Khursheed Alam

Genotoxicity and immunogenicity of DNA-advanced glycation end products formed by methylglyoxal and lysine in presence of Cu2+.

The highly reactive electrophile, methylglyoxal (MG), a break down product of carbohydrates, is a major environmental mutagen having potential genotoxic effects. Previous studies have suggested the reaction of MG with free amino groups of proteins forming advanced glycation end products (AGEs). This results in the generation of free radicals which play an important role in pathophysiology of aging and diabetic complications. MG also reacts with free amino group of nucleic acids resulting in the formation of DNA-AGEs. While the formation of nucleoside AGEs has been demonstrated previously, no extensive studies have been performed to assess the genotoxicity and immunogenicity of DNA-AGEs. In this study we report both the genotoxicity and immunogenicity of AGEs formed by MG-Lys-Cu(2+) system. Genotoxicity of the experimentally generated AGEs was confirmed by comet-assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single-stranded regions and destabilization of hydrogen bonds. Immunogenicity of native and MG-Lys-Cu(2+)-DNA was probed in female rabbits. The modified DNA was highly immunogenic eliciting high titre immunogen specific antibodies, while the unmodified form was almost non-immunogenic. The results show structural perturbations in MG-Lys-Cu(2+)-DNA generating new epitopes that render the molecule immunogenic.

Preferential recognition of Amadori-rich lysine residues by serum antibodies in diabetes mellitus: role of protein glycation in the disease process.

This study analyzes effect of glycation on proteins rich in lysine residues as hyperglycemia induced protein glycation has been mainly reported in diabetes mellitus at the intrachain lysine residues leading to the formation of Amadori modified proteins. We have studied the effect of glucose on poly-l-lysine (PLL), a homopolymer of lysine residues. Levels of Amadori products in the glycated PLL were evaluated by fructosamine assay and the presence of 5-hydroxymethylfurfural (HMF) in the glycated PLL was analyzed by thiobarbituric acid assay. Fluorescence and FT-IR spectroscopy were applied to characterize the modified PLL. Binding characteristics of experimentally induced antibodies against glycated PLL and the presence of antibodies against glycated PLL in the sera of diabetes patients was evaluated by solid phase enzyme immunoassays. The fructosamine assay showed significantly high yield of early glycation (Amadori) products in the glycated PLL, which was confirmed by increased yield of HMF from Amadori products of glycated PLL. Loss in fluorescence intensity and appearance of a new band corresponding to Amadori products were observed in FT-IR spectrum of the glycated PLL. Glycated PLL was found to be highly immunogenic in rabbits as compared to the native form. Serum antibodies from diabetes patients showed appreciably high recognition of the glycated PLL. The results conclusively show the glycation induced damage to the lysine molecules and specific recognition of Amadori-lysine residues by serum antibodies from diabetes patients. The glycated lysine residues may serve as a diagnostic biomarker for early glycation process in diabetes mellitus.

Structural and immunological characterization of Amadori-rich human serum albumin: role in diabetes mellitus.

Proteins modifications in diabetes may lead to early glycation products (EGPs) as well as advanced glycation end products (AGEs). Whereas no extensive studies have been carried out to assess the role of EGPs in secondary complications of diabetes, numerous investigators have demonstrated the role of AGEs. Early glycation involves attachment of glucose on ε-NH2 of lysine residues of proteins leading to generation of the Amadori product (an early glycation species). This study reports the structural and immunological characterization of EGPs of HSA because we believe that during persistent hyperglycemia the HSA, one of the major blood proteins, can undergo fast glycation. Glucose mediated generation of EGPs of HSA was quantitated as Amadori products by NBT assay and authenticated by boronate affinity chromatography and LC/MS. Compared to native HSA changes in glycated-HSA were characterized by hyperchromicity, loss in fluorescence intensity and a new peak in the FTIR profile. Immunogenicity of native- and glycated-HSA was evaluated by inducing antibodies in rabbits. Results suggest generation of neo-epitopes on glycated-HSA rendering it highly immunogenic compared to native HSA. Quantization of EGPs of HSA by authentic antibodies against HSA-EGPs can be used as marker for early detection of the initiation/progression of secondary complications of diabetes.

Hydroxyl radical modification of collagen type II increases its arthritogenicity and immunogenicity.

