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Ever increasing information on genome and proteome has offered fascinating details and new opportunities to understand the molecular biology. It is now known that histone proteins surrounding the DNA play a crucial role in the chromatin structure and function. Histones undergo a plethora of posttranslational enzymatic modifications that influence nucleosome dynamics and affect DNA activity. Earlier research offered insights into the enzymatic modifications of histones; however, attention has been diverted to histone modifications induced by by-products of metabolism without enzymatic engagement in the last decade. Nonenzymatic modifications of histones are believed to be crucial for epigenetic landscape, cellular fate and for role in human diseases. Glycation of histone proteins constitutes the major nonenzymatic modifications of nuclear proteins that have implications in diabetes and cancer. It has emerged that glycation damages nuclear proteins, modifies amino acids of histones at crucial locations, generates adducts affecting histone chromatin interaction, develops neo-epitopes inducing specific immune response and impacts cell function. Presence of circulating antibodies against glycated histone proteins in diabetes and cancer has shown immunological implications with diagnostic relevance. These crucial details make histone glycation an attractive focus for investigators. This review article, therefore, makes an attempt to exclusively summarize the recent research in histone glycation, its impact on structural integrity of chromatin and elaborates on its role in diabetes and cancer. The work offers insights for future scientists who investigate the link between metabolism, biomolecular structures, glycobiology, histone-DNA interactions in relation to diseases in humans.
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Diabetes Mellitus , Neoplasias , Glicosilación , Histonas/metabolismo , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
This study elucidates the immunological implications of methylglyoxal (MGO) modified LDL in diabetes type 2 patients (T2DM). Under in-vitro modifications, MGO altered the tertiary structure of LDL. TNBS and phenanthrenequinone assays confirmed lysine and arginine residues as main targets of MGO in LDL. HPLC and LCMS studies confirmed the generation of Nϵ-(carboxymethyl) lysine in the modified protein. Comet assay showing increased tail length of DNA in lymphocytes inferred the cytotoxicity of MGO-LDL. The easy penetration of MGO-LDL into the nucleus is possibly a consequence of its reduced size, post-modification, as observed from the studies on hydrodynamic radii studies in DLS experiments. MGO-LDL was found to be more immunogenic, as compared to native LDL, in immunological studies conducted on experimental rabbits. Our results reflect the presence of neo-antigenic determinants on modified LDL. Competitive inhibition ELISA suggested the presence of neo-epitopes with marked immunogenicity eliciting specific immune response. Binding studies on purified IgG confirmed the enhanced and specific immunogenicity of MGO-LDL. Studies on interaction of MGO-LDL with the circulating auto-antibodies from T2DM patients showed high affinity of serum-antibodies towards MGO-LDL. This study suggests a potent role of glycoxidatively modified LDL in the generation of auto-immune response in T2DM patients.
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The role of aberrant protein modifications in cancer and its diagnosis have emerged as a promising research field. Nonenzymatic glyco-oxidation of proteins under oxidative stress has been associated with carcinogenesis through advanced glycation end products (AGE)-receptors for advanced glycation end products (RAGE) axis. Modified proteins that are immunogenic and stimulate cellular and humoral immune responses are being studied to develop early detection markers of cancer. This study has probed the structural alternations; leading to the formation of adducts and aggregates, in histone H2A upon in vitro modification by methylglyoxal (MG). The immunogenicity of modified histone H2A and its binding with cancer autoantibodies was also assessed. MG induced lysine side chain modifications, blocking of free amino groups and the formation of condensed cross structures in histone H2A; and its effect was inhibited by carbonyl scavengers. It led to the adduct formation and generation of N-epsilon-(carboxyethyl)lysine (CEL) and its decomposition forms as revealed by Matrix-assisted laser desorption ionization-mass spectrometry, high-performance liquid chromatography and LC-MS. MG-H2A showed amorphous aggregate formation under electron microscopy and altered binding with DNA in circular dichroism studies. The modified histone elicited high titer immunogen-specific antibodies in rabbits when compared with the native, thus pointing toward the generation of neo-epitopes in MG-H2A. The autoantibodies derived from cancer patients exhibited enhanced binding with MG-H2A as compared with the native histone in enzyme-linked immunosorbent assay and gel retardation assay. This reflects sharing of epitopes on MG-H2A and histones in cancer patients. The neo-epitopes on H2A may be responsible for induction and elevated levels of antibodies in cancer patients. Thus, MG-H2A may be considered as potential antigenic candidate for auto-immune response in cancer.
