Thermal aggregation of glycated bovine serum albumin

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.     

Glycation of fibronectin inhibits VEGF‐induced angiogenesis by uncoupling VEGF receptor‐2‐c‐Src crosstalk

Glycation of extracellular matrix proteins has been demonstrated to contribute to the pathogenesis of vascular complications. However, no previous report has shown the role of glycated fibronectin (FN) in vascular endothelial growth factor (VEGF)‐induced angiogenesis. Thus, this study aimed to investigate the effects of glycated FN on VEGF signalling and to clarify the molecular mechanisms involved. FN was incubated with methylglyoxal (MGO) in vitro to synthesize glycated FN, and human umbilical vein endothelial cells (HUVECs) were seeded onto unmodified and MGO‐glycated FN. Then, VEGF‐induced angiogenesis and VEGF‐induced VEGF receptor‐2 (VEGFR‐2) signalling activation were measured. The results demonstrated that normal FN‐positive bands (260 kD) vanished and advanced glycation end products (AGEs) appeared in MGO‐glycated FN and glycated FN clearly changed to a higher molecular mass. The glycation of FN inhibited VEGF‐induced VEGF receptor‐2 (VEGFR‐2), Akt and ERK1/2 activation and VEGF‐induced cell migration, proliferation and tube formation. The glycation of FN also inhibited the recruitment of c‐Src to VEGFR‐2 by sequestering c‐Src through receptor for AGEs (RAGE) and the anti‐RAGE antibody restored VEGF‐induced VEGFR‐2, Akt and ERK1/2 phosphorylation, endothelial cell migration, proliferation and tube formation. Furthermore, the glycation of FN significantly inhibited VEGF‐induced neovascularization in the Matrigel plugs implanted into subcutaneous tissue of mice. Taken together, these data suggest that the glycation of FN may inhibit VEGF signalling and VEGF‐induced angiogenesis by uncoupling VEGFR‐2‐c‐Src interaction. This may provide a novel mechanism for the impaired angiogenesis in diabetic ischaemic diseases.     

Aspartic acid functions as carbonyl trapper to inhibit the formation of advanced glycation end products by chemical chaperone activity

Advanced glycation end products (AGEs) were implicated in pathology of numerous diseases. In this study, we present the bioactivity of aspartic acid (Asp) to inhibit the AGEs. Hemoglobin and bovine serum albumin (BSA) were glycated with glucose, fructose, and ribose in the presence and absence of Asp (100–200 μM). HbA₁c inhibition was investigated using human blood and characterized by micro-column ion exchange chromatography. The effect of methyl glyoxal (MG) on hemoglobin and BSA was evaluated by fluorescence spectroscopy and gel electrophoresis. The effect of MG on red blood cells morphology was characterized by scanning electron micrographs. Molecular docking was performed on BSA with Asp. Asp is capable of inhibiting the formation of fluorescent AGEs by reacting with the reducing sugars. The presence of Asp as supplement in whole blood reduced the HbA₁c% from 8.8 to 6.1. The presence of MG showed an increase in fluorescence and the presence of Asp inhibited the glycation thereby the fluorescence was quenched. MG also affected the electrophoretic mobility of hemoglobin and BSA by forming high molecular weight aggregates. Normal RBCs showed typical biconcave shape. MG modified RBCs showed twisted and elongated shape whereas the presence of ASP tends to protect RBC from twisting. Asp interacted with arginine residues of bovine serum albumin particularly ARG 194, ARG 198, and ARG 217 thereby stabilized the protein complex. We conclude that Asp has dual functions as a chemical chaperone to stabilize protein and as a dicarbonyl trapper, and thereby it can prevent the complications caused by glycation.     

Changes in glycation of fibrous type I collagen during long-term in vitro incubation with glucose

The course of glycation of calf skin fibrous type I collagen was monitored in vitro under physiological conditions during an 8-week incubation period in order to take into account the long half-life of this protein. The formation of glycated compounds was measured by determining fructosamine, pentosidine, and carboxymethyllysine content. The incubation conditions were as physiological as possible in sterile saline phosphate buffer, except glucose concentration. With incubation medium containing 200 mmol glucose, fibrous collagen underwent solubilization; in addition an increase in fructosamine, pentosidine, and carboxymethyllysine content in both solubilized and remaining insoluble collagen was noticed. There was a spontaneous, restricted, and time-dependent native glycated state of collagen; high concentration glucose enhanced the formation of glycated compounds and induced changes in solubility and glycoxidated products. The production of pentosidine during incubation without glucose should be considered as an event resulting from the initial fructosamine. Whereas the production of carboxymethyllysine during long-term incubation with glucose provided indirect proof of an additional oxidative process after early glycated product formation. These experimental observations provide insight into the in vivo context of advanced glycation end product formation in chronic hyperglycemia and aging.     

