Zarina Arif

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.

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.

Glycated-H2A histone is better bound by serum anti-DNA autoantibodies in SLE patients: Glycated-histones as likely trigger for SLE?

Abstract Histones are the most abundant proteins associated with genomic DNA. Recent observations show that histones are quite susceptible to non-enzymatic glycation which results in the generation of free radicals causing structural perturbations. In this study, our aim is to define the role of deoxyribose-modified H2A histone in SLE initiation/progression. Glycation reaction was carried out by incubating H2A histone with 10 mM deoxyribose for 21 days at 37 °C. Structural changes in glycated-H2A were studied by various physico-chemical techniques. The antigen-antibody interaction was studied by direct binding, inhibition ELISA and mobility shift assay. Deoxyribose-modified-H2A histone showed increased hyperchromicity and increased fluorescence intensity. CD results demonstrated almost 50% loss in alpha helix conformation as a consequence of glycation. This was supported by an increase in Tm value vis-à-vis thermal stability. Glycated-H2A showed cross linking in SDS-PAGE. SLE sera positive for anti-nDNA autoantibodies showed preference for deoxyribose-modified-H2A histone compared to native H2A histone or native DNA. Inhibition ELISA supported the above findings. Band shift assay further reiterated the preferential recognition of glycated-H2A over native H2A by SLE IgG autoantibodies. Deoxyribose-modified-H2A histone exhibited damage as revealed by various physico-chemical studies. Glycation of H2A has resulted in the generation of neo-epitopes on H2A histone, which are preferably bound by SLE anti-nDNA autoantibodies. It implies that deoxyribose-modified-H2A may trigger immune response resulting in the generation of anti-glycated H2A antibodies with DNA cross reacting properties.

NATIVE DNA FRAGMENTS PHOTOCROSSLINKED TO PSORALEN BINDS TO ANTI-B AND ANTI-Z DNA ANTIBODIES

Calf thymus DNA fragments of varying size (100-600 bp) were covalently linked to psoralen in the presence of 365 nm UV light. The crosslinked species were separated from the monoadducts by hydroxyapatite column chromatography. The DNA-psoralen crosslink showed binding with naturally occurring anti-DNA autoantibodies. This was substantiated by the recognition of affinity purified IgG by DNA-psoralen crosslinks of varying DNA size. The photo-crosslinks exhibited considerable recognition of both monoclonal and polyclonal anti-Z-DNA antibodies in inhibition ELISA and band shift assay.

Elucidating the impact of glucosylation on human serum albumin: A multi-technique approach

Early glycation products as well as advance glycation end products are involved in pathogenesis of diabetes. Most of studies carried out on AGEs and their possible role in assessing diabetes complications, whereas only a few were focused to highlight the role of Amadori products. In this study, an attempt has been made to investigate a structural and immunological characterizations of Amadori-albumin upon early glucosylation because albumin undergoes fast glycation under hyperglycaemic condition. Amadori-albumin formation was determined by NBT assay and Amadori adducts in glycated samples were confirmed by LC-MS. Structural alterations in Amadori-albumin were characterized by loss in fluorescence intensity, loss in secondary and tertiary structures, exposure of hydrophobic patches, shifting in Amide bands and increment in hydrodynamic radius. Further, presence to autoantibodies against Amadori-albumin in diabetes patients were confirmed by direct binding ELISA and inhibition ELISA. Immunological studies results showed that autoantibodies present in diabetic patients with and without chronic kidney disease (CKD) showed significant binding with Amadori-albumin in comparison to the native protein. Anti Amadori-albumin antibodies predominantly present in CKD patients compare to without CKD patients. Band shift assay results showed true interaction between Amadori-albumin and autoantibodies present in CKD patients. Glucosylation results showed structural alterations in Amadori-albumin and hence generation of neo-epitopes in HSA molecule. Such modifications rendering the protein highly immunogenic that may be recognized as foreign molecule by immune cells and induced autoantibodies in diabetic patients. These finding signify the role of Amadori-albumin in kidney dysfunction in diabetes and raised level of autoantibodies may be used as biomarker for progression of CKD.

Autoantibodies-Like Antigen Binding Characteristics of Induced Antibodies Against Polylysine-Polyglutamate Complex

Rabbits immunized with polylysine-polyglutamate complex induced high titer antibodies. The immune IgG exhibited high degree of specificity towards the immunogen. In a competition assay, nDNA, heat denatured DNA and total buffalo thymus RNA were inhibitory. An appreciable binding with polyglutamate, poly(rG).poly(dC) poly(dG).poly(dC), cardiolipin, poly(dA-dU). Poly(dA-dU) and Z- or Z-like conformations demonstrates the polyspecificity of induced antibodies. The immunological specificity of induced antibodies was comparable to autoantibodies derived from SLE. The results might be of some help in understanding the etiology of autoimmune process.

