Non-enzymatic Glycation Of Proteins Research Articles

Rutin metabolites: Novel inhibitors of nonoxidative advanced glycation end products

Glycation is a nonenzymatic condensation reaction between reducing sugars and amino groups of proteins that undergo rearrangements to stable ketoamines, leading to the formation of advanced glycation end products (AGEs) including fluorescent (argpyrimidine) and nonfluorescent ( N ε-carboxymethyllysine; CML) protein adducts and protein cross-links. AGEs are formed via protein glycation and correlate with processes resulting in aging and diabetes complications. Reactive carbonyl species such as glyoxal and methylglyoxal are ubiquitous by-products of cell metabolism that potently induce the formation of AGEs by nonenzymatic protein glycation and may achieve plasma concentrations of 0.3–1.5 μmol/L. In this in vitro study histone H1 glycation by glyoxal, methylglyoxal, or ADP-ribose was used to model nonoxidative protein glycation, permitting us to distinguish specific AGE inhibition from general antioxidant action. Rutin derivatives were tested as AGE inhibitors because rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, is metabolized by gut microflora to a range of phenolic compounds that are devoid of significant antioxidant activity and achieve blood concentrations in the μmol/L range. Our data show that in a 1:1 stoichiometry with glyoxal or methylglyoxal, 3,4-dihydroxyphenylacetic acid (DHPAA) and 3,4-dihydroxytoluene (DHT) are powerful inhibitors of CML and argpyrimidine histone H1 adduct formation, respectively. Furthermore, when DHPAA and DHT were tested as inhibitors of histone H1 glycation by the powerful glycating agent ADP-ribose, they inhibited glycation as effectively as aminoguanidine. These results suggest that dietary flavonoids may serve as effective AGE inhibitors and suggest mechanisms whereby fruit- and vegetable-rich diets contribute to the prevention of processes resulting in aging and diabetes complications.

Hypothesis: the “metabolic memory”, the new challenge of diabetes

Large randomized studies have established that early intensive glycaemic control reduces the risk of diabetic complications, both micro and macrovascular. However, epidemiological and prospective data support a long-term influence of early metabolic control on clinical outcomes. This phenomenon has recently been defined as “metabolic memory”. Potential mechanisms for propagating this “memory” are the non-enzymatic glycation of cellular proteins and lipids, and an excess of cellular reactive oxygen and nitrogen species, in par ticular originated at the level of glycated-mitochondrial proteins, perhaps acting in concert with one another to maintain stress signaling. Furthermore, the emergence of this “metabolic memory” suggests the need of a very early aggressive treatment aiming to “nor malize” the metabolic control and the addition of agents which reduce cellular reactive species and glycation in addition to normalizing glucose levels in diabetic patients in order to minimize long-term diabetic complications.

Non-enzymatic glycation of proteins: From diabetes to cancer

Non-enzymatic glycation of proteins: From diabetes to cancer

Covalent Binding Antibodies Suppress Advanced glycation: On the Innate Tier of Adaptive Immunity

Non-enzymatic protein glycation is a source of metabolic stress that contributes to cytotoxicity and tissue damage. Hyperglycemia has been linked to elevation of advanced glycation endproducts, which mediate much of the vascular pathology leading to diabetic complications. Enhanced glycation of immunoglobulins and their accelerated vascular clearance is proposed as a natural mechanism to intercept alternative advanced glycation endproducts, thereby mitigating microvascular disease. We reported that antibodies against the glycoprotein KLH have elevated reactivity for glycopeptides from diabetic serum. These reactions are mediated by covalent binding between antibody light chains and carbonyl groups of glycated peptides. Diabetic animals that were immunized to induce reactive antibodies had attenuated diabetic nephropathy, which correlated with reduced levels of circulating and kidney-bound glycation products. Molecular analysis of antibody glycation revealed the preferential modification of light chains bearing germline-encoded lambda V regions. We previously noted that antibody fragments carrying V regions in the germline configuration are selected from a human Fv library by covalent binding to a reactive organophosphorus ester. These Fv fragments were specifically modified at light chain V region residues, which map to the combining site at the interface between light and heavy chains. These findings suggest that covalent binding is an innate property of antibodies, which may be encoded in the genome for specific physiological purposes. This hypothesis is discussed in context with current knowledge of the natural antibodies that recognize altered self molecules and the catalytic autoantibodies found in autoimmune disease.

