Biochemical Studies on Methylglyoxal-Mediated Glycated Histones: Implications for Presence of Serum Antibodies against the Glycated Histones in Patients with Type 1 Diabetes Mellitus
Reactive carbonyl species (RCS) mainly reacts with lysine and arginine residues of proteins to form advanced glycation end products (AGEs). Histone was glycoxidated with glyoxal and methylglyoxal. It was characterized by polyacrylamide gel electrophoresis and quenching studies involving penicillamine and aminoguanidine as carbonyl scavengers. Further characterization of histone modified with methylglyoxal was done by UV, fluorescence, and IR spectrophotometry. Spectral analysis of the protein clearly demonstrates structural perturbation in the histone by methylglyoxal. Methylglyoxal-induces cross-linking in the protein leading to aggregation. Role of methylglyoxal mediated glycoxidation of histone in type 1 diabetes was also undertaken. Antibodies were detected against glycoxidated histone in sera of type 1 diabetes patients by solid-phase enzyme immunoassay. The findings indicate that as a result of structural perturbation in histone by methylglyoxal, the modified histone may be involved in production of serum antibodies in the diabetes patients. 1. Introduction There is overwhelming evidence for involvement of reactive oxygen species (ROS) in a number of pathophysiological conditions such as diabetes, cancer, and aging but the studies linking reactive carbonyl species (RCS) to the conditions are limited [1, 2]. RCS, such as methylglyoxal, is produced by degradation of lipid peroxidation products, early protein glycation adducts, and as a byproduct of glycolysis. RCS modification of histone results in cross-linking of proteins and induces ROS-dependent cleavage of plasmid DNA [3]. The proteasome degradation of RCS products is not complete and remnants may accumulate and cause epigenetic changes as well as further DNA and protein damage [4]. Earlier studies have shown that histones from liver cells of diabetic rats contain high level of AGEs [5]. We [6] have found antigenicity of glycated poly-L-lysine in experimental animals and autoantibodies were also detected against the modified lysine polypeptide in diabetes patients. A recent work has demonstrated in vivo formation of RCS-mediated AGEs in histone H1 using antibodies against oxidative protein adducts [7]. This study characterizes methylglyoxal-modified histone (a lysine-rich protein) and evaluates its role in type 1 diabetes patients. 2. Materials and Methods 2.1. Chemicals Calf thymus whole histones (type II-A) and methylglyoxal were purchased from Sigma (St. Louis, MO, USA). Polystyrene flat bottom UV microtiter plates were obtained from Greiner BioOne (Germany). All other chemicals and reagents
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