Abstract
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•Insulin modified by hydroxyl radical shows loss of α-helix and gain in β content.•Oxidation of insulin generates non-fibrillar and cytotoxic amorphous aggregates.•Oxidized insulin is highly immunogenic and generates specific immune response in immunized rabbits.•Immunological studies confirm the presence of circulating antibodies in type 2 diabetes patients.
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 β 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.