Increased retinopathy occurrence in type 1 diabetes patients with increased serum levels of the advanced glycation endproduct hydroimidazolone

We aimed to investigate associations between serum levels of the advanced glycation endproduct methylglyoxal-derived hydroimidazolone (MG-H1) and retinopathy in a sample of patients with type 1 diabetes. We conducted a cross-sectional study in a Scandinavian ophthalmology outpatient clinic on 61 randomly selected patients with type 1 diabetes. Blood samples and retinal photographs were taken at the same visit. Serum levels of hydroimidazolone immunoreactivity were determined using an immunoassay, and levels of retinopathy were determined from seven standard field stereo photographs of each eye according to the ETDRS method. Results were compared between patients with and without retinopathy. Hydroimidazolone quartiles were significantly associated with retinopathy (p = 0.013). The most profound increase in occurrence of retinopathy was observed from the lowest to the second-lowest hydroimidazolone quartile. Adjusted for duration of diabetes using logistic regression, a significant difference in the presence of retinopathy was found when comparing the lowest quartile with the rest (p = 0.022). In our patients with type 1 diabetes, serum levels of hydroimidazolone were found to be associated with retinopathy. This is in keeping with findings in a larger sample of patients with type 2 diabetes.

The nonenzymatic glycation of proteins and nucleic acids, their importance for the development of diabetic complications, possible molecular basis of aging and autoimmunological processes.

The formation of nonenzymatic glycation products of proteins and nucleic acids appears to be a link between chronic hyperglycaemia and long-term diabetic complications and special forms of aging during normoglycaemia. The major effects of extended glycation include cross-linking of glycated proteins, attachment of soluble proteins to extracellular glycated matrices, conformational changes of proteins followed by altered functions and immunogenicity, and abnormalities in nucleic acid functions.

Diminished adhesion of endothelial aortic cells on fibronectin and collagen layers after nonenzymatic glycation.

Adhesion of bovine endothelial cells on fibronectin and collagen before and after nonenzymatic glycation in vitro has been studied. Nonenzymatic glycation of these proteins reduced their ability to bind endothelial cells. Furthermore, nonenzymatically glycated fibronectin failed to bind to normal and nonenzymatically glycated gelatin and to fibrin. So gelatin and fibrin Sepharoses can be used to separate highly glycated fibronectins from fibronectins with a low degree of nonenzymatic glucose substitution. Sodium dodecylsulfate polyacrylamide gel electrophoresis did not demonstrate a covalent cross-link between nonenzymatically glycated fibronectins. These results present further evidences for the role of nonenzymatic glycation of proteins in the development of vascular complications in long-term diabetes and of atherosclerosis.

Diabetic microvascular complications and growth factors.

Diabetes mellitus is associated with typical patterns of long term vascular complications which vary with the organ involved. The microvascular kidney disease (Olgemoller and Schleicher, 1993) is characterized by thickening of the capillary basement membranes and increased deposition of extracellular matrix components (ECM), while loss of microvessels with subsequent neovascularisation is predominant in the eye and peripheral nerves. On the other hand macrovascular disease is characterized by accelerated atherosclerosis. These complications are dependent on long term hyperglycemia. Specific biochemical pathways linking hyperglycaemia to microvascular changes were proposed: the polyol pathway (Greene et al., 1987), non-enzymatic glycation of proteins (Brownlee et al., 1988), glucose autooxidation and oxidative stress (Hunt et al., 1990), hyperglycemic pseudohypoxia (Williamson et al., 1993) enhanced activation of protein kinase C by de novo-synthesis of diacyl glycerol (Lee et al., 1989; DeRubertis and Craven 1994) and others. These pathways are not mutually exclusive (Larkins and Dunlop, 1992; Pfeiffer and Schatz, 1992). They may be linked to alterations in the synthesis of growth factors particularly since atherosclerosis and angioneogenesis are associated with increased proliferation of endothelial and smooth muscle cells. Increased synthesis of ECM components is stimulated by growth factors like transforming growth factor beta (TGF beta) (Derynck et al., 1984) and insulin-like growth factor I (IGF-I) (Moran et al., 1991). This review will summarize some of the recent evidence for an involvement of growth factors in diabetic vascular complications and will attempt to assign their emergence in the sequence of events leading to vascular complications.

