Methylglyoxal-induced modification of myoglobin: An insight into glycation mediated protein aggregation.

Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with the proteins to form advanced glycation end products (AGEs) following a Maillard-like reaction. In a time-dependent reaction study of MG with the heme protein myoglobin (Mb), MG was found to induce significant structural alterations of the heme protein, such as heme loss, changes in tryptophan fluorescence, and decrease of α-helicity with increased ß-sheet content. These changes were found to occur gradually with increasing period of incubation. Incubation of Mb with MG induced the formation of several AGE adducts, including, carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87, carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139. MG induced amyloid-like aggregation of Mb was detected at a longer period of incubation. MG-derived AGEs, therefore, appear to have an important role as the precursors of protein aggregation, which, in turn, may be associated with pathophysiological complications.

Biophysical and mass spectrometry based characterization of methylglyoxal-modified myoglobin: Role of advanced glycation end products in inducing protein structural alterations.

Methylglyoxal (MG) is a highly reactive α-dicarbonyl compound which reacts with proteins to form advanced glycation end products (AGEs). MG-induced AGE (MAGE) formation is particularly significant in diabetic condition. In the current study, we have undertaken a time-dependant characterization of MG-modified myoglobin following incubation of the heme protein with the α-dicarbonyl compound for different time periods. Interestingly, mass spectrometric studies indicated modifications at two specific lysine residues, Lys-87 and Lys-133. The AGE adducts identified at Lys-87 were carboxymethyllysine and carboxyethyllysine, while those detected at Lys-133 included pyrraline-carboxymethyllysine and carboxyethyllysine, respectively. Far-UV CD studies revealed a decrease in the native α-helical content of the heme protein gradually with increasing time of MG incubation. In addition, MG modification was found to induce changes in tertiary structure as well as surface hydrophobicity of the heme protein. MG-derived AGE adducts thus appear to alter the structure of Mb considerably. Considering the increased level of MG in diabetic condition, the current study appears physiologically relevant in terms of understanding AGE-mediated protein modification and subsequent structural changes.

Modification with N-benzylisatin restricts stress-induced aggregation of hen egg white lysozyme: Anti-amyloidogenic property of isatin derivative with possible clinical implications.

Hen egg white lysozyme (HEWL) is a structural homolog of human lysozyme and is widely used as a model protein to investigate protein aggregation. The effect of N-benzylisatin on stress-induced aggregation of HEWL has been investigated in the present study. Interaction of the isatin derivative with HEWL induced changes in protein secondary and tertiary structural conformation as evident from different biophysical and spectroscopic studies. In addition, modification with N-benzylisatin was found to increase the conformational stability of HEWL and afford considerable resistance to the protein to stress-induced aggregation as indicated from subsequent experimental studies, including thioflavin T fluorescence, microscopic imaging and dynamic light scattering analysis. Protein modification was analysed and confirmed by MALDI-TOF and ESI-MS studies. The results highlight possible clinical implications of isatin derivative in the treatment of protein misfolding and conformational disorders.

Effect of glyoxal and 1-methylisatin on stress-induced fibrillation of Hen Egg White Lysozyme: Insight into the anti-amyloidogenic property of the compounds with possible therapeutic implications.

Systemic amyloidosis is a hereditary disorder that mostly arises as a result of specific point mutations to the wild type gene of lysozyme, forming mutant lysozyme variants leading to aggregation of the protein. The small monomeric protein Hen Egg White Lysozyme (HEWL) is a structural homolog of Human Lysozyme and is widely used as a model protein to investigate protein aggregation. In the present study, we have investigated the effect of 1-methylisatin, an indole derivative and glyoxal, a reactive dicarbonyl compound, on stress-induced aggregation of HEWL. Interaction of the compounds with HEWL induced changes in structure and surface hydrophobicity of the protein as evident from CD spectroscopy, tryptophan fluorescence and ANS binding studies. Additional experiments (Thioflavin T fluorescence, AFM imaging and DLS studies) demonstrate that stress induces amyloid-like fibrillation of HEWL, however, prior modification of the protein with glyoxal or 1-methylisatin significantly reduces its susceptibility to aggregation. High resolution mass spectrometric analysis indicated that 1-methylisatin primarily complexes with the protein in the form of a dimer. On the other hand, glyoxal-mediated modification of the protein induces formation of glycated adducts (carboxymethyllysine, hydroimidazolone). The results highlight possible clinical implications of the compounds in treatment of systemic amyloidosis and protein conformational disorder.

