the compounds were confirmed by IR, NMR, mass spectrometry, and elemental analysis. Most of the title compounds exhibited promising activity. Best antiglycation activity was found for Tyr analogue with methoxy group as a substituent particularly at the para position with IC₅₀ value of 1.9 μM against the positive control, Rutin, with IC₅₀ = 41.9 μM. Thus, the title compounds represent novel class of potent antiglycating agents.     

Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of αA- and αB-crystallins

Earlier studies have shown significant loss of chaperone activity in α-crystallin from diabetic lenses. In vitro glycation studies have suggested that glycation of α-crystallin could be the major cause of chaperone activity loss. The following lysine (K) residues in α-crystallin have been identified as the major glycation sites: K11, K78, and K166 in αA-crystallin and K90, K92, and K166 in αB-crystallin. The present study was aimed to assess the contribution of each of the above glycation site in the overall glycation and loss of chaperone activity by mutating them to threonine followed by in vitro glycation with fructose. Level of glycated protein (GP) was determined by phenylboronate affinity chromatography, advanced glycation end products (AGEs) by direct ELISA using anti-AGE polyclonal antibody, and chaperone activity by using alcohol dehydrogenase as the target protein. K11T, K78, and K166T mutants of αA showed 33, 17, and 27% decrease in GP and 32, 18, and 21% decrease in AGEs, respectively, as compared to αA-wt. Likewise, K90T, K92T, K90T/K92T, and K166T mutants of αB showed 18, 21, 29, and 12% decrease in GP and 22, 24, 32, and 16% decrease in AGEs, respectively. Chaperone activity also showed concomitant increase with decreasing glycation and AGEs formation. αA-K11T and αB-K90T/K92T mutants showed the largest decrease in glycation and increase in chaperone activity.     

Ellagic acid, a new antiglycating agent: its inhibition of Nϵ-(carboxymethyl)lysine

Non-enzymatic glycation is a complex series of reactions between reducing sugars and amino groups of proteins. Accumulation of AGEs (advanced glycation end-products) due to non-enzymatic glycation has been related to several diseases associated with aging and diabetes. The formation of AGEs is accelerated in hyperglycaemic conditions, which alters the structure and function of long-lived proteins, thereby contributing to long-term diabetic complications. The present study describes AGE inhibition and the mechanism of action of a new antiglycating agent, EA (ellagic acid), a flavonoid present in many dietary sources. Inhibition of AGE formation by EA was demonstrated with different proteins, namely eye lens TSP (total soluble protein), Hb (haemoglobin), lysozyme and BSA, using different glycating agents such as fructose, ribose and methylglyoxal by a set of complementary methods. These results suggest that the antiglycating action of EA seems to involve, apart from inhibition of a few fluorescent AGEs, predominantly inhibition of CEL [N?-(carboxyethyl)lysine] through scavenging of the dicarbonyl compounds. Furthermore, MALDI-TOF-MS (matrix-assisted laser-desorption ionisation-time-of-flight MS) analysis confirms inhibition of the formation of CEL on lysozyme on in vitro glycation by EA. Prevention of glycation-mediated β-sheet formation in Hb and lysozyme by EA confirm its antiglycating ability. Inhibition of glycosylated Hb formation in human blood under ex vivo high-glucose conditions signifies the physiological antiglycating potential of EA. We have also determined the effectiveness of EA against loss of eye lens transparency through inhibition of AGEs in the lens organ culture system. These findings establish the antiglycating potential of EA and its in vivo utility in controlling AGE-mediated diabetic pathologies.     

Chemical modifications of ribonuclease U1.

In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.     

The presence of essential histidine residues in manganese(III)-containing acid phosphatase from sweet potato

Chemical modification studies of manganese(III)-containing acid phosphatase [EC 3.1.3.2] were carried out to investigate the contributions of specific amino-acid side-chains to the catalytic activity. Incubation of the enzyme with N-ethylmaleimide at pH 7.0 caused a significant loss of the enzyme activity. The inactivation followed pseudo-first-order kinetics. Double log plots of pseudo-first-order rate constant vs. concentration gave a straight line with a slope of 1.02, suggesting that the reaction of one molecule of reagent per active site is associated with activity loss. The enzyme was protected from inactivation by the presence of molybdate or phosphate ions. Amino acid analyses of the N-ethylmaleimide-modified enzyme showed that the 96%-inactivated enzyme had lost about one histidine and one-half lysine residue per enzyme subunit without any significant decrease in other amino acids, and also demonstrated that loss of catalytic activity occurred in parallel with the loss of histidine residue rather than that of lysine residue. Molybdate ions also protected the enzyme against modification of the histidine residue. The enzyme was inactivated by photooxidation mediated by methylene blue according to pseudo-first-order kinetics. The pH profile of the inactivation rates of the enzyme showed that an amino acid residue having a pKa value of approximately 7.2 was involved in the inactivation. These studies indicate that at least one histidine residue per enzyme subunit participates in the catalytic function of Mn(III)-acid phosphatase.     

Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose

Summary. We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N^ε-(carboxymethyl)lysine; CML) were analyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N^ε-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.     

Mass spectrometric determination of early and advanced glycation in biology

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N^ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.