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Anticuerpos Antineoplásicos/inmunología , Autoanticuerpos/inmunología , Neoplasias Gastrointestinales/inmunología , Histonas/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias de la Próstata/inmunología , Piruvaldehído/metabolismo , Adolescente , Adulto , Animales , Estudios de Casos y Controles , Epítopos/inmunología , Femenino , Histonas/química , Histonas/metabolismo , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Persona de Mediana Edad , ConejosRESUMEN
This study is to probe the pattern of antibiotic resistance against aminoglycosides and its mechanism in E. coli obtained from patients from Chennai, India. Isolation and identification of pathogens were done on MacConkey agar. Antimicrobial sensitivity testing was done by disc diffusion test. The identification of genes encoding aminoglycoside modifying enzymes was done by Polymerase Chain Reaction (PCR). Out of 98 isolates, 71 (72.45%) isolates were identified as E. coli and the remaining 27 (27.55%) as other bacteria. Disc diffusion method results showed a resistance level of 72.15% for streptomycin, 73.4% for gentamicin, 63.26% for neomycin, 57.14% for tobramycin, 47.9% for netilmicin, and 8.16% for amikacin in E. coli. PCR screening showed the presence of four genes, namely, rrs, aacC2, aacA-aphD, and aphA3, in their plasmid DNA. The results point towards the novel mechanism of drug resistance in E. coli from UTI patients in India as they confirm the presence of genes encoding enzymes that cause resistance to aminoglycoside drugs. This could be an alarm for drug prescription to UTI patients.
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Aminoglicósidos/genética , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Infecciones Urinarias/microbiología , Antibacterianos/farmacología , Escherichia coli/enzimología , Humanos , India , Pruebas de Sensibilidad MicrobianaRESUMEN
Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 µM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients.
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Anticuerpos Antinucleares/inmunología , ADN/química , Diabetes Mellitus Tipo 1/inmunología , Ácido Peroxinitroso/química , Plásmidos/genética , Aductos de ADN/genética , Roturas del ADN , Epítopos/inmunología , Guanina/análogos & derivados , Guanina/biosíntesis , Guanina/sangre , Guanina/química , Humanos , Inmunoglobulina G/inmunología , Nitritos/sangre , Nitritos/química , Ácido Peroxinitroso/sangre , Plásmidos/química , Plásmidos/inmunologíaRESUMEN
The deleterious impact of toxic constituents of hair dyes over the human health has gained immense attention in the recent past. Their oncogenicity, mutagenicity, role in protein modification, impact on cellular metabolism has been documented. There is little information on the mechanism of reactivity of hair dye components with the nucleic acids and its implications. This work, therefore, uses computational, biophysical/biochemical, microscopic and cell-based study to analyze the interaction of monocyclic aromatic amine and a hair dye component, 4-chloro-orthophenylenediamine (4-Cl-OPD) with the DNA, its impact on DNA structure and cell survival. The results suggest that 4-Cl-OPD binds with the DNA in minor groove of the duplex involving three base pairs preferentially the G-C residues, induces strand breaks and makes DNA thermally labile through loss of hydrogen bonding/base unstacking. 4-Cl-OPD causes fragmentation of DNA, reduction in size of the molecule, alters B-DNA conformation and disrupts its secondary structure. The modified DNA gives fragmented appearance, shows broken strands and aggregation in ultra-structural analysis. 4-Cl-OPD induces ROS generation in lymphocytes, increases the comet's average tail length and reduces the viability of lymphocytes. This study forms a base for establishing the direct toxicity of 4-Cl-OPD at the molecular and cellular level through direct production of superoxide radicalCommunicated by Ramaswamy H. Sarma.
