Analysis of glycated serum proteins in type 2 diabetes patients with nephropathy

The aim of this study was to screen for proteins that are susceptible to glycation under hyperglycemic conditions in patients with type 2 diabetic nephropathy. Serum proteins were analyzed by a proteomic approach using two-dimensional electrophoresis (2-DE) and ESI-Q-TOF MS/MS. Gels were stained with Pro-Q Emerald 488 to analyze the serum glycoproteome, followed by silver nitrate to examine the total serum proteome. Patient sera were divided into four groups according to their microalbuminuria index: type 2 diabetics with normoalbuminuria, microalbuminuria, and overt nephropathy, and healthy subjects. When the HbA1c levels of the diabetic groups were examined, groups with higher HbA1c exhibited higher fructosamine levels, suggesting that the loss of glycemic control affected the glycation of serum proteins. The proteins that became glycated under poor glycemic control were PEDF, apolipoprotein J precursor, hemopexin, immunoglobulin mu heavy chain, and immunoglobulin kappa chain. As albuminuria increased, a marker of kidney damage, the levels of glycated prekallikrein and complement factor C4B3 also increased. The glycated proteins identified in this study may provide the foundation for the development of novel markers of diabetes, hyperglycemia, and diabetic complications.     

Comprehensive identification of glycated peptides and their glycation motifs in plasma and erythrocytes of control and diabetic subjects

Nonenzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semiquantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies.     

Early glycation products of endothelial plasma membrane proteins in experimental diabetes

The participation of glucose and two intermediates of glucose metabolism: glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate (Gald3P) to the formation of early glycation products was comparatively evaluated in the endothelial plasma membrane of streptozotocin-induced diabetic rats. Antibodies risen to a carrier protein reductively glycated by each of the sugars mentioned above were used to probe by immunoblotting the proteins of the lung microvascular endothelium plasmalemma purified from normal and diabetic rats. The amount of glycated endothelial plasma membrane proteins was below the limit of detection in normoglycemic animals but increased dramatically in diabetic animals for glucose and G6P. In contrast, no signal was found in diabetic rats for Gald3P, indicating that either the contribution of this phosphotriose to the glycation of intracellular proteins is negligible in vivo, or the Schiff base generated by this sugar transforms very rapidly into products of advanced glycation. Globally, the endothelial plasma membrane proteins bound on average 300 times more glucose than G6P proving that, in spite of its low in vitro potency as glycating agent, glucose represents the main contributor to the intracellular formation of early glycation products. The most abundant glycated proteins of the lung endothelial plasma membrane were separated by two dimensional electrophoresis and identified by mass spectrometry.     

Proteomic profiling of nonenzymatically glycated proteins in human plasma and erythrocyte membranes

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this work, we report the first proteomics-based characterization of nonenzymatically glycated proteins in human plasma and erythrocyte membranes from individuals with normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes mellitus. Phenylboronate affinity chromatography was used to enrich glycated proteins and glycated tryptic peptides from both human plasma and erythrocyte membranes. The enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation-tandem mass spectrometry, resulting in the confident identification of 76 and 31 proteins from human plasma and erythrocyte membranes, respectively. Although most of the glycated proteins could be identified in samples from individuals with normal glucose tolerance, slightly higher numbers of glycated proteins and more glycation sites were identified in samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

Low plasma albumin levels are associated with increased plasma protein glycation and HbA1c in diabetes

Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.     

Glycation of Brain Actin in Experimental Diabetes

Abstract: Actin is a neuronal protein involved in axonal transport and nerve regeneration, both of which are known to be impaired in experimental diabetes. To determine if actin is subject to glycation, we rendered rats diabetic by injection of streptozotocin. Two or 6 weeks later brains were removed and a preparation of cytoskeletal proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Brains from diabetic animals contained an extra polypeptide that migrated close to actin and reacted with monoclonal antibody C4 against actin. It was also found in a preparation of soluble synaptic proteins from diabetic rat brain, indicating that it was at least partly neuronal in origin. This polypeptide could be produced by incubation of cytoskeletal proteins from brains of nondiabetic rats with glucose-6-phosphate in vitro. The appearance of this glycated actin in diabetic animals was prevented by administration of insulin for a period of 6 weeks. We could not detect any effect of glycation in vitro on the ability of muscle G-actin to form F-actin filaments and its significance for the function of actin remains to be determined. The finding that glycation of platelet-derived actin from diabetic patients was significantly increased implies that the abnormality may also occur in clinical diabetes.     

