Glycoxidation of mammalian whole histone generates highly immunogenic aggregates: Sera of SLE patients contain autoantibodies against aggregates.

Non-enzymatic glycation and oxidation of self-proteins, causing formation and accumulation of advanced glycation end products (AGEs), have been reported in an array of pathologies, including systemic lupus erythematosus (SLE). Such modifications may generate neo-epitopes, break immunological tolerance, and induce antibody response. In this study, we have first analysed the structural modifications of whole histone in the presence of deoxyribose followed by oxidation with hydroxyl radicals. Changes in the secondary and tertiary structure of the whole histone were determined by spectroscopic techniques and biochemical assays. Fluorescence spectroscopy and UPLC-MS showed the generation of AGEs such as carboxymethyl lysine and pentosidine, while DLS and TEM indicated the presence of amorphous AGE-aggregates. Moreover, rabbits immunized with these histone-AGEs exhibited enhanced immunogenicity and ELISA and western immunoblot of IgG antibodies from SLE patients' sera showed a significantly higher specificity towards modified histone-AGEs than the native histone.

Role of Carbamylated Biomolecules in Human Diseases.

Carbamylation (or carbamoylation) is a non-enzymatic modification of biomolecules mediated by cyanate, a dissociation product of urea. Proteins are more sensitive to carbamylation. Two major sites of carbamylation reaction are: Nα -amino moiety of a protein N-terminus and the Nɛ -amino moiety of proteins' lysine residues. In kidney diseases, urea accumulates and the burden of carbamylation increases. This may lead to alteration in the structure and function of many important proteins relevant in maintenance of homeostasis. Carbamylated proteins namely, carbamylated-haemoglobin and carbamylated-low density lipoprotein (LDL) have been implicated in hypoxia and atherosclerosis, respectively. Furthermore, carbamylation of insulin, oxytocin, and erythropoietin have caused changes in the action of these hormones vis-à-vis the metabolic pathways they control. In this short review, authors have compiled the data on role of carbamylated proteins, enzymes, hormones, LDL, and so on, in human diseases. © 2018 IUBMB Life, 70(4):267-275, 2018.

Methylglyoxal-induces multiple stable changes in human serum albumin before forming nephrotoxic advanced glycation end-products: Injury demonstration in human embryonic kidney cells.

The minor fraction of methylglyoxal that is not metabolized in healthy humans reacts with macromolecules to form AGEs. In diabetics, the formation of MG is accelerated; its level may be enhanced multifold. The glyoxalase enzymes responsible for the regular and effective clearance of excess methylglyoxal may become defective in diabetes mellitus leading to its retention in cells and plasma. The methylglyoxal-modified-HSA was prepared, characterised by multiple biophysical techniques and biochemical (s) and its damaging effect was examined on embryonic kidney cell line HEK 293. The UV results showed hyperchromicity in MG-modified-HSA while nitroblue tetrazolium and fluorescence data suggested AGEs formation in comparison to control HSA. Upward shift of negative peaks in CD suggested reduction in α-helicity. Accelerated mobility and diffused broad bands observed in native and SDS polyacrylamide gel, respectively suggest neutralization of some of the positive charges on MG-modified-HSA as well as generation of cross-links. As observed by trypan blue assay, MTT, LDH activity assay, acridine orange, propidium iodide, ethidium bromide, 4',6-diamidino-2-phenylindole (DAPI) staining and ROS measurements, the MG-HSA AGEs caused damage to human embryonic kidney cells. The data suggest that MG-HSA AGEs may trigger powerful inflammatory responses at cellular level which might set the stage for nephrotoxicity in diabetics.

Carbamylation of human serum albumin generates high-molecular weight aggregates: fine characterization by multi-spectroscopic methods and electron microscopy.

Carbamylation is the non-enzymatic reaction between isocyanic acid and macromolecules (mainly proteins) which results in carbamylation-derived products (CDPs) generation, wherein the macromolecules show altered structure and function. In this study, we examined the modifications caused in human serum albumin (HSA) upon interaction with potassium cyanate (KCNO). HSA was incubated with varying concentrations of KCNO for 6 h at 37 °C. The resultant product was characterized by biochemical and biophysical techniques. Among other changes, the carbamylated-HSA showed homocitrulline generation (LC-MS), increase in mass (DLS), and amyloidogenic aggregate formation (Congo red, SEM, TEM). The Gibb's free energy was calculated to be -2.91 to -3.95 kcal mol-1, suggesting that the binding was spontaneous and energetically favourable. The results indicate that in chronic kidney disease patients, elevated levels of isocyanic acid (formed from urea) may modify the albumin structure and lead to its conversion into amyloidogenic aggregates, thus accelerating kidney damage.

Impact of endogenous stress on albumin structure in systemic lupus erythematosus (SLE) patients.