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Tinturas para el Cabello , Humanos , ADN/química , Fenilendiaminas , Conformación de Ácido NucleicoRESUMEN
4-Chloro-1,2-phenylenediamine (4-Cl-OPD) is a halogenated aromatic diamine used as a precursor in permanent hair color production. Despite its well-documented mutagenic and carcinogenic effects in various in vitro and in vivo models, its role in fibrillar aggregate formation and their genotoxic effect in therapeutic proteins has received less attention. The significance of human serum albumin (HSA) arises from its involvement in bio-regulatory and transport processes. HSA misfolding and aggregation are responsible for some of the most frequent neurodegenerative disorders. We used various complementary approaches to track the formation of amyloid fibrils and their genotoxic effect. Molecular dynamics study demonstrated the complex stability. The impact of 4-Cl-OPD on the structural dynamics of HSA was confirmed by Raman spectroscopy, X-ray diffraction, HPLC and SDS-PAGE. Fibrilllar aggregates were investigated using Congo red assay, DLS, and SEM. The genotoxic nature of 4-Cl-OPD was confirmed using plasmid nicking assay and DAPI staining, which revealed DNA damage and cell apoptosis. 4-Cl-OPD provides a model system for studying fibrillar aggregation and their genotoxic potential in the current investigation. Future studies should investigate the inhibition of the aggregation/fibrillation process, which may yield valuable clinical insights.
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Methyl methanesulfonate (MMS) is a highly toxic DNA-alkylating agent that has a potential to damage the structural integrity of DNA. This work employed multiple biophysical and computational methods to report the MMS mediated structural alterations in the DNA (MMS-DNA). Spectroscopic techniques and gel electrophoresis studies revealed MMS induced exposure of chromophoric groups of DNA; methylation mediated antiâsyn conformational change, DNA fragmentation and reduced nucleic acid stability. MMS induced single-stranded regions in the DNA were observed in nuclease S1 assay. FT-IR results indicated MMS mediated loss of the assigned peaks for DNA, partial loss of C-O ribose, loss of deoxyribose region, C-O stretching and bending of the C-OH groups of hexose sugar, a progressive shift in the assigned guanine and adenine peaks, loss of thymine peak, base stacking and presence of C-O-H vibrations of glucose and fructose, indicating direct strand breaks in DNA due to backbone loss. Isothermal titration calorimetry showed MMS-DNA interaction as exothermic with moderate affinity. Dynamic light scattering studies pointed towards methylation followed by the generation of single-stranded regions. Electron microscopy pictured the loss of alignment in parallel base pairs and showed the formation of fibrous aggregates in MMS-DNA. Molecular docking found MMS in close contact with the ribose sugar of DNA backbone having non-bonded interactions. Molecular dynamic simulations confirmed that MMS is capable of interacting with DNA at two levels, one at the level of nitrogenous bases and another at the DNA backbone. The study offers insights into the molecular interaction of MMS and DNA.Communicated by Ramaswamy H. Sarma.
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ADN , Ribosa , Daño del ADN , Reparación del ADN , Metilmetanosulfonato/toxicidad , Simulación del Acoplamiento Molecular , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
AIMS: This study aims to investigate the role of glyoxal modified LDL in the immunopathology of diabetes and cardiovascular disease. BACKGROUND: Glycoxidation of proteins is widely studied in relation to diabetes and cardiovascular disease. OBJECTIVE: This study probed the glyoxal mediated modifications in LDL, analyzed the immunogenicity of the glycated LDL and ascertained the presence of circulating antibodies against modified LDL in patients with type 2 diabetes mellitus (T2DM), coronary artery disease (CAD) and patients with both (T2DM+CAD). METHODS: Glyoxal mediated modifications in LDL were studied by multiple spectroscopic techniques, high-performance liquid chromatography and electron microscopy. Immunization studies were carried in New Zealand rabbits. The presence of antibodies against glyoxal modified LDL in immunized rabbits and human subjects was analyzed by ELISA. RESULTS: Glyoxal altered the structural integrity of LDL and led to the formation of AGEs. It decreased the alpha-helix content of LDL; increased ß sheet formation, increased carbonyl content and decreased free lysine and arginine content. Modified LDL showed aggregation, generation of of Nε- (Carboxymethyl) lysine and the formation of amorphous type aggregates. It exhibited high antigenicity and generated a specific immune response that shared common antigenic determinants with other glycated proteins. Direct binding data showed the presence of anti-glyoxal modified LDL antibodies in patients with T2DM, CAD and patients with both T2DM and CAD. Further analysis in competitive binding assay revealed specific binding characteristics of auto-antibodies. Sera from patients with T2DM+CAD exhibited the highest binding with glyoxal modified LDL. CONCLUSION: Glyoxal-modified LDL has neo-antigenic determinants that cause the generation of circulating antibodies in diabetes and coronary artery disease. The study might have potential relevance in biomarker development.