Fisetin lowers methylglyoxal dependent protein glycation and limits the complications of diabetes

The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.     

Back-regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy

Diabetic nephropathy (DN) is a major cause of morbidity and mortality in diabetic patients. To prevent the development of this disease and to improve advanced kidney injury, effective therapies directed toward the key molecular target are required. Grape seed proanthocyanidin extracts (GSPE) have been reported to be effective in treating DN, while little is known about the functional protein changes. In this study, we used streptozotocin (STZ) to induce diabetic rats. GSPE (250 mg/kg body weight/day) were administrated to diabetic rats for 24 weeks. Serum glucose, glycated hemoglobin, and advanced glycation end products were determined. Consequently, 2-D difference gel electrophoresis and mass spectrometry were used to investigate kidney protein profiles among the control, untreated and GSPE treated diabetic rats. Twenty-five proteins were found either up-regulated or down-regulated in the kidneys of untreated diabetic rats. Only nine proteins in the kidneys of diabetic rats were found to be back-regulated to normal levels after GSPE therapy. These back-regulated proteins are involved in oxidative stress, glycosylation damage, and amino acids metabolism. Our findings might help to better understanding of the mechanism of DN, and provide novel targets for estimating the effects of GSPE therapy.     

Localization of the ezrin binding epitope for advanced glycation endproducts

Glycated proteins/advanced glycation endproducts contribute to the development of diabetic complications but the precise pathway from glycated proteins to complications is still being delineated. The ezrin, radixin and moesin protein family is a new class of advanced glycation endproduct-binding protein and we hypothesize that advanced glycation endproducts mediate some of their detrimental effects leading to diabetic complications by inhibiting ezrin's actions. Our previous study revealed that glycated proteins bind to the N-terminal domain of ezrin (aa 1–324) and this study further defines the ezrin binding epitope. Binding of glycated albumin to recombinant N-ezrin deletion constructs (aa 1–280, 1–170 and 1–144) and glutathione-S-transferase-N-ezrin fusion proteins, (aa 200–324 and 270–324) was analysed using ligand and far Western blotting, and surface plasmon resonance. Glycated albumin binding was markedly reduced on removal of amino acids 280–324, while binding was preserved in the fusion proteins. A series of peptides based on residues 280–324 was synthesized and those containing residues 277–299 of ezrin bound maximally. Peptide binding to glycated albumin was glycation-specific. An ezrin peptide (aa 277–299) dose-dependently reversed the inhibitory effect of glycated albumin on ezrin (1–324) phosphorylation in vitro, suggesting that binding of advanced glycation endproducts to ezrin changes the conformation of the latter sufficiently to alter binding interactions distant from the advanced glycation endproduct-binding site. This may have consequences for subcellular ezrin localization and signalling pathways. Altogether, these studies provide important structural knowledge for developing peptide antagonists that may be therapeutically useful in preventing advanced glycation endproduct:ezrin interactions in diabetes.     

Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESIMS) was used for relative quantification of glycated Cu-Zn superoxide dismutase (SOD-1) in human erythrocytes. SOD-1 samples were prepared from erythrocytes by removing hemoglobin using hemoglobind gel followed by ethanol and chloroform extraction. The reproducibility in measurement of the relative percentage of glycated protein was good, and the standard deviation of each measurement was 4.0%. From the mass spectral analysis of a mixture of commercial SOD-1 and in vitro partially glycated SOD-1 in several ratios, it was found that free and glycated SOD-1 have the same ionization efficiencies. The percentage of glycation on SOD-1 was measured in 30 individuals, including patients with diabetes mellitus. The glycation levels ranged from 4.5% to below the detection limit. The SOD-1 sample extracted from erythrocytes was fractionated by Glyco-Gel B chromatography, and the separated fractions were analyzed by MS. The mass spectra of absorbed fraction showed significant amounts of non-specific binding of non-glycated proteins to Glyco-Gel B.     

Does enhanced expression of the Na+-CA2+ exchanger increase myocardial vulnerability to ischemia/reperfusion injury in rabbit hearts?