Background The oxidation of proteins by endogenously generated free radicals causes structural modifications in the molecules that lead to generation of neo-antigenic epitopes that have implications in various autoimmune disorders, including rheumatoid arthritis (RA). Collagen induced arthritis (CIA) in rodents (rats and mice) is an accepted experimental model for RA. Methodology/Principal Findings Hydroxyl radicals were generated by the Fenton reaction. Collagen type II (CII) was modified by •OH radical (CII-OH) and analysed by ultraviolet-visible (UV-VIS), fluorescence and circular dichroism (CD) spectroscopy. The immunogenicity of native and modified CII was checked in female Lewis rats and specificity of the induced antibodies was ascertained by enzyme linked immunosorbent assay (ELISA). The extent of CIA was evaluated by visual inspection. We also estimated the oxidative and inflammatory markers in the sera of immunized rats. A slight change in the triple helical structure of CII as well as fragmentation was observed after hydroxyl radical modification. The modified CII was found to be highly arthritogenic and immunogenic as compared to the native form. The CII-OH immunized rats exhibited increased oxidative stress and inflammation as compared to the CII immunized rats in the control group. Conclusions/Significance Neo-antigenic epitopes were generated on •OH modified CII which rendered it highly immunogenic and arthritogenic as compared to the unmodified form. Since the rodent CIA model shares many features with human RA, these results illuminate the role of free radicals in human RA.

Fine characterization of glucosylated human IgG by biochemical and biophysical methods.

Nonenzymatic glycosylation of proteins finally generates advanced glycation end products (AGEs). The Schiffs base and Amadori adduct are stages of early glycation. AGE-modified IgG may undergo conformational alterations and the final entity of the process may be involved in the pathogenesis of Rheumatoid Arthritis (RA). In this study, glycation of human IgG was carried out with varying concentrations of glucose. Effect of incubation period on glycation of IgG has also been studied. Amadori adduct was detected by nitroblue tetrazolium (NBT) dye. The glucose mediated structural alterations in IgG were studied by UV, fluorescence, CD, FT-IR, DLS and DSC spectroscopy, and SDS-PAGE. Glycation-induced aggregation in AGE-IgG was reported in the form of binding of thioflavin T and congo red. Furthermore, AGE-modified IgG exhibited hyperchromicity, decrease of tryptophan fluorescence accompanied by increase in AGE specific fluorescence, loss of β-sheet, appearance of new peak in FT-IR, increase in hydrodynamic size and melting temperature. SDS-PAGE results showed decrease in the band intensity of glycosylated-IgG compared to native IgG. Glycation-induced modifications and aggregation of IgG might be important in the pathogenesis of RA.

Methylglyoxal mediated conformational changes in histone H2A—generation of carboxyethylated advanced glycation end products

Methylglyoxal, an oxo-aldehyde has been implicated as a potential precursor in non enzymatic glycation reactions. Its role in the modification of extra cellular proteins has been extensively reported, but little is known about its modification of nuclear proteins, like histones. Here, we report the methylglyoxal induced modification of histone H2A which forms an essential part of intact core nucleosome. In this study commercially available histone H2A was subjected to in vitro non-enzymatic glycation by methylglyoxal. The structural alterations in the histone were characterised by various biophysical and biochemical techniques. The modified histone showed hyperchromicity at 276nm, loss in intrinsic tyrosine fluorescence intensity at 305nm along with a red shift, cross linking and dimer formation in SDS PAGE, induction of α-helix in CD spectroscopy, reduced hydrophobicity in ANS binding studies, accumulation of AGE products, increased carbonyl content, and appearance of a novel peak showing carboxyethylation complemented by a shift in amide I and amide II bands in ATR-FTIR spectroscopy. The modified histone exhibited increased melting temperatures (Tm) and enhanced heat capacities (Cp) in differential scanning calorimetric analysis. The results suggest that methylglyoxal significantly altered the structure of the nuclear histone H2A by non enzymatic glycation reaction. The conformational changes in histone H2A may influence the chromatin integrity which may have implications in various pathological conditions.

Human DNA damage by the synergistic action of 4-aminobiphenyl and nitric oxide: an immunochemical study.

4‐Aminobiphenyl (4‐ABP), an aromatic amine is a major environmental carcinogen found mainly in cigarette smoke. It has been vastly implicated in mutagenesis and cancer development. In this study, commercially available human placental DNA was exposed to 4‐ABP (1.3 mM) in presence of sodium nitroprusside (SNP; 8 mM) at 37°C for 3 h. The 4‐ABP + SNP‐mediated structural changes in human DNA were studied by ultraviolet, circular dichroism and fluorescence spectroscopy, thermal melting profile, agarose gel electrophoresis, and nuclease S1 digestibility assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single‐stranded regions and destabilization of hydrogen bonds. Modification was also visualized in agarose gel electrophoresis. Furthermore, nuclease S1 digestibility confirmed the generation of single strand breaks. Rabbits challenged with 4‐ABP‐SNP‐modified human DNA‐induced high‐titer immunogen‐specific antibodies, which showed Cross‐reaction with modified/unmodified DNA bases and ss‐DNA in competitive inhibition assay. The immunogen specificity of induced antibodies against 4‐ABP‐SNP‐modified human DNA was further confirmed in gel retardation assay. It may be concluded that induction of anti‐modified DNA antibodies could be due to perturbation in the DNA structure and its subsequent recognition by immunoregulatory cells as a foreign molecule.