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Enfermedad de la Arteria Coronaria , Diabetes Mellitus Tipo 2 , Animales , Autoanticuerpos , Epítopos , Glioxal , Humanos , ConejosRESUMEN
Reactive oxygen species (ROS) cause oxidative damage to proteins and generate deleterious by-products which induce a breakdown of immune tolerance and produce antibodies against host macromolecules with implication in human diseases. This study characterizes the hydroxyl radical (OH) modifications of insulin, evaluates its cytotoxicity and immunogenicity, and probes its role in type 2 diabetes (T2DM) autoimmunity. The results demonstrate susceptibility of insulin to modifications induced by OH, causing exposure of its chromophoric aromatic amino acid residues, quenching of tyrosine fluorescence intensity, loss of α-helix and gain in ß content. Modification causes re-arrangement of native interactions of the aromatic residues in insulin. It enhanced the carbonyl content in insulin, exposed its hydrophobic patches and generated non-fibrillar, amorphous type of aggregates that are cytotoxic in nature. Native insulin induced low titre antibodies in immunized rabbits, whereas OH modified insulin generated a strong immune response. Competitive ELISA studies showed high specificity of antibodies generated against OH modified insulin towards the modified protein. Cross reaction studies showed the presence of common antigenic determinants on various oxidised proteins. Since T2DM patients show increased ROS production, oxidation of insulin is expected to occur, which might amplify autoimmune reactions against insulin. True to the assumption, direct binding ELISA showed the presence of anti-OH insulin circulating antibodies in T2DM patients which are specific for the oxidized insulin. In conclusion, insulin loses structural integrity to OH, forms cytotoxic amorphous aggregates, turns highly immunogenic and elicits humoral response in T2DM patients.
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Diabetes Mellitus Tipo 2 , Radical Hidroxilo , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Inmunidad , Insulina , Conejos , Especies Reactivas de OxígenoRESUMEN
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Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α2M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α2M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α2M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in ß-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α2M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α2M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α2M as compared to the native α2M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α2M and makes it immunogenic. The presence of neo-antigenic determinants on modified α2M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease. Communicated by Ramaswamy H. Sarma.
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Artritis Reumatoide , Autoanticuerpos/inmunología , alfa-Macroglobulinas/inmunología , Animales , Epítopos/inmunología , Humanos , Ácido Peroxinitroso , ConejosRESUMEN
Glycation and oxidation induce structural alterations in the proteins in an interdependent manner with consequent pathological implications. The published literature presents wide range of modifications in conformational characteristics of proteins by glycation and oxidation; however, there is little data that could elaborate the cumulative effect of both the processes. This study has analysed the modifications in IgG by methylglyoxal (MG) (glycative stress), hydroxyl radical ([Formula: see text]) (oxidative stress) and by their combined action i.e. [Formula: see text] treatment of MG glycated IgG (glycoxidation). It further addresses the implications of the altered structural integrity of IgG on its immunological characteristics and impact on haematological parameters in rabbits. Using circular dichroism, FTIR, SDS-PAGE analysis, thioflavin-T fluorescence assay, congo red absorbance analysis, dynamic light scattering, transmission electron microscopy, ELISA, blood cell counts and rectal temperature studies, we report that the glycoxidative modification caused maximum alteration in the IgG as compared to the glycatively and oxidatively modified protein. Far-UV CD results confirmed the highest decline in the beta-pleated sheet content of the protein by glycoxidation. The damage led to the reduced flexibility and enhanced electronic interactions in IgG as observed by near-UV CD. Modifications caused cross-linking and adduct formation in the serum protein. The electron micrograph confirmed amorphous aggregation in modified IgG. The modifications increased the hydrodynamic radius of IgG by allowing the attachment of [Formula: see text] and MG residues. The glycoxidatively modified IgG induced the maximum antibody titres that showed high specificity towards the altered IgG. The glycoxidation of IgG leads to activation of inflammatory pathways.
