Talha Mohd, Mir Abdul Rouf, Habib Safia, Abidi Minhal, Warsi Mohd Sharib, Islam Sidra
Department of Biochemistry, Jawaharlal Nehru Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh, Uttar Pradesh, India.
Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.
Spectrochim Acta A Mol Biomol Spectrosc. 2021 Jul 5;255:119640. doi: 10.1016/j.saa.2021.119640. Epub 2021 Mar 9.
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.
活性氧（ROS）会对蛋白质造成氧化损伤，并产生有害的副产物，这些副产物会导致免疫耐受的破坏，并产生针对宿主大分子的抗体，从而引发人类疾病。本研究对胰岛素的羟基自由基（OH）修饰进行了表征，评估了其细胞毒性和免疫原性，并探究了其在2型糖尿病（T2DM）自身免疫中的作用。结果表明胰岛素易受OH诱导的修饰影响，导致其发色芳香族氨基酸残基暴露、酪氨酸荧光强度淬灭、α-螺旋丧失以及β含量增加。修饰导致胰岛素中芳香族残基的天然相互作用重新排列。它增加了胰岛素中的羰基含量，暴露了其疏水区域，并产生了非纤维状的无定形聚集体，这些聚集体具有细胞毒性。天然胰岛素在免疫兔中诱导产生低滴度抗体，而OH修饰的胰岛素则产生强烈的免疫反应。竞争性ELISA研究表明，针对OH修饰胰岛素产生的抗体对修饰后的蛋白质具有高度特异性。交叉反应研究表明，各种氧化蛋白质上存在共同的抗原决定簇。由于T2DM患者的ROS产生增加，预计胰岛素会发生氧化，这可能会放大针对胰岛素的自身免疫反应。正如所料，直接结合ELISA显示T2DM患者中存在针对OH胰岛素的循环抗体，这些抗体对氧化胰岛素具有特异性。总之，胰岛素对OH失去结构完整性，形成具有细胞毒性的无定形聚集体，变得具有高度免疫原性，并在T2DM患者中引发体液反应。