New insights into deleterious impacts of in vivo glycation on albumin antioxidant activities

Background

Albumin constitutes the most abundant circulating antioxidant and prevents oxidative damages. However, in diabetes, this plasmatic protein is exposed to several oxidative modifications, which impact on albumin antioxidant properties.

Methods

Most studies dealing on albumin antioxidant activities were conducted on in vitro modified protein. Here we tried to decipher whether reduced antioxidant properties of albumin could be evidenced in vivo. For this, we compared the antioxidant properties of albumin purified from diabetic patients to in vitro models of glycated albumin.

Results

Both in vivo and in vitro glycated albumins displayed impaired antioxidant activities in the free radical-induced hemolysis test. Surprisingly, the ORAC method (Oxygen Radical Antioxidant Capacity) showed an enhanced antioxidant activity for glycated albumin. Faced with this paradox, we investigated antioxidant and anti-inflammatory activities of our albumin preparations on cultured cells (macrophages and adipocytes). Reduced cellular metabolism and enhanced intracellular oxidative stress were measured in cells treated with albumin from diabetics. NF-kB –mediated gene induction was higher in macrophages treated with both type of glycated albumin compared with cells treated with native albumin. Anti inflammatory activity of native albumin is significantly impaired after in vitro glycation and albumin purified from diabetics significantly enhanced IL6 secretion by adipocytes. Expression of receptor for advanced glycation products is significantly enhanced in glycated albumin-treated cells.

Conclusions and general significance

Our results bring new evidences on the deleterious impairments of albumin important functions after glycation and emphasize the importance of in vivo model of glycation in studies relied to diabetes pathology.     

Hypochlorous acid generates Nε-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.     

Glycation modulates alpha-synuclein fibrillization kinetics: A sweet spot for inhibition

Glycation is a nonenzymatic posttranslational modification (PTM) known to be increased in the brains of hyperglycemic patients. Alpha-synuclein (αSN), a central player in the etiology of Parkinson’s disease, can be glycated at lysine residues, thereby reducing αSN fibril formation in vitro and modulating αSN aggregation in cells. However, the molecular basis for these effects is unclear. To elucidate this, we investigated the aggregation of αSN modified by eight glycating agents, namely the dicarbonyl compound methylglyoxal (MGO) and the sugars ribose, fructose, mannose, glucose, galactose, sucrose, and lactose. We found that MGO and ribose modify αSN to the greatest extent, and these glycation products are the most efficient inhibitors of fibril formation. We show glycation primarily inhibits elongation rather than nucleation of αSN and has only a modest effect on the level of oligomerization. Furthermore, glycated αSN is not significantly incorporated into fibrils. For both MGO and ribose, we discovered that a level of ∼5 modifications per αSN is optimal for inhibition of elongation. The remaining sugars showed a weak but optimal inhibition at ∼2 modifications per αSN. We propose that this optimal level balances the affinity for the growing ends of the fibril (which decreases with the extent of modification) with the ability to block incorporation of subsequent αSN subunits (which increases with modification). Our results are not only relevant for other αSN PTMs but also for understanding PTMs affecting other fibrillogenic proteins and may thus open novel avenues for therapeutic intervention in protein aggregation disorders.     

Cyperus rotundus suppresses AGE formation and protein oxidation in a model of fructose-mediated protein glycoxidation