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Fenómenos Biofísicos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Animales , Antígenos/metabolismo , Benzotiazoles/metabolismo , Temperatura Corporal , Dicroismo Circular , Rojo Congo , Reacciones Cruzadas , Dispersión Dinámica de Luz , Femenino , Fluorescencia , Glicosilación , Inmunoglobulina G/química , Inmunoglobulina G/ultraestructura , Oxidación-Reducción , Unión Proteica , Conejos , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The detection of autoantibodies generated against modified proteins that stimulate cellular and humoral immune response has developed a lot of interest in the recent years and a search for biomarkers for the early detection of diseases has increased. IgG protein has earned attention for its possible modifications under hyperglycaemic conditions in rheumatoid arthritis, wherein dicarbonyl stress has been reported to alter the structural integrity of the protein. This report suggests that the interaction of the methylglyoxal with the IgG has consequences in the autoimmunopathology of rheumatoid arthritis. Our molecular docking analysis of methylglyoxal and IgG revealed a close interaction between the two molecules. TNBS studies confirmed the interaction by showing a decline in free lysine-arginine content post-MG modifications in IgG. The modified IgG was thermally more stable and showed the generation of glycation adducts N-epsilon-carboxyethyllysine. Rheumatoid arthritis patients showed enhanced carbonyl stress which was expected to induce structural changes in the epitope makeup of IgG. The ELISA studies and gel retardation assay confirmed auto-antibodies against MG modified IgG (MG-IgG) pointing towards the generation of neoepitopes upon IgG after interaction with MG. This study establishes the IgG modification in RA patients under alter carbonyl concentrations.
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Artritis Reumatoide/inmunología , Inmunidad Celular , Inmunoglobulina G/inmunología , Piruvaldehído/inmunología , Artritis Reumatoide/sangre , Artritis Reumatoide/patología , Autoanticuerpos/sangre , Autoanticuerpos/química , Autoanticuerpos/inmunología , Biomarcadores/sangre , Epítopos/sangre , Epítopos/inmunología , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/inmunología , Humanos , Inmunoglobulina G/química , Simulación del Acoplamiento Molecular , Estrés Oxidativo/inmunología , Conformación Proteica , Piruvaldehído/químicaRESUMEN
Human serum albumin (HSA) - the most abundant plasma protein plays an important role in the transport of endogenous and exogenous molecules in the body. Its modifications have been implicated in a variety of pathological disorders. We have studied the interaction of HNE with HSA at a molecular level by docking experiment and the results suggest a strong interaction between HNE and HSA. Immunological studies revealed that the circulating auto-antibodies in rheumatoid arthritis (RA) patients have a stronger affinity towards HNE-modified HSA. The HSA isolated from RA patients (RA-HSA) exhibited HNE mediated damage in its secondary and tertiary structure when compared to HSA derived from healthy human subjects (NH-HSA). RA patients presented a significant rise in carbonyls and a considerable decline in free thiol content. Preferential binding of experimentally induced anti-HNE-HSA antibodies to RA-HSA over NH-HSA was observed by ELISA. The results suggest HNE induced structural perturbations in HSA with neoepitopes that generate anti-HNE-HSA antibodies in RA. Hence, HNE-HSA may provide lead towards the development of a biomarker for the disease.
