Crowding modulates the glycation of plasma proteins: In vitro analysis of structural modifications to albumin and transferrin and identification of sites of modification

Protein modification occurs in biological milieus that are characterized by high concentrations of (macro)mol-ecules (i.e. heterogeneous and packed environments). Recent data indicate that crowding can modulate the extent and rate of protein oxidation, however its effect on other post-translational modifications remains to be explored. In this work we hypothesized that crowding would affect the glycation of plasma proteins. Physiologically-relevant concentrations of albumin (35 mg mL-1) and transferrin (2 mg mL-1) were incubated with methylglyoxal and glyoxal (5 mu M-5 mM), two alpha-oxoaldehyde metabolites that are elevated in the plasma of people with diabetes. Crowding was induced by adding dextran or ficoll polymers. Electrophoresis, electron microscopy, fluorescence spectroscopy and mass spectrometry were employed to investigate the structural consequences of glycation under crowded conditions. Our data demonstrate that crowding modulates the extent of formation of transferrin cross-links, and also the modification pathways in both albumin and transferrin. Arginine was the most susceptible residue to modification, with lysine and cysteine also affected. Loss of 0.48 and 7.28 arginine residues per protein molecule were determined on incubation with 500 mu M methylglyoxal for albumin and transferrin, respectively. Crowding did not influence the extent of loss of arginine and lysine for either protein, but the sites of modification, detected by LC-MS, were different between dilute and crowded conditions. These data confirm the relevance of studying modification processes under conditions that closely mimic biological milieus. These data unveil additional factors that influence the pattern and extent of protein modification, and their structural consequences, in biological systems.

Automated summary

This study explores the effect of crowding on protein glycation using high concentrations of proteins and alpha-oxoaldehyde metabolites. The results show that crowding modulates the extent of transferrin cross-links formation and the modification pathways in albumin and transferrin. Arginine is the most susceptible residue for modification, and crowding does not affect the extent of arginine and lysine loss, but the modification sites differ under dilute and crowded conditions.