Abstract
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.
Highlights
Human serum albumin (HSA) is most abundant serum protein
Direct binding enzyme linked immune sorbent assay (ELISA) results showed induction of high titre antibodies (>1:12800) when albumin modified with 75mM glucose was used as immunogen
Antisera against 25mM and 50mM glucose modified Amadorialbumin showed antibody titre of >1:6400 and
Summary
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 [1]. 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 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 [3]. 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 [15]
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