Methylglyoxal-Modified Human Serum Albumin Binds to Leukocyte Myeloperoxidase and Inhibits its Enzymatic Activity

Abstract

Hyperglycemia in diabetes mellitus induces modification of proteins by glucose and its derivative methylglyoxal (MG). Neutrophils perform their bactericidal activity mainly via reactive halogen (RHS) and oxygen (ROS) species generation catalyzed by myeloperoxidase (MPO) stored in neutrophil azurophilic granules (AGs) and membrane NADPH oxidase, respectively. Herein, we study the binding of human serum albumin (HSA) modified with MG (HSA-MG) to MPO and its effects on MPO activity and release by neutrophils. Peroxidase activity of MPO was registered by oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and chlorinating activity by decolorization of Celestine blue B dye. Binding of HSA-MG to MPO was studied by affinity chromatography, disc-electrophoresis, ligand Western blotting and enzyme-linked solid phase immunoassay using monoclonal antibodies (mAbs) to MPO. ROS and RHS generation were detected by lucigenin (Luc) and luminol (Lum) chemiluminescence (CL), respectively. Neutrophil degranulation was assessed by flow cytometry using fluorescent labeled antibodies to the marker proteins CD63 from AGs and CD11b from peroxidase-negative granules (PNGs). NETosis was assayed by quantifying DNA network-like structures (NET-like structures) in blood smears stained by Romanowsky. HSA-MG bound to MPO, giving a stable complex (Kd = 1.5 nM) and competing with mAbs, and non-competitively inhibited peroxidase and chlorinating MPO activity and induced degranulation of PNGs but not of AGs. HSA-MG enhanced Luc-CL per se or following PMA, unlike Lum-CL, and did not affect spontaneous or PMA-stimulated NETosis. Thus, HSA modified under hyperglycemia-like conditions stimulated NADPH oxidase of neutrophils but dampened their functions dependent on activity of MPO, with no effect on its release via degranulation or NETosis. This phenomenon could underlie the downregulation of bactericidal activity of MPO and neutrophils, and hence of innate immunity, giving rise to wound healing impairment and susceptibility to infection in patients with hyperglycemia.

Transcriptional Insights of Oxidative Stress and Extracellular Traps in Lung Tissues of Fatal COVID-19 Cases

“…Immune responses associated with "cytokine storms" [59] could activate ROS, which may result in the consumption of nitric oxide (NO), a critical vasodilation regulator. Neutrophils execute their microbicidal activity mainly through reactive halogen species (RHS) and ROS generation catalyzed by MPO [60]. We used the term "ROS" throughout the manuscript instead of ROS and RHS for simplicity.…”

Incorporation of Pectin into Vaterite Microparticles Prevented Effects of Adsorbed Mucin on Neutrophil Activation

“…The formation of neutrophil extracellular traps (NETosis) was detected using flow cytometry as described elsewhere [41]. Monoclonal anti-MPO antibodies were obtained after the immunization of mice [42] and labelled with the NHS-ester of Cy5 (Lumiprobe, Moscow, Russia) according to the recommendations of the manufacturer. The neutrophils were incubated with pectin and CC or CCP microparticles in a concentration of 1 mg/mL for 30 min at 37 • C, and then, if necessary, PMA was added (25 nM, the standard NET formation inducer) and the solution was incubated for another 30 min at 37 • C. Then, the DNA-binding dye SYTOX Green (50 nM), which is impermeant to live cells, and anti-MPO antibodies (14.5 µg/mL) conjugated with the Cy5 fluorescent label were added to the samples, which were incubated for 5 min at room temperature in the dark.…”