Pyridoxamine protects proteins from damage by hypohalous acids in vitro and in vivo.

Diabetes is characterized, in part, by activation of toxic oxidative and glycoxidative pathways that are triggered by persistent hyperglycemia and contribute to diabetic complications. Inhibition of these pathways may benefit diabetic patients by delaying the onset of complications. One such inhibitor, pyridoxamine (PM), had shown promise in clinical trials. However, the mechanism of PM action in vivo is not well understood. We have previously reported that hypohalous acids can cause disruption of the structure and function of renal collagen IV in experimental diabetes (K.L. Brown et al., Diabetes 64:2242-2253, 2015). In the present study, we demonstrate that PM can protect protein functionality from hypochlorous and hypobromous acid-derived damage via a rapid direct reaction with and detoxification of these hypohalous acids. We further demonstrate that PM treatment can ameliorate specific hypohalous acid-derived structural and functional damage to the renal collagen IV network in a diabetic animal model. These findings suggest a new mechanism of PM action in diabetes, namely sequestration of hypohalous acids, which may contribute to known therapeutic effects of PM in human diabetic nephropathy.

Hypohalous acids contribute to renal extracellular matrix damage in experimental diabetes.

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid-derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of α1ß1 integrin consistent with the modification of collagen IV by hypochlorous (HOCl) and hypobromous acids. Noncollagenous (NC1) hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W(28) and W(192) are significantly elevated compared with nondiabetic controls. Molecular dynamics simulations predicted a more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/refolding. Our results suggest that hypohalous acid-derived modifications of renal ECM, and specifically collagen IV networks, contribute to functional protein damage in diabetes.

Glucose autoxidation induces functional damage to proteins via modification of critical arginine residues.

Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)ß(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.