Screening for glucose‐triggered modifications of glutathione

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. These structures may undergo further Amadori rearrangement and free radical-mediated oxidation to finally generate irreversible advanced glycation end products (AGEs). One of the factors known to modulate the glycation of proteins is glutathione, the most abundant nonprotein thiol tripeptide with the gamma-linkage, H-Glu(Cys-Gly-OH)-OH (GSH). Screening for products formed by GSH with D-glucose is an essential step in understanding the participation of GSH in glycation (the Maillard) reaction. Under the conditions used in these studies we observed N-(1-deoxy-D-fructos-1-yl)-pyroglutamic acid as the major glycation product formed in the mixtures of GSH and glucose in vitro. A RP HPLC/MS and tandem MS analyses of the GSH/glucose mixtures revealed that cleavage of the N-terminal glutamic acid and the formation of pyroglutamic acid-related Amadori product were accompanied by generation of Cys-Gly-derived Amadori and thiazolidine compounds.

Enrichment of Amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography

Amadori peptides were enriched using boronate affinity tips and measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF/MS). As demonstrated by electrochemical measurements, the tips show the highest binding efficiency for glucose at pH 8.2 employing ammonium chloride/ammonia buffer with ionic strength of 150 mM, exceeding taurine buffer at the same concentration. The bound constituents were released by sorbitol and formic acid. It was also demonstrated that elution with sorbitol at 1.2 M is superior to acidic media. Comparison of results was based on the numbers of detected peptides and their glycated sites. Using sorbitol for elution requires desalting prior to analysis. Therefore, three different sorbents were tested: fullerene-derivatized silica, ZipTip (C18), and C18 silica. Fullerene-derivatized silica and ZipTip showed the same performance regarding the numbers of glycated peptides, and sites were better than C18 silica. The elaborated off-line method was compared with liquid chromatography–tandem mass spectrometry (LC–MS/MS) measurements, by which considerable less modified peptides were detected. Affinity tips used under optimized conditions were tested for the analysis of human serum albumin (HSA) from sera of healthy and diabetic individuals. A peptide with a mass of 1783.9 Da could be detected only in samples of diabetic patients and, therefore, could be a very interesting biomarker candidate.

The receptor for advanced glycation endproducts (RAGE) and cardiovascular disease

Recent and compelling investigation has expanded our view of the biological settings in which the products of nonenzymatic glycation and oxidation of proteins and lipids - the advanced glycation endproducts (AGEs) - form and accumulate. Beyond diabetes, natural ageing and renal failure, AGEs form in inflammation, oxidative stress and in ischaemia-reperfusion. The chief signal transduction receptor for AGEs - the receptor for AGEs (RAGE) - is a multiligand-binding member of the immunoglobulin superfamily. In addition to AGEs, RAGE binds certain members of the S100/calgranulin family, high-mobility group box 1 (HMGB1), and beta-amyloid peptide and beta-sheet fibrils. Recent studies demonstrate beneficial effects of RAGE antagonism and genetic deletion in rodent models of atherosclerosis and ischaemia-reperfusion injury in the heart and great vessels. Experimental evidence is accruing that RAGE ligand generation and release during ischaemia-reperfusion may signal through RAGE, thus suggesting that antagonism of this receptor might provide a novel form of therapeutic intervention in heart disease. However, it is plausible that innate, tissue-regenerative roles for these RAGE ligands may also impact the failing heart - perhaps through RAGE and/or distinct receptors. In this review, we focus on RAGE and the consequences of its activation in the cardiovasculature.

The “Metabolic Memory”: Is More Than Just Tight Glucose Control Necessary to Prevent Diabetic Complications?

The concept of a "metabolic memory," that is of diabetic vascular stresses persisting after glucose normalization, has been supported both in the laboratory and in the clinic and in both type 1 and type 2 diabetes. Using PubMed, we searched for publications on diabetic micro- and macrovascular complications using terms such as persistence, prolongation, sustained, and "memory" and focusing on the mechanistic basis behind this metabolic memory. We found that as early as the mid-1980s this memory phenomenon was described in diabetic animals and isolated cells exposed to high glucose followed by normalized glucose and then, beginning around 2002, in results from large clinical trials such as the Diabetes Complications and Control Trial-Epidemiology of Diabetes Interventions and Complications and the United Kingdom Prospective Diabetes Study. Furthermore, mechanisms for propagating this memory appear focused on the nonenzymatic glycation of cellular proteins and lipids and on an excess of cellular reactive oxygen and nitrogen species, in particular originating at the level of glycated mitochondrial proteins and perhaps acting in concert with one another to maintain stress signaling independent of glucose levels. The emergence of this metabolic memory suggests the need for early aggressive treatment aiming to "normalize" metabolic control together perhaps with the addition of agents which reduce cellular reactive species and glycation in order to minimize long-term diabetic complications.

Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin

Escherichia coli ( E. coli) that expresses galactose-reactive lectins, like K88 adhesin, causes high mortality among piglets. Carbohydrates that compete for adhesion could serve as an alternative for disease prevention. Porcine serum albumin (PSA) was modified by non-enzymatic glycation with lactose to produce PSA-Lac or PSA-Glc β (1–4) Gal, as confirmed by reduction of available free amino groups, increased molecular mass and by Ricinus communis lectin recognition. E. coli K88 binds to PSA-Lac treatments containing three and four lactoses, respectively. In addition, PSA-Lac partially inhibited K88 strain adherence to mucins. These results suggest that neoglycoconjugates obtained by non-enzymatic glycation of proteins may serve in the prophylaxis of piglets’ diarrhea.

264: In severe preeclampsia, activation of the receptor for advanced glycation end-products (RAGE) system occurs independent of antigenic N-carboxymethyl-lysine modified proteins

Severe preeclampsia (sPE) is characterized by RAGE system activation, which leads to inflammation, oxidative stress and endothelial injury. The putative stimulus remains unknown. One candidate is the antigenic N-carboxymethyl-lysine (CML), an advanced glycation end-product (AGE) formed by the nonenzymatic glycation of proteins. This study was conducted to determine if CML and CML auto-antibodies may be responsible for activation of the RAGE system in women with sPE.

Improved Methods for the Enrichment and Analysis of Glycated Peptides

Nonenzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. Herein we report improved methods for the enrichment and analysis of glycated peptides using boronate affinity chromatography and electron-transfer dissociation mass spectrometry, respectively. The enrichment of glycated peptides was improved by replacing an off-line desalting step with an online wash of column-bound glycated peptides using 50 mM ammonium acetate, followed by elution with 100 mM acetic acid. The analysis of glycated peptides by MS/MS was improved by considering only higher charged (> or = 3) precursor ions during data-dependent acquisition, which increased the number of glycated peptide identifications. Similarly, the use of supplemental collisional activation after electron transfer (ETcaD) resulted in more glycated peptide identifications when the MS survey scan was acquired with enhanced resolution. Acquiring ETD-MS/MS data at a normal MS survey scan rate, in conjunction with the rejection of both 1+ and 2+ precursor ions, increased the number of identified glycated peptides relative to ETcaD or the enhanced MS survey scan rate. Finally, an evaluation of trypsin, Arg-C, and Lys-C showed that tryptic digestion of glycated proteins was comparable to digestion with Lys-C and that both were better than Arg-C in terms of the number of glycated peptides and corresponding glycated proteins identified by LC-MS/MS.

ASSOCIATION BETWEEN SERUM CARBOXYMETHYL‐LYSINE, A DOMINANT ADVANCED GLYCATION END PRODUCT, AND ANEMIA IN ADULTS: THE BALTIMORE LONGITUDINAL STUDY OF AGING

To the Editor: The pathophysiology of anemia in older adults is incompletely understood, and a substantial proportion of anemia in this population remains unexplained.1 The factors that play a role in anemia in adults are incompletely identified. Advanced glycation end products (AGEs) are a heterogeneous group of bioactive molecules formed by the nonenzymatic glycation of proteins, lipids, and nucleic acids.2 AGEs have been widely implicated in the pathogenesis of cardiovascular and renal disease, and diabetes.2,3

Non-Enzymatic Glycation of Human Fibrillin-1

Non-enzymatic glycation of proteins is one of the key mechanisms in the pathogenesis of diabetic complications and may be significant in the age-related changes of tissues. We isolated and investigated the in vitro glycation of human aortic fibrillin-1. Fibrillin-1 was prepared from thoracic aortas of 9 accident victims distributed in three age groups. The purity of isolated fibrillin-1 was proved. It was glycated by incubating with different glucose concentrations in 0.2 M phosphate buffer, pH 7.4, for 30 days, at 37°C. The degree of early products of glycation was measured by two colorimetric methods, i.e. nitroblue tetrazolium and 2-thiobarbituric acid. Advanced glycation end products (AGEs) were determined by fluorescence measurement. The highest level of early products of glycation was found on day 2 after the beginning of incubation for the fibrillin-1 isolated from the youngest group. Fluorescence in the age groups, as an index of advanced glycation, consistently increased between days 6 and 24. The fibrillin-1 isolated from the youngest group had the highest capacity to form fructosamine and AGEs under glycation in vitro. The capacity of glycation of the ‘oldest’ fibrillin did not increase significantly during the incubation. Investigation of the properties of glycated fibrillin-1 will help to understand the importance of this long-lived protein to age-related changes in tissues and for diabetic complications.