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Aldehídos/química , Artritis Reumatoide/sangre , Biomarcadores/sangre , Albúmina Sérica Humana/química , Aldehídos/sangre , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Peroxidación de Lípido/efectos de los fármacos , Estrés Oxidativo/genética , Unión Proteica , Albúmina Sérica Humana/inmunología , Albúmina Sérica Humana/aislamiento & purificaciónRESUMEN
Structural alterations in proteins under oxidative stress have been widely implicated in the immuno-pathology of various disorders. This study has evaluated the extent of damage in the conformational characteristics of IgG by hydroxyl radical (OH) and studied its implications in the immuno-pathology of rheumatoid arthritis (RA). Using various biophysical and biochemical techniques, changes in aromatic microenvironment of the IgG and the protein aggregation became evident after treatment with OH. The SDS-PAGE study confirmed the protein aggregation while far ultraviolet circular dichroism spectroscopy (Far-UV CD) and fourier transform infrared spectroscopy (FTIR) inferred towards the alterations in secondary structure of IgG under OH stress. Dynamic light scattering showed that the modification increased the hydrodynamic radius and polydispersity of IgG. The free arginine and lysine content reduced upon modification. OH induced aggregation was confirmed by enhanced thioflavin-T (ThT) fluorescence and red shift in the congo red (CR) absorbance. The study on experimental animals reiterates the earlier findings of enhanced immunogenicity of OH treated IgG (OH-IgG) compared to that of native IgG. OH-IgG strongly interacted with the antibodies derived from the serum of 80 rheumatoid arthritis (RA) patients. The overwhelming and strong tendency of OH-IgG to bind the antibodies derived from the serum of RA patients points towards the modification of IgG under patho-physiological conditions in RA that generate neo-epitopes and eventually cause the generation of auto antibodies that circulate in the patient sera. Further studies on this aspect may possibly lead to the development of a biomarker for RA.
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Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Radical Hidroxilo/química , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Autoanticuerpos/sangre , Estudios de Casos y Controles , Humanos , Oxidación-Reducción , Estrés OxidativoRESUMEN
Hyperglycaemic influence on carcinogenesis and tumour progression is emerging as a link between diabetes and cancer. This work establishes the disturbed structural integrity of nucleosomal linker histone H1 by methyglyoxal (MG) and then correlates the role of modified H1 in the auto-immunopathogenesis of multiple cancers. MG modification caused a loss of free ε-amino groups in H1 and raised its ß-sheet structural component with a consequence of non amyloid aggregation. It changed the folding-unfolding denaturation pattern of H1 and attached itself to the lysine residues of the protein eventually making up Nε-(carboxyethyl) lysine. The structural variations act as extra antigenic determinants on H1 that yield aggressive antibody response, when immunised in rabbits. The ELISA tests proved the immunoglobulin response very specific and gel based studies established the preferential binding of antibodies generated against MG-H1 with the modified protein. Cross reaction analysis inferred the multiple specific natures of immunoglobulins with binding tendencies against different inhibitors. The immunoglobulin content in blood sera derived from human subjects with tumours of oesophagus, stomach, gall bladder and ovary confirmed the antibody presence against MG-H1 and competitive ELISA showed their high specificity. This may suggest a link between nucleosomal linker H1, hyperglycaemia, glycoxidation and cancer.
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Histonas/inmunología , Histonas/metabolismo , Neoplasias/inmunología , Adulto , Animales , Reacciones Cruzadas , Epítopos/inmunología , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/metabolismo , Femenino , Neoplasias de la Vesícula Biliar/inmunología , Neoplasias de la Vesícula Biliar/metabolismo , Histonas/química , Humanos , Masculino , Neoplasias/metabolismo , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Piruvaldehído/metabolismo , Conejos , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismoRESUMEN
Structural rearrangements and condensations of proteins under hyperglycemic stress have been implicated in various pathological disorders. This study aims to probe the role of methylglyoxal (MG) modified human immunoglobulin G (MG-IgG) in immuno-pathology of type 2 diabetes mellitus (T2DM). MG was found to perturb the structural integrity of IgG, affect its aromatic micro-environment and cause the generation of advanced glycation end products (AGEs) and aggregate adducts. It liberated the hydrophobic pockets of the protein, reduced its ß pleated sheet structure and affected its tertiary conformation. Transition from ß sheet to α helix and random coil was also observed in IgG upon modification by MG. It acted with strong oxidative potential and caused oligomerisation and disordered or amorphous type aggregation in the modified protein. Modified IgG had a cytotoxic and genotoxic impact. The MG modified IgG presented novel antigenic determinants that lead to an aggressive immune response. The antibodies had high affinity towards the immunogen. Auto-antibodies derived from T2DM patients exhibited strong affinity towards the modified IgG in comparison to the unmodified protein. Specificity of serum antibodies from T2DM patients was further confirmed by competitive-inhibition ELISA. The potential role of MG-IgG in the immunopathogenesis of T2DM has been discussed.
