Abstract:This study was carried out to compare the enzymatic and bactericidal activity of mature, dimeric myeloperoxidase (MPO) and its monomeric form. Dimeric MPO was isolated from HL-60 cells. Hemi-MPO obtained from dimeric MPO by reductive cleavage of a disulfide bond between protomeric subunits was used as the monomeric form. Both peroxidase and halogenating (chlorinating) activities of MPO were assayed, each of them by two methods. Bactericidal activity of the MPO/Н2О2/Cl- system was tested using the Escherichia c… Show more
Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to inluence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulide bond but in inlammatory foci as a result of disulide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct efects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band three protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, signiicant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the irst time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer lealet of RBC membrane. However, the efects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These indings suggest that the ability of MPO protein to inluence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inlammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.
Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to inluence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulide bond but in inlammatory foci as a result of disulide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct efects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band three protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, signiicant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the irst time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer lealet of RBC membrane. However, the efects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These indings suggest that the ability of MPO protein to inluence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inlammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.
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.
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