Non-enzymatic glycation, as the chain reaction between reducing sugars and the free amino groups of proteins, has been shown to correlate with severity of diabetes and its complications. Cyperus rotundus (Cyperaceae) is used both as a food to promote health and as a drug to treat certain diseases. In this study, considering the antioxidative effects of C. rotundus, we examined whether C. rotundus also protects against protein oxidation and glycoxidation. The protein glycation inhibitory activity of hydroalcoholic extract of C. rotundus was evaluated in vitro using a model of fructose-mediated protein glycoxidation. The C. rotundus extract with glycation inhibitory activity also demonstrated antioxidant activity when a ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays as well as metal chelating activity were applied. Fructose (100 mM) increased fluorescence intensity of glycated bovine serum albumin (BSA) in terms of total AGEs during 14 days of exposure. Moreover, fructose caused more protein carbonyl (PCO) formation and also oxidized thiol groups more in glycated than in native BSA. The extract of C. rotundus at different concentrations (25–250 μg/ml) has significantly decreased the formation of AGEs in term of the fluorescence intensity of glycated BSA. Furthermore, we demonstrated the significant effect of C. rotundus extract on preventing oxidative protein damages including effect on PCO formation and thiol oxidation which are believed to form under the glycoxidation process. Our results highlight the protein glycation inhibitory and antioxidant activity of C. rotundus. These results might lead to the possibility of using the plant extract or its purified active components for targeting diabetic complications.     

Glycation improves the thermostability of trypsin and chymotrypsin

A novel glycation procedure, in vacuo glycation, was used to attach glucose covalently to the lysine residues of trypsin and chymotrypsin. Glycated trypsin and glycated chymotrypsin have greatly increased thermostability compared to the native enzymes. For example, glycated bovine trypsin, incubated at 50 degrees C and pH 8.0 for 3 h, retained more than 50% of its original activity whereas the native enzyme was inactivated under the same conditions. Similarly, after incubation at 50 degrees C and pH 8.0, glycated bovine chymotrypsin retained 45% of its original activity and the native enzyme was inactivated. Glycated porcine trypsin is exceptionally thermostable and could be used to digest native ribonuclease at 70 degrees C without the need for prior denaturation. The apparent increase in the thermal stability of the glycated proteins observed in activity measurements is also reflected by an increase in the T(m) values determined with differential scanning calorimetry (DSC) and circular dichroism (CD). The glycation does not alter the activity or specificity of these enzymes.     

Studies on advanced glycation end products by recent mass spectrometric techniques

Summary The results obtained by different mass spectrometric approaches in the field of advanced glycation of proteins are reported and discussed in detail in comparison with those obtained by other analytical methodologies (fluorescence and absorbance spectroscopies, radioimmunoassay, enzyme-linked immunosorbent assay). They have been subdivided in three main groups: analysis on degraded glycated proteins, direct analysis of glycated proteins and studies on the reaction between protected lysine and glucose. The general overview so achieved indicate mass spectrometry as a particularly valid analytical method in this field of research.     

Non-enzymatic glycation of elastin

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 investigated thein vitro glycation of human aortic -elastin, and chose and adapted methods for evaluating the degree and kinetics of glycation. -Elastin was prepared from thoracic aortas of young accident victims and glycated by incubating with different glucose concentrations (25, 50, 75 and 100 mmol/l) in 0.2 M phosphate buffer, pH 7.8 for 30 days, at 37°C. The degree of glycation was measured by three colorimetric methods,i.e. Nitroblue tetrazolium, 2-thiobarbituric acid and hydrazine; by aminophenyl-boronate affinity chromatography which determines Amadori products; and by a fluorescence method which determines advanced glycosylation end products. The highest degree of glycation was found on day 3 after the beginning of incubation. Fluorescence, as an index of advanced glycation, consistently increased from days 5 to 24. Investigation of the properties of glycated elastin may help in understanding the importance of this long-lived protein for the age-related changes in tissues and for diabetic complications.     

An autoantibody against N-(carboxyethyl)lysine (CEL): Possible involvement in the removal of CEL-modified proteins by macrophages

Advanced glycation end products (AGEs) are believed to play a significant role in the development of diabetic complications. In this study, we measured the levels of autoantibodies against several AGE structures in healthy human plasma and investigated the physiological role of the autoantibodies. A high titer of the autoantibody against N^ε-(carboxyethyl)lysine (CEL) was detected in human plasma compared with other AGE structures such as CML and pentosidine. The purified human anti-CEL autoantibody reacted with CEL-modified human serum albumin (CEL-HSA), but not CML-HSA. A rabbit polyclonal anti-CEL antibody, used as a model autoantibody against CEL, accelerated the uptake of CEL-HSA by macrophages, but did not enhance the uptake of native HSA. Furthermore, when ¹²⁵I-labeled CEL-HSA was injected into the tail vein of mice, accumulation of ¹²⁵I-CEL-HSA in the liver was accelerated by co-injection of the rabbit anti-CEL antibody. These results demonstrate that the autoantibody against CEL in plasma may play a role in the macrophage uptake of CEL-modified proteins.     