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Diabetes Mellitus Tipo 2/inmunología , Inmunoglobulina G/metabolismo , Piruvaldehído/metabolismo , Daño del ADN , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Voluntarios Sanos , Humanos , Linfocitos/metabolismo , Adulto JovenRESUMEN
Glycoxidation plays a crucial role in diabetes and its associated complications. Among the glycoxidation agents, methylglyoxal (MG) is known to have very highglycationpotential witha concomitant generation of reactive oxygen species (ROS) during its synthesis and degradation. The presentstudy probes the MG and ROSinduced structural damage to immunoglobulin G (IgG) and alterations in its immunogenicity in diabetes type 2 patients (T2DM). Human IgG was first glycated with MG followed by hydroxyl radical (OHâ¢) modification. Glycoxidation mediated effects on IgG were evaluated by various physicochemical techniques likeultraviolet (UV) and fluorescence spectroscopy, 8-anilinonaphthalene-1-sulfonic acid (ANS) binding studies, carbonyl andfree sulfhydryl groups assay, matrix assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF), red blood cell (RBC) haemolysis assay, Congored (CR) staining analysis and scanning electron microscopy (SEM). The results revealed hyperchromicityin UV, advanced glycation end product (AGE)specific and ANS fluorescence, quenching in tyrosine and tryptophan fluorescence intensity,enhanced carbonyl content,reduction in free sulfhydryl groups,pronounced shift in m/z value of IgGand decrease in antioxidant activity in RBC induced haemolysis assayupon glycoxidation. SEM and CRstaining assay showed highly altered surface morphology in glycoxidised sample as compared to the native. Enzyme linked immunosorbent assay (ELISA) and band shift assay were performed to assess the changes in immunogenicity of IgG upon glyoxidation and its role in T2DM. The serum antibodies derived from T2DM patients demonstrated strong affinity towards OH⢠treated MG glycatedIgG (OHâ¢-MG-IgG) when compared to native IgG (N-IgG) or IgGs treated with MG alone (MG-IgG) or OH⢠alone (OHâ¢-IgG). This study shows the cumulating effect of OH⢠on the glycation potential of MG. The results point towards the modification of IgG in diabetes patients under the effect of glycoxidative stress, leading to the generation of neo-epitopes on theIgG molecule and rendering it immunogenic.
Asunto(s)
Diabetes Mellitus Tipo 2/inmunología , Epítopos/inmunología , Radical Hidroxilo/química , Inmunoglobulina G/química , Antioxidantes/metabolismo , Autoanticuerpos/inmunología , Estudios de Casos y Controles , Eritrocitos/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Hemólisis , Humanos , Oxidación-Reducción , Piruvaldehído/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Novel immunological epitopes upon glyoxidation modified proteins have been discovered and multi-specific natural antibodies against them have been identified. The association of glyoxidation with cancer has also been reported. We probed the link of glyoxidation of histone H2A with autoimmune response in cancer. METHODS: We report the formation of thermo stable amorphous aggregate formation in histone H2A upon methylglyoxal modification using circular dichroism (CD) analysis and transmission electron microscopy. RESULTS: The modified histone was found to be highly immunogenic that generates specific immune response in rabbits as analysed by cross reaction studies by competitive ELISA and Western blotting technique. The anti-methylglyoxal modified H2A antibodies were found in the circulating autoantibodies in various types of cancer patients by immunosorbent assay and gel retardation studies. The results clearly indicate the formation of highly specific antibodies in cancer patients against glyoxidation modified histone H2A with cross reactive tendencies with other glycated proteins and nucleic acids. CONCLUSIONS: The results are important because a link between AGE-RAGE axis (Advanced glycation end products and Receptors for AGEs) and carcinogenesis is emerging and the role of glyoxidation of proteins is expected in the development of biomarkers for the early detection of cancer.