Hyperglycemia induced reactive species trigger structural changes in human serum albumin of type 1 diabetic subjects

Chronic oxidative stress fuels pathogenesis of a large set of diseases. Oxidative stress is the cause and consequence of numerous diseases including type 1 diabetes mellitus (T1DM), in which there is selective destruction of insulin producing pancreatic β-cells. Studies have documented that hyperglycemia produces profound stress. In vivo production of numerous reactive oxygen, nitrogen, chlorine species and lipid/sugar oxidation products in T1DM patients may be the result of persistent hyperglycemia. Post-translational modifications by reactive species may create new antigenic epitopes and play a role in the development of autoimmune response. In this paper our main focus was to establish the effect of existing hyperglycemia induced oxido-nitrosative stress in T1DM patients on the integrity of human serum albumin. Raised nitric oxide, carbonyl, RBC hemolysis, lowered ferric reducing antioxidant power (FRAP), thiol and deformed RBC in T1DM are all highly suggestive of persistent oxido-nitrosative stress. Hyperglycemia induced generation of advanced glycation end products (AGEs) was established by LCMS. Chronic oxido-nitrosative stress can modify HSA in T1DM patients, producing immunologically active albumin. Therefore, it is speculated that the aberrant HSA may play a role in the initiation/progression of T1DM.

Non-enzymatic glucosylation induced neo-epitopes on human serum albumin: A concentration based study

Hyperglycaemia induced non enzymatic glycation is accelerated in diabetic patients and aggressively involved in diabetes progression. Human serum albumin (HSA) is the most abundant protein in blood circulation. In hyperglycaemia, it undergoes fast glycation and results in the impairment of structure. Our previous study has demonstrated structural alterations in Amadori-albumin modified with different glucose concentrations from physiological to pathophysiological range. Here, we focused on immunological characterization of Amadori-albumin. Immunogenicity of Amadori-albumin was analysed by direct binding and competitive ELISA. Amadori-albumin was found to be highly immunogenic (expect albumin modified with 5mM) and induced high titre antibodies depending upon the extent of modification. Very high titre antibodies were obtained with albumin modified with 75mM glucose as compared to native albumin. Anti-Amadori-albumin-IgG from rabbit sera exhibited increased recognition of Amadori-albumin than native albumin in competitive immunoassay. Alteration induced in albumin after glucosylation has made it highly immunogenic. Induced antibodies were quite specific for respective immunogens but showed cross-reaction with other Amadori/native proteins. It suggests that glucosylation has generated highly immunogenic epitopes on albumin. Formation of high molecular weight immune complex with retarded mobility further supports specificity of anti-Amadori-albumin-IgG towards Amadori-albumin. It may be concluded that due to early glycation, an array of modification occurred in HSA structure. Such gross structural changes might favour polymerization of most of the native epitopes into potent immunogenic neo-epitopes, but some original epitopes were still active and has contributed in the immunogenicity. It could be concluded that induction of anti-Amadori-albumin antibodies may be due to protection of glucose modified albumin from protiolytic breakdown. We assumed that this type of protein modifications might occur in diabetic patients in hyperglycaemic conditions that may be recognised as foreign molecules and can induce autoantibodies. Increased level of anti-Amadori-albumin autoantibodies may be used as a biomarker in disease diagnosis and its progression.

Relevance of Nitroxidation of Albumin in Rheumatoid Arthritis: A Biochemical and Clinical Study

Objective: To study the role of peroxynitrite-modified human serum albumin (nitroxidized-albumin) in rheumatoid arthritis. Methods: Human serum albumin was exposed to peroxynitrite and changes in albumin structure were monitored by UV-visible, fluorescence and circular dichroism spectroscopy, thioflavin T, Congo red binding and attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR). Antioxidant properties of nitroxidized-albumin were evaluated by free radical induced RBC hemolysis test. Markers of protein oxidation like carbonyl, thiol, dityrosine and RBC hemolysis were evaluated in RA patients’ sera. Binding of autoantibodies in RA sera (n=50) with nitroxidized-albumin was studied by direct binding, inhibition ELISA and electrophoretic mobility shift gel assay. Results: The nitroxidized-albumin indicated the generation of nitrotyrosine, nitrotryptophan, carbonyl, dityrosine and reduction in tyrosine and tryptophan fluorescence and α-helicity. Fluorescence emission intensities of thioflavin T and Congo red got augmented upon binding with nitroxidized-albumin. Furthermore, secondary and tertiary structures of nitroxidized-albumin were altered as evident by ATR- FTIR, far and near-UV CD. Autoantibodies in RA sera (or IgG purified from sera) showed enhanced binding with nitroxidized-albumin as determined by direct binding and inhibition ELISA. Protein carbonyls, dityrosine and RBC hemolysis were significantly high, but thiol was significantly low in RA sera compared to age- and sex- matched control. Conclusion: Endogenously available peroxynitrite can nitrate and oxidize albumin, leading to protein nitration/ oxidation and subsequent formation of crosslinks, aggregates and immunogenic nitroxidized-albumin. Therefore, nitroxidized-albumin may be a potential trigger for autoantibodies in RA patients.

Nucleic acid‐8‐methoxypsoralen crosslinks bind monoclonal anti‐Z‐DNA antibody

Native calf thymus DNA and poly(dA‐dT) poly(dA‐dT) were photo‐adducted with 8‐methoxypsoralen and characterized by thermal denaturation (Tm) and hydroxyapatite column chromatography. The data demonstrated the formation of interstrand photo‐crosslinks. It has been shown by competition ELISA and band shift assays that crosslinked species of DNA‐8‐MOP and poly(dA‐dT)‐8‐MOP photoadducts recognize previously defined monoclonal anti‐Z‐DNA antibody (Z22). The results indicate the possible presence of Z‐ or Z‐like epitopes on nucleic acid‐8‐MOP crosslinks as Z22 antibody does not recognize other nucleic acid conformations. These studies also point out that conformational changes in DNA arising from the photo‐addition could induce antibodies to DNA or could cause autoimmune disease.