Acquired immunogenicity of human DNA damaged by N‐hydroxy‐N‐acetyl‐4‐aminobiphenyl

4‐Aminobiphenyl, a known carcinogen, has many environmental sources like cigarette smoke, industrial waste, and so forth. It can be metabolized to form a potent mutagen, N‐hydroxy‐N‐acetyl‐4‐aminobiphenyl (N‐OH‐AABP) that undergoes further processing to form electrophilic nitrenium ions which interact with DNA‐forming covalent adducts, thereby exerting genotoxic effects. While the mutagenicity of N‐OH‐AABP has been amply reported, no extensive studies have been performed to assess the immunogenicity of N‐OH‐AABP‐modified DNA. In this study, human placental DNA was modified with N‐OH‐AABP, and the structural perturbations in the DNA molecule were evaluated by ultraviolet spectroscopy and nuclease S1 digestion. Native and N‐OH‐AABP‐modified DNA were used as antigens for immunizing female rabbits. The modified DNA was found to be highly immunogenic, eliciting high titer immunogen‐specific antibodies, while the native form was almost nonimmunogenic. The induced antibodies exhibited wide range of heterogeneity in recognizing various nucleic acid conformers and DNA bases. We also detected deposits of immune complex in glomerular basement membrane in rabbits immunized with N‐OH‐AABP‐DNA. Possible role of N‐OH‐AABP‐DNA in the induction of antibodies in cancer patients and the related consequences have been discussed.

Physicochemical and immunological studies on 4-hydroxynonenal modified HSA: implications of protein damage by lipid peroxidation products in the etiopathogenesis of SLE.

4-Hydroxynonenal (HNE) is the most abundant and toxic aldehyde generated by the oxidation of plasma membrane polyunsaturated fatty acids. Systemic lupus erythematosus (SLE), a chronic autoimmune disease, is primarily characterized by increased levels of autoantibodies, predominantly against ds-DNA. However, the initial antigenic stimulus for the disease etiopathogenesis has remained elusive. HNE has been extensively used as a biomarker of oxidative stress. It can form adduct with proteins, making them highly immunogenic. Increased levels of such aldehyde-protein adducts have been reported in various pathological states, including autoimmune disorders like SLE and arthritis. In the present study, HNE-mediated structural changes in human serum albumin (HSA) were characterized by UV, fluorescence, CD and FT-IR spectroscopy as well as by polyacrylamide gel electrophoresis. Furthermore, immunogenicity of native and HNE-modified HSA was probed in female rabbits. The HNE-modified HSA was highly immunogenic eliciting high titre immunogen specific antibodies. Binding of SLE anti-DNA antibodies was analyzed by direct binding and competition ELISA. The data show preferential binding of SLE autoantibodies to HNE-modified HSA as compared to native HSA or native DNA. Our results suggest that HNE modification generates neoepitopes on HSA causing enhanced autoantibodies production. The results point towards the possible role of HNE-modified HSA in SLE etiopathogenesis.

Peroxynitrite-induced modification of H2A histone presents epitopes which are strongly bound by human anti-DNA autoantibodies: Role of peroxynitrite-modified-H2A in SLE induction and progression

Peroxynitrite is a potent oxidant and nitrating agent and has in vivo existence. It is a powerful proinflammatory substance and may increase vascular permeability in inflamed tissues. Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease of unknown etiology. Since its discovery, numerous self- and non-self, nuclear, and cytoplasmic antigens have been suggested as stimuli for SLE initiation, but the exact trigger is yet to be identified. In this study, an attempt has been made to investigate the binding characteristics of SLE anti-DNA autoantibodies to native DNA and native and peroxynitrite-modified H2A histone to explore the possible role of modified protein antigen(s) in SLE initiation and progression. The nuclear protein (H2A histone) was modified by peroxynitrite synthesized in our laboratory. The peroxynitrite-modified H2A revealed generation of nitrotyrosine, dityrosine, and carbonyls when subjected to investigation by physicochemical methods. Binding characteristics and specificity of SLE anti-DNA antibodies were analyzed by direct binding and inhibition enzyme-linked immunosorbent assay. The data show preferential binding of SLE autoantibodies to peroxynitrite-modified H2A histone in comparison with native H2A histone or native DNA. A band shift assay further substantiated the enhanced recognition of peroxynitirite-modified H2A histone by anti-DNA autoantibodies. The results suggest that peroxynitrite modification of self-antigen(s) can generate neoepitopes capable of inducing SLE characteristic autoantibodies. The preferential binding of peroxynitrite-modified H2A histone by SLE anti-DNA antibodies points out the likely role of oxidatively modified and nitrated H2A histone in the initiation/progression of SLE. Moreover, oxidatively modified and nitrated nuclear protein antigen, rather than nucleic acid antigens, appear to be more suitable as a trigger for SLE.