抗体药物糖化检测方法及其生物学功能研究进展

糖化是一个重要的蛋白质修饰过程,可能影响治疗性蛋白药物（如单克隆抗体药物）的生物活性及分子稳定性。许多研究表明糖化血红蛋白水平升高与心血管疾病及动脉粥样硬化有着密切关系。人体的血浆蛋白,如白蛋白、球蛋白、纤维蛋白和胶原蛋白也可能被糖化,进而形成AGEs,蛋白药物的生产、储存以及药物在体内循环过程中都可能发生糖化反应。综述了治疗性抗体药物糖化的原因、分析方法,以及糖化对抗体药物生物学功能的影响,以期为临床抗体药物的开发、优化及贮存条件研究提供参考。     

Detection of oxidized and glycated proteins in clinical samples using mass spectrometry — A user's perspective

Background

Proteins in human tissues and body fluids continually undergo spontaneous oxidation and glycation reactions forming low levels of oxidation and glycation adduct residues. Proteolysis of oxidised and glycated proteins releases oxidised and glycated amino acids which, if they cannot be repaired, are excreted in urine.

Scope of Review

In this review we give a brief background to the classification, formation and processing of oxidised and glycated proteins in the clinical setting. We then describe the application of stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of oxidative and glycation damage to proteins in clinical studies, sources of error in pre-analytic processing, corroboration with other techniques – including how this may be improved – and a systems approach to protein damage analysis for improved surety of analyte estimations.

Major conclusions

Stable isotopic dilution analysis LC-MS/MS provides a robust reference method for measurement of protein oxidation and glycation adducts. Optimised pre-analytic processing of samples and LC-MS/MS analysis procedures are required to achieve this.

General significance

Quantitative measurement of protein oxidation and glycation adducts provides information on level of exposure to potentially damaging protein modifications, protein inactivation in ageing and disease, metabolic control, protein turnover, renal function and other aspects of body function. Reliable and clinically assessable analysis is required for translation of measurement to clinical diagnostic use. Stable isotopic dilution analysis LC-MS/MS provides a “gold standard” approach and reference methodology to which other higher throughput methods such as immunoassay and indirect methods are preferably corroborated by researchers and those commercialising diagnostic kits and reagents. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Acetoacetate promotes the formation of fluorescent advanced glycation end products (AGEs)

Acetoacetate (AA) is an important ketone body, which produces reactive oxygen species (ROS). Advanced glycation end products (AGEs) are defined as final products of glycation process whose production is influenced by the levels of ROS. The accumulation of AGEs in the body contributes to pathogenesis of many diseases including complications of diabetes, and Alzheimer’s and Parkinson’s disease. Here, we evaluated the impact of AA on production of AGEs upon incubation of human serum albumin (HSA) with glucose. The effect of AA on the AGEs formation of HSA was studied under physiological conditions after incubation with glucose for 35 days. The physical techniques including circular dichroism (CD) and fluorescence spectroscopy were used to assess the impact of AA on formation and structural changes of glycated HSA (GHSA). Our results indicated that the secondary and tertiary structural changes of GHSA were increased in the presence of AA. The fluorescence intensity measurements of AGEs also showed an increase in AGEs formation. Acetoacetate has an activator effect in formation of AGEs through ROS production. The presence of AA may result in enhanced glycation in the presence of glucose and severity of complications associated with accumulation of AGEs.     

Novel application of surface plasmon resonance biosensor chips for measurement of advanced glycation end products in serum of Zucker diabetic fatty rats

Advanced glycation end products (AGEs) have been implicated in diabetic complications. To measure AGEs, especially N-(carboxymethyl)lysine (CML), in sera from Zucker diabetic fatty rats (ZDF) and Zucker lean rats (ZL), we used a novel method of protein chip and surface plasmon resonance imaging (SPRI). Serum samples were obtained from male ZDF and ZL rats at 20 weeks of age. Antibodies to AGEs or CML were immobilized on a gold surface, which was modified by cysteine-tagged, protein-G constructs. The gold chip upon which the serum was spotted was optically coupled with a prism coupler. The reflected images from the gold chip were obtained using a charge-coupled device (CCD) camera. The direct analysis of the glycated proteins and products using SPRI showed that AGEs and CML levels were elevated in ZDF serum, compared with ZL serum. The lowest detection limit of AGEs was 10 ng/ml, with a working range covering the physiological range. These results indicate that the protein chip and SPRI system is very suitable for the measurement of glycated proteins and end products in serum samples. This system offers high sensitivity without any fluorescent or other labeling of the components and saves a substantial amount of time, resources, and labor. Our results suggest that SPRI systems can be used as a tool to diagnose diabetic complications.     