Glyoxal modification mediates conformational alterations in silk fibroin: Induction of fibrillation with amyloidal features.

Silkwormsilk protein fibroin is widely exploited to develop novel silk-based biomaterials due to its stable ß-sheet structure, providing high crystallinity and tensile strength. The polymorphic behaviour of silk fibroin provides a window to modulate its structural transitions during self-assembly for different functional outcomes. Most studies are therefore mainly focused on formation of well-developed ß-sheet structure and self-assembly of silk fibroin which are regulated by many parameters. Glyoxal, a highly reactive α-oxoaldehyde, reacts with different proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significance of protein modification by glyoxal-derived AGEs, in the present study the effect of glyoxal (250, 500 and 1000 µM) on the structure of silk fibroin has been investigated. CD and fluorescence studies reveal that higher concentrations of the α-oxoaldehyde induce considerable alterations of secondary and tertiary structure of the protein leading to aggregation following incubation with for 3 weeks. The aggregates exhibit fibrillar morphology with amyloidal nature as evident from SEM, FTIR and XRD experiments. The findings highlight that glycationinduced modification can be a possible approach for modulating the conformation of the silk protein which may be relevant in connection to clinical, biomedical or synthetic biology based applications.

Effect of Glyoxal Modification on a Critical Arginine Residue (Arg-31α) of Hemoglobin: Physiological Implications of Advanced Glycated end Product an in vitro Study.

BACKGROUND: Non-enzymatic protein glycation is involved in structure and stability changes that impair protein functionality, resulting in several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer's disease, Parkinson's disease and Andrade's syndrome). Glyoxal, an endogenous reactive oxoaldehyde, increases in diabetes and reacts with several proteins to form advanced glycation end products through Maillard-like reaction. OBJECTIVE: Human hemoglobin, the most abundant protein in blood cells is subjected to nonenzymatic modification by reactive oxoaldehydes in diabetic condition. In the present study, the effect of a low concentration of glyoxal (5 µM) on hemoglobin (10 µM) has been investigated following a period of 30 days incubation in vitro. METHODS: Different techniques, mostly biophysical and spectroscopic (e.g. circular dichroism, differential scanning calorimetric study, dynamic light scattering, mass spectrometry, etc.) were used to study glyoxal-induced changes of hemoglobin. RESULTS: Glyoxal-treated hemoglobin exhibits decreased absorbance around 280 nm, decreased fluorescence and reduced surface hydrophobicity compared to normal hemoglobin. Glyoxal treatment enhances the stability of hemoglobin and lowers its susceptibility to thermal aggregation compared to control hemoglobin as seen by different studies. Finally, peptide mass fingerprinting study showed glyoxal to modify an arginine residue of α-chain of hemoglobin (Arg-31α) to hydroimidazolone. CONCLUSION: Increased level of glyoxal in diabetes mellitus as well as its high reactivity may cause modifications of the heme protein. Thus, considering the significance of glyoxal-induced protein modification under physiological conditions, the observation appears clinically relevant in terms of understanding hydroimidazolone-mediated protein modification under in vivo conditions.

Methylglyoxal modification reduces the sensitivity of hen egg white lysozyme to stress-induced aggregation: Insight into the anti-amyloidogenic property of α-dicarbonyl compound.

