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Methylglyoxal-Modified Human Serum Albumin Binds to Leukocyte Myeloperoxidase and Inhibits its Enzymatic Activity
Oleg M Panasenko1,2, Viktor A Ivanov1, Elena V Mikhalchik1
1Department of Biophysics, Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia.
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.
Insights
Methylglyoxal-modified human serum albumin (HSA-MG) binds to myeloperoxidase (MPO), inhibiting its activity and enhancing neutrophil NADPH oxidase. This impairs neutrophil bactericidal function, potentially explaining infections in diabetes.
Area of Science:
- Immunology
- Biochemistry
- Diabetology
Background:
- Hyperglycemia in diabetes mellitus causes protein modifications, including methylglyoxal (MG) modification of human serum albumin (HSA).
- Neutrophils utilize myeloperoxidase (MPO) and NADPH oxidase for bactericidal activity, generating reactive oxygen (ROS) and halogen (RHS) species.
- The interaction between modified proteins and neutrophil components in hyperglycemia is not fully understood.
Purpose of the Study:
- To investigate the binding of MG-modified HSA (HSA-MG) to MPO.
- To determine the effects of HSA-MG on MPO activity and neutrophil function.
- To explore the implications for innate immunity and infection susceptibility in diabetic patients.
Main Methods:
- Studied HSA-MG binding to MPO using affinity chromatography, disc-electrophoresis, ligand Western blotting, and ELISA.
- Assessed MPO peroxidase and chlorinating activities using specific substrates.
- Measured ROS and RHS generation via chemiluminescence, neutrophil degranulation by flow cytometry, and NETosis by DNA staining.
Main Results:
- HSA-MG formed a stable complex with MPO, non-competitively inhibiting its enzymatic activities.
- HSA-MG induced degranulation of peroxidase-negative granules and enhanced ROS generation, but not RHS or NETosis.
- HSA-MG binding to MPO dampened MPO-dependent functions while stimulating NADPH oxidase activity.
Conclusions:
- HSA modification under hyperglycemia impairs neutrophil bactericidal capacity by inhibiting MPO activity.
- The stimulation of NADPH oxidase suggests a complex effect on neutrophil oxidative burst.
- These findings may explain impaired innate immunity, delayed wound healing, and increased infection risk in diabetic individuals.