The present study focused on examining the efficacy of feeding a rutin-glucose derivative (G-rutin) to inhibit glycation reactions that can occur in muscle, kidney and plasma proteins of diabetic rats. Both thiobarbituric acid-reactive substance levels and protein carbonyl contents in muscle and kidney were significantly (p < 0.05) reduced in streptozotocin-induced diabetic rats fed G-rutin supplemented diet, compared to diabetic rats fed control diet. The N -fructoselysine content in muscle and kidney, a biomarker of early glycation reaction, was markedly (p < 0.05) increased by diabetes, but significantly (p < 0.05) reduced in diabetic rats fed G-rutin. Advanced glycation end-products (AGEs) in serum and kidney protein were measured by immunoblot using anti-AGE antibody, and were also reduced in diabetic rats fed dietary G-rutin. Feeding G-rutin also slightly inhibited aldose reductase activity in these animals. These results demonstrate for the first time that dietary G-rutin consumption can provide potential health benefits that are related to the inhibition of tissue glycation reactions common to diabetes.     

Identification of preferential protein targets for carbonylation in human mature adipocytes treated with native or glycated albumin

Oxidative modifications in proteins can participate in the regulation of cellular functions and are frequently observed in numerous states of diseases. Albumin can undergo increased glycation during diabetes. An accumulation of oxidatively modified proteins in human mature adipocytes incubated with glycated albumin has previously been described. This study herein reports the identification of specifically carbonylated targets following separation of the cell proteins by 2D gels, Western blotting and mass spectrometry analyses. It identified eight oxidatively modified proteins, two of which (ACTB and Annexin A2) appeared as significantly more carbonylated in adipocytes treated with glycated albumin than with native albumin. Intracellular stress, evaluated in SW872 cell line, showed an impairment in the protective antioxidant action exerted by native BSA after the glycation of the protein. Decreased proteasome peptidase activities were found in glycated BSA-treated mature adipocytes. The data suggest an association of oxidative damage with the progression of diabetes disorders at the adipocytes level.     

Non-enzymatic glycation of proteins: From diabetes to cancer

Incubation of proteins with glucose leads to their non-enzymatic glycation and formation of Amadori products known as an early glycation product. Oxidative cleavage of Amadori products is considered as a major route to advanced glycation endproducts (AGEs) formation in vivo. Non-enzymatic glycation of proteins or Maillard reaction is increased in diabetes mellitus due to hyperglycemia and leads to several complications such as blindness, heart disease, nerve damage, and kidney failure. The early and advanced glycation products are accumulated in plasma and tissues of diabetic patients and cause production of autoantibodies against corresponding products. The advanced glycation products are also associated with other diseases like cancer. This review summarizes current knowledge of these stage specific glycated products as common and early diagnostic biomarkers for the associated diseases and the complications with the aim of a novel therapeutic target for the diseases.     

Identification of preferential protein targets for carbonylation in human mature adipocytes treated with native or glycated albumin

Oxidative modifications in proteins can participate in the regulation of cellular functions and are frequently observed in numerous states of diseases. Albumin can undergo increased glycation during diabetes. An accumulation of oxidatively modified proteins in human mature adipocytes incubated with glycated albumin has previously been described. This study herein reports the identification of specifically carbonylated targets following separation of the cell proteins by 2D gels, Western blotting and mass spectrometry analyses. It identified eight oxidatively modified proteins, two of which (ACTB and Annexin A2) appeared as significantly more carbonylated in adipocytes treated with glycated albumin than with native albumin. Intracellular stress, evaluated in SW872 cell line, showed an impairment in the protective antioxidant action exerted by native BSA after the glycation of the protein. Decreased proteasome peptidase activities were found in glycated BSA-treated mature adipocytes. The data suggest an association of oxidative damage with the progression of diabetes disorders at the adipocytes level.     

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.     

Chronic hyperglycemia in experimental diabetes mellitus of short duration does not contribute to muscle capillary basement membrane thickening

The effect of chronic hyperglycemia on the relationship of nonenzymatic glycation and capillary basement membrane thickness in muscle was studied in streptozotocin-induced diabetic rats early in the course of diabetes mellitus. Diabetic animals were placed on either standard (24%) or restricted (8%) protein diet. The animals on 8% protein diet had elevated glycated hemoglobin levels (p less than 0.01) and increased levels of nonenzymatic glycation of basement membrane (p less than 0.01) as compared to insulin-treated diabetic (euglycemic), age-matched control, and streptozotocin-injected nondiabetic animals also on 8% protein diet. In contrast, diabetic animals on restricted (8%) protein diet and those on standard (24%) protein diet showed no statistical differences between them with regards to the above parameters. Moreover, there were no statistical differences among diabetic and control animals on either 8 or 24% protein diet with respect to muscle capillary membrane thickness. Even though the peripheral muscle biopsy study of capillary basement membrane is less invasive than kidney biopsy, the results of this study suggest that neither nonenzymatic glycation nor basement membrane thickness can be utilized as predictors of renal dysfunction during early onset of diabetes mellitus.     