The reactive α-oxoaldehyde, methylglyoxal reacts with different proteins to form Advanced Glycation End Products (AGEs) through Maillard reaction. Its level increases significantly in diabetic condition. Here, we have investigated the effect of different concentrations of methylglyoxal (200-400 µM) on the monomeric protein, hen egg white lysozyme (HEWL) following incubation for 3 weeks. Reaction of methylglyoxal with HEWL induced considerable changes in tertiary structure of the protein, but no significant alteration in secondary structure, as evident from different spectroscopic and biophysical studies. Interestingly, methylglyoxal modification was found to enhance the thermal stability of the protein and reduce its sensitivity to stress-induced aggregation. Finally, peptide mass fingerprinting revealed modification of arginine (Arg-45, Arg-14, Arg-68 or Arg-72) and lysine (Lys-116) residues of the protein to AGE adducts, namely, hydroimidazolone, tetrahydropyrimidine, and carboxyethyllysine. Methylglyoxal-derived AGE adducts (MAGE) appear to be responsible for the observed changes in protein. As demonstrated in the present study, the findings may highlight a possible therapeutic potential of the α-oxoaldehyde against protein misfolding and conformational disorder.Communicated by Ramaswamy H. Sarma.

Glyoxal-induced modification enhances stability of hemoglobin and lowers iron-mediated oxidation reactions of the heme protein: An in vitro study.

Glyoxal, a reactive α-oxoaldehyde, increases in diabetic condition. It reacts with different proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significance of AGE-mediated protein modification by glyoxal, here we have investigated the in vitro effect of the reactive α-oxoaldehyde (10, 20µM) on the heme protein hemoglobin (HbA0) (100µM) after incubation for one week at 25°C. In comparison with HbA0, glyoxal-treated HbA0 exhibited decreased absorbance around 280nm, reduced intrinsic fluorescence and lower surface hydrophobicity. Glyoxal treatment was found to increase the stability of HbA0 without significant perturbation of the secondary structure of the heme protein. In addition, H2O2-mediated iron release and subsequent iron-mediated oxidative (Fenton) reactions were found to be lower in presence of glyoxal-treated HbA0 compared to HbA0. Mass spectrometric studies revealed modification of arginine residues of HbA0 (Arg-31α, Arg-40ß) to hydroimidazolone adducts. AGE-induced modifications thus appear to be associated with the observed changes of the heme protein. Considering the increased level of glyoxal in diabetes mellitus as well as its high reactivity, glyoxal-derived AGE adducts might thus be associated with modifications of the protein including physiological significance.

Formation of Pentosidine Cross-Linking in Myoglobin by Glyoxal: Detection of Fluorescent Advanced Glycation End Product.

Glyoxal, a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significance of protein modification by glyoxal-derived AGEs, we investigated the in vitro effect of glyoxal (200 µM) on the monomeric heme protein myoglobin (Mb) (100 µM) after incubation for one week at 25 °C. Glyoxal-treated Mb exhibited increased absorbance around the Soret region, decreased α-helicity and thermal stability compared to control Mb. Intrinsic fluorescence spectrum of the treated Mb showed an additional signal in the 400-500 nm region on excitation at 280 nm that was absent in control Mb. When excited at 335 nm, the glyoxal-treated sample gave a strong fluorescence indicating AGE formation. Mass spectrometric studies revealed formation of glyoxal-derived fluorescent AGE adduct pentosidine between Lys-145 and Arg-139 residues of Mb. Other than pentosidine, additional AGE adducts, namely, carboxymethyllysine at Lys-133, hydroimidazolone at Arg-31 and pyrrolidone-carboxymethyllysine at Lys-145 were also detected. Lys-145 was thus found to contain two different types of AGE adducts, indicating the heterogeneous nature of in vitro glycation reaction. AGE-induced protein modifications might be associated with complications in disease conditions.

Methyglyoxal administration induces modification of hemoglobin in experimental rats: An in vivo study.

