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)molecules (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 μM-5 mM), two α-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 μM 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.
Competing Interests: Declaration of competing interest M.J.D. declares commercial consultancy contracts with Novo Nordisk A/S. This funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish these results. The other authors declare no conflicts of interest with regard to the data presented.
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