Systemic lupus erythematosus (SLE) is an inflammatory, autoimmune disorder of unknown etiology. The inflammatory stress in SLE patients may modify macromolecules and produce structural/functional abnormalities. The present study is aimed at examining the consequences of stresses on the structure of albumin in SLE patients. Albumin was isolated from the sera of SLE/healthy subjects. Multiple physicochemical techniques were used to elucidate, structure of albumin. Advanced glycation end products in SLE patients' albumin were identified by the AGE specific fluorescence. Quenching of tryptophan, tyrosine fluorescence and surface protein hydrophobicity was observed in SLE patients' albumin. Protein-bound carbonyls were elevated while free thiol, lysine, arginine, and alpha helicity was found to be decreased in SLE albumin. Furthermore, changes in the secondary structure of SLE albumin were observed as shift in the position of amide I/II bands. Functionality of SLE albumin was also compromised as its cobalt-binding ability was substantially declined. Adduction of moieties was detected by dynamic light scattering (DLS) and confirmed by matrix assisted laser desorption/ionization. DLS, thioflavin T and transmission electron microscopy results confirmed aggregates in SLE patients' albumin. This study may be helpful in understanding the role of modified albumin in the cofounding pathologies associated with SLE.

Physicochemical characterization of carbamylated human serum albumin: an in vitro study.

Carbamylation is an ubiquitous process in which cyanate (OCN-) reacts with the N-terminal amino or ε-amino moiety and generates α-carbamyl amino acids and ε-carbamyl-lysine (homocitrulline). The process leads to irreversible changes in protein charge, structure and function. In this study, we have investigated the effect of carbamyl (generated from potassium cyanate) on human serum albumin (HSA) structure and function. The carbamylated-HSA (c-HSA) showed various modifications when examined by UV, fluorescence, FT-IR and far-UV CD spectroscopies. c-HSA exhibited hypochromicity, loss in α-helical content, changes in the amide I and amide II band, etc. Native-PAGE showed increase in the mobility of c-HSA compared to native-HSA. Aggregate(s) formation in c-HSA was detected by thioflavin T dye. The biochemical investigations carried out on c-HSA suggested increase in carbonyl content and decreased binding of TNBS (trinitrobenzenesulphonic acid) and Sakaguchi reagent. The attachment of the carbamyl moiety to HSA was confirmed from MALDI-TOF results. The functional defects in c-HSA were confirmed from the low binding of bilirubin. Taken together, carbamylation of albumin caused changes in the structural and functional properties of HSA. To the best of our knowledge, this is the first report on detailed biophysical characterization of carbamylated-HSA.

SLE autoantibodies are well recognized by peroxynitrite-modified-HSA: Its implications in the pathogenesis of SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disorder where the role of inflammatory processes in the etiopathogenesis is well documented. Despite extensive research, the trigger for initiation of the disease has not been identified. Peroxynitrite, a strong nitrating/oxidizing agent has been reported in SLE and other autoimmune diseases. In this study, human serum albumin (HSA) was exposed to peroxynitrite for 30min at 37°C. The structure of HSA was grossly perturbed when examined by various physico-chemical techniques. Peroxynitrite mediated nitration of HSA was confirmed by LCMS/MS. Furthermore, increase in hydrodynamic radius of peroxynitrite-modified-HSA suggests the attachment of nitro group(s). Aggregation in peroxynitrite-modified-HSA was evident in a TEM scan. Nitration, oxidation, cross linking, aggregation etc conferred immunogenicity on peroxynitrite-modified-HSA. High titre antibodies were elicited in rabbits immunized with peroxynitrite-modified-HSA. Induced antibodies were highly specific for peroxynitrite-modified-HSA but showed considerable binding with other nitrated molecules. Direct binding/inhibition ELISA carried out with autoantibodies in SLE sera showed preferential binding with peroxynitrite-modified-HSA. Anti-nDNA positive IgG from SLE sera showed preference for peroxynitrite-modified-HSA when subjected to immunoassay (direct binding and inhibition) and mobility shift assay. Our results reinforce the role of augmented inflammation in SLE progression.

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 ß-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.

Effect of peroxynitrite on human serum albumin: a multi technique approach.

In this study, human serum albumin (HSA), the most abundant protein of blood plasma, was modified with varying concentrations of peroxynitrite. The peroxynitrite-induced changes in HSA was monitored by spectroscopy, SDS-PAGE, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal denaturation studies, and matrix-assisted laser desorption/inonization-time of flight mass spectrometry (MALDI-TOF MS). Aggregate formation was studied by thioflavin T binding and scanning electron microscopy (SEM). The results indicated formation of 3-nitrotyrosine, 6-nitrotryptophan, dityrosine, and carbonyls in modified samples and showed retarded mobility in SDS-polyacrylamide gel. Reduction in α-helicity and surface protein hydrophobicity confirmed the secondary and tertiary structure alterations in peroxynitrite-modified-HSA. Also, attachment of nitro group and increase in melting temperature was observed in modified sample. Furthermore, significant enhancement in the fluorescence intensity of ThT upon binding with peroxynitrite-modified-HSA and images under scanning electron microscope are suggestive of protein aggregation. It is, therefore, speculated that HSA modified by endogenously formed peroxynitrite might act as a trigger for nitration/aggregation and suggested the role of peroxynitrite-modified-HSA in SLE.