Methylglyoxal, a highly reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. In the present study, the effect of methylglyoxal on experimental rat hemoglobin in vivo has been investigated with respect to structural alterations and amino acid modifications, after external administration of the α-dicarbonyl compound in animals. Different techniques, mostly biophysical, were used to characterize and compare methylglyoxal-treated rat hemoglobin with that of control, untreated rat hemoglobin. In comparison with methylglyoxal-untreated, control rat hemoglobin, hemoglobin of methylglyoxal-treated rats (32mg/kgbodywt.dose) exhibited slightly decreased absorbance around 280nm, reduced intrinsic fluorescence and lower surface hydrophobicity. The secondary structures of hemoglobin of control and methylglyoxal-treated rats were more or less identical with the latter exhibiting slightly increased α-helicity compared to the former. Compared to control rat hemoglobin, methylglyoxal-treated rat hemoglobin showed higher stability. Peptide mass fingerprinting analysis revealed modifications of Arg-31α, Arg-92α and Arg-104ß of methylglyoxal-treated rat hemoglobin to hydroimidazolone adducts. The modifications thus appear to be associated with the observed structural alterations of the heme protein. Considering the increased level of methylglyoxal in diabetes mellitus as well as its high reactivity, AGE-induced modifications may have physiological significance.

Methylglyoxal modification enhances the stability of hemoglobin and lowers its iron-mediated oxidation reactions: An in vitro study.

Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. In the present study, we have investigated the in vitro effect of methylglyoxal (200, 300µm) on the heme protein hemoglobin (HbA0) (100µm) after incubation for one week at 25°C. Compared to HbA0, MG-treated HbA0 exhibited decreased absorbance around 280nm, reduced intrinsic fluorescence and lower surface hydrophobicity. MG treatment was not found to significantly affect the secondary structure of HbA0. The stability of MG-treated HbA0 was found to be higher compared to HbA0. Moreover, H2O2-mediated iron release and subsequent iron-mediated oxidation (Fenton) reactions were found to be lower in presence of MG-treated HbA0 compared to HbA0. As shown by mass spectrometric studies, MG modified Arg-92α, Arg-104ß, Arg-31α and Arg-40ß of HbA0 to hydroimidazolone adducts. The modifications thus appear to be associated with the observed structural alterations of the heme protein. Considering the increased level of MG in diabetes mellitus as well as its high reactivity, AGEs might be associated with structural and functional modifications of the protein including physiological significance.

Glyoxal administration induces formation of high molecular weight aggregates of hemoglobin exhibiting amyloidal nature in experimental rats: An in vivo study.

Glyoxal, a highly reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs). In the present study, we have investigated the effect of glyoxal on experimental rat hemoglobin in vivo after external administration of the α-dicarbonyl compound in animals. Gel electrophoretic profile of hemolysate collected from glyoxal-treated rats (32mg/kg body wt. dose) after one week exhibited the presence of some high molecular weight protein bands that were found to be absent for control, untreated rats. Mass spectrometric and absorption studies indicated that the bands represented hemoglobin. Further studies revealed that the fraction exhibited the presence of intermolecular cross ß-sheet structure. Thus glyoxal administration induces formation of high molecular weight aggregates of hemoglobin with amyloid characteristics in rats. Aggregated hemoglobin fraction was found to exhibit higher stability compared to glyoxal-untreated hemoglobin. As evident from mass spectrometric studies, glyoxal was found to modify Arg-30ß and Arg-31α of rat hemoglobin to hydroimidazolone adducts. The modifications thus appear to induce amyloid-like aggregation of hemoglobin in rats. Considering the increased level of glyoxal in diabetes mellitus as well as its high reactivity, the above findings may be physiologically significant.

Methylglyoxal-induced modification causes aggregation of myoglobin.

Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. We have investigated the in vitro effect of MG (200µM) on the monomeric heme protein myoglobin (Mb) (100µM) in a time-dependent manner (7 to 18days incubation at 25°C). MG induces significant structural alterations of the heme protein, including heme loss, changes in tryptophan fluorescence, decrease of α-helicity with increased ß-sheet content etc. These changes occur gradually with increased period of incubation. Incubation of Mb with MG for 7days results in formation of the AGE adducts: carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87 and carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133. On increasing the period of incubation up to 14days, additional AGEs namely, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139 have been detected. MG also induces aggregation of Mb, which is clearly evident with longer period of incubation (18days), and appears to have amyloid nature. MG-derived AGEs may thus have an important role as the precursors of protein aggregation, which, in turn, may be associated with physiological complications.