Delay of diabetic cataract in rats by the antiglycating potential of cumin through modulation of α-crystallin chaperone activity

α-Crystallin, a molecular chaperone of the eye lens, plays an important role in maintaining the transparency of the lens by preventing the aggregation/inactivation of several proteins and enzymes in addition to its structural role. α-Crystallin is a long-lived protein and is susceptible to several posttranslational modifications during aging, more so in certain clinical conditions such as diabetes. Nonenzymatic glycation of lens proteins and decline in the chaperone-like function of α-crystallin have been reported in diabetic conditions. Therefore, inhibitors of nonenzymatic protein glycation appear to be a potential target to preserve the chaperone activity of α-crystallin and to combat cataract under hyperglycemic conditions. In this study, we investigated the antiglycating potential of cumin in vitro and its ability to modulate the chaperone-like activity of α-crystallin vis-à-vis the progression of diabetic cataract in vivo. Aqueous extract of cumin was tested for its antiglycating ability against fructose-induced glycation of goat lens total soluble protein (TSP), α-crystallin from goat lens and a nonlenticular protein bovine serum albumin (BSA). The antiglycating potential of cumin was also investigated by feeding streptozotocin (STZ)-induced diabetic rats with diet containing 0.5% cumin powder. The aqueous extract of cumin prevented in vitro glycation of TSP, α-crystallin and BSA. Slit lamp examination revealed that supplementation of cumin delayed progression and maturation of STZ-induced cataract in rats. Cumin was effective in preventing glycation of TSP and α-crystallin in diabetic lens. Interestingly, feeding of cumin to diabetic rats not only prevented loss of chaperone activity but also attenuated the structural changes of α-crystallin in lens. These results indicated that cumin has antiglycating properties that may be attributed to the modulation of chaperone activity of α-crystallin, thus delaying cataract in STZ-induced diabetic rats.

Possible link of food-derived advanced glycation end products (AGEs) to the development of diabetes

China Da Qing diabetes prevention study has recently shown that group-based lifestyle interventions over six years can prevent or delay the development of diabetes in patients with impaired glucose tolerance (IGT) for up to 14 years after the active intervention. These findings suggest the sustained beneficial effects of lifestyle interventions to prevent diabetes in at-risk patients for diabetes. Therefore, the clinical study suggests that so-called 'metabolic memory' is involved in the development of diabetes. Potential mechanisms for propagating this 'metabolic memory' are the non-enzymatic glycation of proteins. The formation and accumulation of advanced glycation end products (AGEs) have been known to progress at an accelerated rate under diabetes, and there is accumulating evidence that AGEs play a role in the development of diabetes by inducing islet beta cell damage and/or insulin resistance. Further, there are several animal studies to suggest that dietary AGEs are involved in insulin resistance, visceral obesity and the development of diabetes. These findings led us to speculate that the long-term beneficial influence of early lifestyle interventions on the development of diabetes could be ascribed, at least in part, to their inhibitory effects on AGEs. That is, intake of food-derived AGEs may be suppressed in the lifestyle intervention group, which could reduce the risk for the development of diabetes in high-risk patients with IGT. Therefore, it is an interesting issue to clarify whether food-derived AGEs are actually restricted during the active intervention period and if circulating or tissue AGE levels at the closure of the China Da Qing diabetes prevention study could predict the risk for the development of diabetes 14 years after the trial. This additional, clinical investigation may provide us more information about whether restriction of food-derived AGEs is beneficial for the prevention of the development of diabetes in high-risk patients and may be a novel therapeutic target to prevent diabetes and its related complications.

Analysis of non‐enzymatically glycated peptides: neutral‐loss‐triggered MS3 versus multi‐stage activation tandem mass spectrometry

Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. While electron transfer dissociation (ETD) has been shown to outperform collision-induced dissociation (CID) in sequencing glycated peptides by tandem mass spectrometry, ETD instrumentation is not yet widely available and often suffers from significantly lower sensitivity than CID. In this study, we evaluated different advanced CID techniques (i.e., neutral-loss-triggered MS(3) and multi-stage activation) during liquid chromatography/multi-stage mass spectrometric (LC/MS(n)) analyses of Amadori-modified peptides enriched from human serum glycated in vitro. During neutral-loss-triggered MS(3) experiments, MS(3) scans triggered by neutral losses of 3 H(2)O or 3 H(2)O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3 H(2)O resulted in significantly more glycated peptide identifications during multi-stage activation experiments. Overall, the multi-stage activation approach produced more glycated peptide identifications, while the neutral-loss-triggered MS(3) approach resulted in much higher specificity. Both techniques are viable alternatives to ETD for identifying glycated peptides.