Date Published: February 13, 2017
Publisher: Public Library of Science
Author(s): Km Neelofar, Zarina Arif, Jamal Ahmad, Khursheed Alam, Jamshidkhan Chamani.
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.
Human serum albumin (HSA) is most abundant serum protein. Structurally, it is single chain globular protein with 585 amino acids, contains 1 free cysteine, 1 tryptophan, 59 lysine and other amino acid residues . The crystal structure of HSA shows that it is a three domain, heart shaped molecule. It is a multifunctional protein in human blood and plays an important protective role as an antioxidant . The epsilon amino group lysine and arginine and free amino group of proteins can be non-enzymatically attached to the reducing sugar to form Schiff base which via intermolecular rearrangement forms stable, covalently bonded Amadori products and finally converted into advanced glycation end products (AGEs). This process occurs in individuals with normal plasma glucose concentrations, but HSA is typically 2–3 times more glycated than the rest of the serum proteins in hyperglycaemic condition . Sustained hyperglycaemia leads to glycation of serum proteins preferably at epsilon amino group of lysine residues. Serum glycated proteins represent a potential marker for hyperglycaemia in diabetes mellitus and its complications . Proteins modification in diabetes may lead to Amadori as well as AGEs. The glycation of HSA may have a variety of important physiological effects and the in vitro modifications of protein by glucose is regarded as an appropriate model for changes in structure and function relevant to diabetes mellitus [5–7]. Proteins interactions with ligands changed their secondary and tertiary structure that was determined by various techniques [8–9]. Structural stability is the main factor to carry out all its functions otherwise it can involve in diseases progression [10–11]. Such modifications on proteins may lead to generation of neo-epitopes which could in turn be more immunogenic . Immunogenic properties of proteins have been widely used to study their structure. Many research articles have revealed that proteins upon glycation have become immunogenic because of conformational changes that gave the titre of antibodies against the modified protein when injected in experimental animals [13–14]. Glycated poly-L-lysine has been used as an antigen to induce antibodies in experimental animal and was reported to be highly immunogenic and specific towards the corresponding antigen .
Glucose is a ubiquitous natural metabolite present in all human tissues and reacts non-enzymatically with nucleophilic groups on proteins. Proteins modified by non-enzymatic glycation or glycoxidation processes are immunologically active and can induce significant immune response. Protein modifications in diabetes and its secondary complications may lead to Amadori as well as AGEs. Structural stability of macromolecules is the main factor to carry out all its functions, otherwise it can be involved in the development of diseases. Maryam et al reported that interaction of carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) with HSA induced structural changes on HSA . During persistent hyperglycaemia, HSA a lysine rich protein can undergo fast glycation. Elaheh et al reported that glycated HSA has less binding affinity for metformin than normal HSA . Our previous findings have shown that with increase in glucose concentrations, conformational changes in albumin were also increased. We have established changes in secondary and tertiary structure of albumin modified with different glucose concentrations, with the help of various techniques . However, in this study we have explored the immunological properties of structurally modified albumin by different glucose concentrations. Albumin in its native form is also immunogenic  but due to glucose induced structural changes in albumin, it became highly immunogenic which in turn produced highly specific antibodies in experimental animal. In our experimental conditions, Amadori-albumin modified with 5mM glucose showed similar induction of antibodies as native albumin which indicate that at physiological glucose concentration HSA is not structurally perturbed. However, immunogenicity of albumin was increased as glucose concentration was increased from hyperglycaemic (25 mM and 50 mM) to chronic hyperglycaemic range (75 mM). This clearly demonstrates that an array of modifications in albumin structure depends on glucose concentration and it might favour polymerization of native epitopes of albumin into potent immunogenic neo-epitopes. Albumin modified with 75 mM glucose was found to be a potent immunogen inducing high titre antibodies in rabbits. Interpretation of our result is that glucose induced conformational alterations in structure of HSA that leads to the generation of neo-epitopes thus enhancing the immunogenicity of Amadori-albumin. Immunogenicity of modified HSA was totally dependent on glucose concentrations. Furthermore, antigenic specificity of affinity purified anti-Amadori-albumin IgG reiterated that the antibodies preferentially recognized the modified epitopes on Amadori-albumin. Notable feature of the anti-Amadori-albumin IgG was that the maximum inhibition in antibody binding was caused when Amadori-albumin (75 mM) was used as inhibitor followed by 25 mM and 50 mM and least with the 5 mM and native-albumin. Lysine residues play an important role in the enhancement of immunogenicity of albumin . Many studies have reported that macromolecules undergo structural perturbations upon glycation which lead to the generation of a new form or neo-epitopes on protein that are recognized as foreign molecule by the immune system and are able to elicit antibody responses . Our findings also indicate that chronic hyperglycaemic glucose concentration has caused changes in albumin structure and generated neo-epitopes on albumin that can induce immune system to produce antibodies. This suggests a role of Amadori-albumin (in chronic condition of hyperglycaemia) in the development and progression of diabetes and related complications. The induced antibodies also exhibited polyspecificity with respect to antigen binding as determined by inhibition assay. The binding of induced antibodies was tested against native and glycated forms of different proteins. It showed variable degree of recognition of the Amadori forms of other proteins. These results, therefore, indicate sharing of common epitopes between Amadori-albumin and Amadori forms of other proteins [28–29]. Moreover, anti-Amadori-albumin IgG antibodies also showed binding with native albumin. It indicates that all epitopes typical of native albumin have not been converted into neo-epitopes upon early glycation. Hence immunization with Amadori-albumin may produce polyspecific antibodies which can recognize both old and neo-epitopes. Gel retardation data further substantiated the preferential recognition of Amadori-albumin over native albumin by anti-Amadori-albumin antibodies. The formation of high molecular weight immune complex and appreciable decrease in the unbound antigen observed with Amadori-albumin, compared to native albumin, shows that the major antibodies were directed against the modified epitopes. These results indicate that native albumin binds to the anti-Amadori-albumin IgG to some extent due to the presence of native epitopes. In addition, the cross reactivity of anti-native-albumin IgG antibodies with Amadori-albumin reveals that modified HSA has got both old and neo-epitopes. Amadori-albumin was found to be immunogenic and it has been reported that type 1 diabetic patients with or without complications have higher level of Amadori-albumin . Serum autoantibodies in type 2 diabetes patients showed preference of Amadori-albumin that suggests a causal role for Amadori-albumin in the pathogenesis of diabetes mellitus.
Collectively, on the basis of our results, we propose that different concentrations glucose modified albumin might be playing a role in presenting unique antigenic determinants. Thus eliciting immune response that were are not ordinarily present on the native molecule, especially albumin modified with high concentration of glucose (75 mM). Changes observed in the immunological properties of Amadori-albumin might be due to conformational alterations in albumin structure upon glycation. Generation of neo-epitopes on albumin might be involved in the induction of autoantibodies in type 2 diabetes. So inhibition of Amadori-albumin production may be useful to reduce diabetes progression.