Long-term incubation of myoglobin with glyoxal induces amyloid like aggregation of the heme protein: Implications of advanced glycation end products in protein conformational disorders

Abstract

Protein glycation is a post-translational modification of proteins which is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. The reactive α-dicarbonyl compound, glyoxal, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. In the current study, the effect of glyoxal on the monomeric protein myoglobin has been investigated following incubation for a period of 30 days. Long-term incubation with glyoxal induced alteration of secondary structure of the heme protein from α-helix to β-sheet resulting in aggregation of the protein. The aggregates were found to exhibit amyloidal features with amorphous morphology as evident from subsequent biophysical spectroscopic and microscopic studies. Finally, mass spectrometric studies revealed glyoxal-induced modification of Lys-96, Lys-118, Lys-145, Arg-31 and Arg-139 residues of myoglobin to AGE adducts. The AGEs detected and identified were carboxymethyllysine (Lys-96), pyrrolidone-carboxymethyllysine (Lys-96, Lys-118 and Lys-145), arginine-derived hydroimidazolone (Arg-31) and the cross-linking adduct pentosidine (Lys-145 and Arg-139). Thus glyoxal-derived AGE adducts appear to induce structural constrain and subsequent aggregation of the heme protein. Considering the increased level of glyoxal in diabetes and the significance of AGE-mediated protein modification, the present study appears clinically relevant in understanding AGE-induced protein modification and subsequent conformational disorders.