Structural alterations of hemoglobin and myoglobin by glyoxal: a comparative study.

Glyoxal, a highly reactive oxoaldehyde, increases in diabetic condition. It reacts with different proteins to form advanced glycation end products (AGEs). Here we have studied the structural alterations as well as the sites and nature of amino acid modifications of two heme proteins, hemoglobin and myoglobin on incubation with glyoxal for seven days at 25°C. In comparison with normal hemoglobin (HbA0), glyoxal-treated hemoglobin (GHbA0) exhibits decreased absorbance around 280 nm, reduced intrinsic fluorescence and lower surface hydrophobicity. However, glyoxal-treated myoglobin (GMb) exhibits the opposite effects in these respects when compared to normal myoglobin (Mb). Glyoxal increases the thermal stability of hemoglobin, while it decreases the stability of myoglobin. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-mass spectrometry reveals modifications of Arg-31α, Arg-40ß and Arg-104ß of hemoglobin by glyoxal to hydroimidazolone adducts. On the other hand, glyoxal modifies Lys-133 and Lys-145 to carboxymethyllysine and Arg-31 to hydroimidazolone adducts in myoglobin. Thus the same oxoaldehyde exerts different effects on hemoglobin and myoglobin and may be associated with different structural properties of the proteins.

In vitro study on structural alteration of myoglobin by methylglyoxal.

Methylglyoxal (MG), a reactive α-oxoaldehyde, reacts with proteins to form irreversible advanced glycation end products (AGEs) following Maillard-like reaction. MG-induced AGE (MAGE) formation may be significant, particularly in diabetic condition with increased level of MG. Although myoglobin (Mb) is known to react with sugars to form AGEs, its interaction with MG is not known. Here we have studied interaction of Mb with MG. After in vitro reaction between Mb and MG at 25 °C for 7 days, the unchanged Mb and modified Mb (MG-Mb) were separated by ion exchange chromatography. Compared to Mb, MG-Mb exhibited higher electrophoretic mobility in native polyacrylamide gel electrophoresis, increased absorbance around 280 nm and more α-helical content, indicating structural changes of the modified protein. As shown by MALDI-mass spectrometry, MG converted Lys-16 and Lys-133 to carboxyethyllysine in MG-Mb. MAGE thus formed in MG-Mb may be associated with its enhanced mobility in native gel due to neutralization of positive charges and the observed structural changes in comparison with Mb.

Methylglyoxal-induced modifications of hemoglobin: structural and functional characteristics.

Methylglyoxal (MG) reacts with proteins to form advanced glycation end products (AGEs). Although hemoglobin modification by MG is known, the modified protein is not yet characterized. We have studied the nature of AGE formed by MG on human hemoglobin (HbA(0)) and its effect on structure and function of the protein. After reaction of HbA(0) with MG, the modified protein (MG-Hb) was separated and its properties were compared with those of the unmodified protein HbA(0). As shown by MALDI-mass spectrometry, MG converted Arg-92α and Arg-104ß to hydroimidazolones in MG-Hb. Compared to HbA(0), MG-Hb exhibited decreased absorbance around 280nm, reduced tryptophan fluorescence (excitation 285nm) and increased α-helix content. However, MG modification did not change the quaternary structure of the heme protein. MG-Hb appeared to be more thermolabile than HbA(0). The modified protein was found to be more effective than HbA(0) in H(2)O(2)-mediated iron release and oxidative damages involving Fenton reaction. MG-Hb exhibited less peroxidase activity and more esterase activity than HbA(0). MG-induced structural and functional changes of hemoglobin may enhance oxidative stress and associated complications, particularly in diabetes mellitus with increased level of MG.