Controversial behavior of aminoguanidine in the presence of either reducing sugars or soluble glycated bovine serum albumin

The elucidation of the controversial inhibitory effect of aminoguanidine (AG) on the cross-linking and fluorescent advanced glycation end products (AGEs) formation during long-term in vitro glycation of type I collagen with 250 mM reducing sugars or 0.5 mg/ml soluble glycated bovine serum albumin (AGE-BSA) was researched.Chromatographic and SDS–PAGE analyses revealed the formation of aggregates during collagen glycation. AG at all concentrations (5–80 mM) prevented the cross-linking of collagen peptides with monosaccharides but an increase in fluorescence with a maximum value at 10 mM AG was noticed. In the presence of AGE-BSA, AG prevented the cross-linking process and decreased the fluorescence levels in a concentration-dependent manner.Our results suggest that AG is an efficient inhibitor of collagen cross-linking and the highest increase in fluorescence due to reducing sugars and AG can be explained by the competition between guanidine group of AG and arginine residues of some protein-bound dideoxyosones, which could form fluorescent compounds.     

Inhibition of protein glycation and advanced glycation end products by ascorbic acid and other vitamins and nutrients

Nonenzymatic glycation, the reaction of glucose and other reducing sugars with protein, reversibly produces Amadori products and over a long period irreversible advanced glycation end products. In diabetes, these reactions are greatly accelerated and are important in the pathogenesis of diabetic complications.

In vitro glycation was studied with bovine albumin as the model protein. A mixture of 25 mM glucose/fructose was used as the glycating agent. The Amadori product was quantitated by thiobarbituric acid colorimetry after hydrolysis. Advanced glycation end products were measured by their intrinsic fluorescence. A number of vitamins and nutrients were found to be potent inhibitors of both the glycation reaction and the subsequent end products. The nutrients were effective at physiological concentrations and exhibited dose-response relationships. The inhibitors included ascorbic acid, tocopherol, pyridoxal, niacinamide, sodium selenite, selenium yeast, and carnosine. A significant correlation was found between the inhibition of glycation and the inhibition of AGE formation (P < 0.001). One of the nutrients, ascorbic acid, was used in a pilot study. Eighteen normal subjects, 7 college age and 10 middle age, were supplemented with 1,000 mg of ascorbic acid in the form of Re-Natured Vitamin C^® for a period of 4 weeks. Serum protein glycation was decreased an average of 46.8% (P < 0.01). These results underline the importance of nutrition in diabetes and indicate the possibility of therapeutic use of these nutrients for the prevention of diabetic complications.     

Hypochlorous acid generates N epsilon-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.     

Biochemical,biophysical, and thermodynamic analysis of <Emphasis Type="Italic">in vitro</Emphasis> glycated human serum albumin

Glycated human serum albumin (HSA) is known to be involved in the pathogenesis of several diseases, and we have therefore investigated possible alterations in HSA on glycation. HSA was incubated for 5 and 20 weeks independently with constant glucose concentration at 37 degrees C under aerobic conditions. Biochemical, spectral, electrophoretic, circular dichroism spectropolarimetric, and thermodynamic analyses confirmed that the structure and stability of HSA is significantly affected on glucose modification. Glycated HSA-AGE-20w showed appreciable elevation (15.8%) in beta-sheet structure and decrease in alpha-helix (10.4%) and random coil (5.7%) structures. Slight changes have also been observed in turns (3.2%) of HSA-AGE-20w. Quenching studies with antioxidants diethylene triaminepentaacetic acid and superoxide dismutase showed inhibition in glycation to the extent of 50-65 and 30-40%, respectively. The novelty of present study is that glycation of HSA can cause induction of secondary and tertiary structure changes that may generate thermodynamically more stable high molecular weight aggregates having remarkably increased beta-sheet structure than its non-glycated form. This may interfere with the normal function of HSA, thus contributing to diabetic complications.