Yeast protein glycation in vivo by methylglyoxal. Molecular modification of glycolytic enzymes and heat shock proteins

Protein glycation by methylglyoxal is a nonenzymatic post-translational modification whereby arginine and lysine side chains form a chemically heterogeneous group of advanced glycation end-products. Methylglyoxal-derived advanced glycation end-products are involved in pathologies such as diabetes and neurodegenerative diseases of the amyloid type. As methylglyoxal is produced nonenzymatically from dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate during glycolysis, its formation occurs in all living cells. Understanding methylglyoxal glycation in model systems will provide important clues regarding glycation prevention in higher organisms in the context of widespread human diseases. Using Saccharomyces cerevisiae cells with different glycation phenotypes and MALDI-TOF peptide mass fingerprints, we identified enolase 2 as the primary methylglyoxal glycation target in yeast. Two other glycolytic enzymes are also glycated, aldolase and phosphoglycerate mutase. Despite enolase's activity loss, in a glycation-dependent way, glycolytic flux and glycerol production remained unchanged. None of these enzymes has any effect on glycolytic flux, as evaluated by sensitivity analysis, showing that yeast glycolysis is a very robust metabolic pathway. Three heat shock proteins are also glycated, Hsp71/72 and Hsp26. For all glycated proteins, the nature and molecular location of some advanced glycation end-products were determined by MALDI-TOF. Yeast cells experienced selective pressure towards efficient use of d-glucose, with high methylglyoxal formation as a side effect. Glycation is a fact of life for these cells, and some glycolytic enzymes could be deployed to contain methylglyoxal that evades its enzymatic catabolism. Heat shock proteins may be involved in proteolytic processing (Hsp71/72) or protein salvaging (Hsp26).     

Effects of oxidative modifications induced by the glycation of bovine serum albumin on its structure and on cultured adipose cells

Non-enzymatic glycosylation (glycation) and oxidative damages represent major research areas insofar as such modifications of proteins are frequently observed in numerous states of disease. Albumin undergoes structural and functional alterations, caused by increased glycosylation during non insulin-dependent diabetes mellitus, which is closely linked with the early occurrence of vascular complications. In this work, we first characterized structural modifications induced by the glycation of bovine serum albumin (BSA). A pathophysiological effect of glycated BSA was identified in primary cultures of human adipocytes as it induces an accumulation of oxidatively modified proteins in these cells. BSA was incubated in the presence or absence of physiological, pathological or supra-physiological concentrations of glucose at 37 degrees C for 7 weeks. Enhanced BSA glycation percentages were determined using boronate affinity columns. The occurrence of oxidative modifications was found to be enhanced in glycated BSA, after determination of the free thiol groups content, electrophoretic migration and infrared spectrometry spectra. An accumulation of carbonyl-modified proteins and an increased release of isoprostane were observed in cell media following the exposure of adipocytes to glycated albumin. These results provide a new possible mechanism for enhanced oxidative damages in diabetes.     

Reduced alkaline phosphatase activity in diabetic rat bone: a re-evaluation

We found previously that human bone alkaline phosphatase (AP) was glycated by aseptic incubation with glucose, and partially broken down by reactive oxygen species. In this study, we examined whether selective in vivo glycation of AP molecules occurred in bone tissue, using experimental diabetic rats induced by streptozotocin and spontaneously diabetic rats. Additionally, the effects of hyperlipidemia on bone AP activity were examined. Serum AP activity was significantly elevated after incipient onset of diabetes, and the increased activity originated from the intestinal isozyme. High levels of intestinal AP activity were also observed in rats with hyperlipidemia induced by feeding high-fat or high-fructose chow, but the AP activity in bone tissues was maintained at a constant level. AP activity in bone was reduced after the onset of diabetes. The resulting bone AP molecule bound to an aminophenylboronic acid column, which had affinity for glycated proteins, and contained smaller molecular sizes than the native bone AP. These results suggest that elevated levels of serum AP activity originated from the intestinal isozyme accompanied with hyperlipidemia induced by diabetes. In contrast, the reduced serum levels of AP activity in diabetic rats might be dependent on inactivation of bone AP, which was glycated, followed by partial breakdown of bone AP molecules, possibly due to reactive oxygen species.