Methylglyoxal-Modified Human Serum Albumin Binds to Leukocyte Myeloperoxidase and Inhibits its Enzymatic Activity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents Used
2.2. Modification of HSA
2.3. MPO Isolation
2.4. Anti-MPO Monoclonal Antibodies
2.5. Neutrophil Isolation
2.6. Affinity Chromatography of HSA-MG on MPO–Sepharose
2.7. Detection of MPO and HSA-MG Interaction Using Disc-Electrophoresis without Detergent and Ligand Western Blotting Assay
2.8. Detection of MPO and HSA-MG Interaction by ELISA
2.9. Evaluation of Dissociation Constant (Kd) Value of MPO-HSA-MG Complex and MPO-Binding Capacity Assay
2.10. MPO Enzymatic Activity
2.11. Neutrophil Chemiluminescence
2.12. Assays for Degranulation of Human Neutrophils
2.13. Spontaneous and PMA-Stimulated NETosis
2.14. Statistical Analysis
3. Results
3.1. Characteristics of HSA-MG
3.2. HSA-MG Binding with MPO
3.3. Effects of HSA-MG on Enzymatic Activity of MPO
3.4. Effects of HSA-MG on Activation of Neutrophils
3.5. Effects of HSA-MG on Degranulation of Neutrophils
3.6. Effects of HSA-MG on NETosis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Mixture | Parameters | Control | HSA | HSA-MG | Ki HSA-MG, μM |
---|---|---|---|---|---|
2–50 μM H2O2 (1 mM ABTS, 5 nM MPO) | KM, µM | 21.3 ± 0.5 | 21.9 ± 0.6 | 21.5 ± 0.5 | 0.84 |
Vmax, s−1 | 65.1 ± 1.3 | 64.4 ± 1.4 | 21.5 ± 0.5 | ||
0.4–2 mM ABTS (50 μM H2O2, 5 nM MPO) | KM, mM | 1.06 ± 0.08 | 1.05 ± 0.09 | 1.04 ± 0.07 | 1.08 |
Vmax, s−1 | 87.2 ± 1.4 | 86.7 ± 1.5 | 59.7 ± 1.3 | ||
5–80 μM H2O2 (150 mM NaCl, 10 nM MPO) | KM, µM | 27.4 ± 1.4 | 26.1 ± 1.5 | 25.4 ± 1.3 | 0.80 |
Vmax, s−1 | 51.2 ± 1.1 | 49.9 ± 1.2 | 31.5 ± 1.1 | ||
25–150 mM NaCl (50 μM H2O2, 10 nM MPO) | KM, mM | 85.7 ± 1.8 | 85.8 ± 1.8 | 85.8 ± 1.7 | 0.94 |
Vmax, s−1 | 49.6 ± 1.0 | 50.2 ± 1.0 | 32.4 ± 0.9 |
Effector | Time of Incubation, h | NET-like Structures, % | Leukocytes, Cells per µL |
---|---|---|---|
Control | 1 | 7.4 ± 0.9 | 5300 ± 290 |
3 | 7.1 ± 0.8 | 5200 ± 290 | |
1 mg/mL HSA | 1 | 7.4 ± 0.8 | 5200 ± 290 |
3 | 7.8 ± 0.8 | 5000 ± 290 | |
0.5 mg/mL HSA-MG | 1 | 8.6 ± 0.8 | 5000 ± 280 |
3 | 8.8 ± 0.9 | 4900 ± 260 | |
1 mg/mL HSA-MG | 1 | 7.2 ± 0.7 | 5200 ± 300 |
3 | 7.4 ± 0.9 | 5200 ± 310 |
Effector | NET-like Structures, % | Leukocytes, Cells per µL |
---|---|---|
Control | 12.8 ± 1.1 | 3900 ± 240 |
1 mg/mL HSA | 12.1 ± 1.0 | 4000 ± 250 |
0.5 mg/mL HSA-MG | 10.9 ± 1.0 | 4000 ± 280 |
1 mg/mL HSA-MG | 11.8 ± 1.0 | 4000 ± 270 |
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Panasenko, O.M.; Ivanov, V.A.; Mikhalchik, E.V.; Gorudko, I.V.; Grigorieva, D.V.; Basyreva, L.Y.; Shmeleva, E.V.; Gusev, S.A.; Kostevich, V.A.; Gorbunov, N.P.; et al. Methylglyoxal-Modified Human Serum Albumin Binds to Leukocyte Myeloperoxidase and Inhibits its Enzymatic Activity. Antioxidants 2022, 11, 2263. https://doi.org/10.3390/antiox11112263
Panasenko OM, Ivanov VA, Mikhalchik EV, Gorudko IV, Grigorieva DV, Basyreva LY, Shmeleva EV, Gusev SA, Kostevich VA, Gorbunov NP, et al. Methylglyoxal-Modified Human Serum Albumin Binds to Leukocyte Myeloperoxidase and Inhibits its Enzymatic Activity. Antioxidants. 2022; 11(11):2263. https://doi.org/10.3390/antiox11112263
Chicago/Turabian StylePanasenko, Oleg M., Viktor A. Ivanov, Elena V. Mikhalchik, Irina V. Gorudko, Daria V. Grigorieva, Liliya Yu. Basyreva, Ekaterina V. Shmeleva, Sergey A. Gusev, Valeria A. Kostevich, Nikolay P. Gorbunov, and et al. 2022. "Methylglyoxal-Modified Human Serum Albumin Binds to Leukocyte Myeloperoxidase and Inhibits its Enzymatic Activity" Antioxidants 11, no. 11: 2263. https://doi.org/10.3390/antiox11112263