Sickle hemoglobin disturbs normal coupling among erythrocyte O2 content, glycolysis, and antioxidant capacity

Abstract

Key Points• Hb-conformation-dependent interaction with band 3 protein regulates glycolysis in RBCs.• In hypoxia, HbS disrupts this system, disabling RBC antioxidant defense. Energy metabolism in RBCs is characterized by O 2 -responsive variations in flux through the Embden Meyerhof pathway (EMP) or the hexose monophosphate pathway (HMP). Therefore, the generation of ATP, NADH, and 2,3-DPG (EMP) or NADPH (HMP) shift with RBC O 2 content because of competition between deoxyhemoglobin and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxygenated RBCs favors HMP flux and NADPH generation (maximizing glutathione-based antioxidant systems). We tested the hypothesis that sickle hemoglobin disrupts cdB3-based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia, we demonstrate in the present study constrained HMP flux, NADPH, and glutathione recycling and reduced resilience to oxidative stress manifested by membrane protein oxidation and membrane fragility. Using a novel, inverted membrane-on-bead model, we illustrate abnormal (O 2 -dependent) association of sickle hemoglobin to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. This finding was confirmed by immunofluorescent imaging during RBC O IntroductionSickle cell anemia (SCA) arises from a single amino acid substitution (Glu6Val) in the ␤-globin chain. Although the change to hemoglobin (Hb) is simple and uniform, SCA is characterized by broad differences in clinical manifestation. Phenotype variation in SCA is thought to arise from both environmental and genetic factors (eg, ␤-gene cluster haplotype, degree of HbF expression, or effects of other epistatic genes). The environmental factor that most clearly influences SCA phenotype is hypoxia, which drives sickle Hb (HbS) polymerization and the resulting well-characterized alterations in RBC physiology and the microcirculation. However, the influence of hypoxia on the SCA phenotype appears to be insufficiently explained by HbS polymerization alone. 1 Moreover, we lack a clear mechanistic understanding of the significant oxidative stress complicating SCA, a key feature of phenotype variation, both at rest and in association with hypoxia. 2 Nonpolymerized, solution-phase HbS may promote oxidative stress, even in RBCs under normal physiologic O 2 gradients. 3 Specifically, the low redox potential for heme in HbS 4 and avid binding affinity of HbS for the cytoplasmic regulatory domain of the Band 3 membrane protein (cdB3) 5,6 strongly affect RBC energetics and antioxidant systems [7][8][9] and, notably, do so as a function of RBC O 2 content. Therefore, both the genesis and the disposal of reactive oxygen species are abnormal in SCA, creating a baseline state of oxidative stress, which worsens in hypoxia.In particular, consideration of metabolic control in RBCs suggests O 2 -dependent HbS-cdB3 interaction as a relatively ...

Metabolomics of ADSOL (AS-1) Red Blood Cell Storage

“…This may be due to a combination of reduced pH and ATP. Additionally, the interactions between PFK and the Band 3 membrane protein, which normally regulate enzyme activity, could be disrupted during RBC storage [49]. Suppression of glycolysis reduces production of ATP and 2,3-DPG, metabolites critical for RBC viability and function.…”

Ebselen exhibits glycation‐inhibiting properties and protects against osmotic fragility of human erythrocytes in vitro

“…NPSH from blood cell suspension increased significantly when the concentration of glucose was raised from 5 to 30 mmol/L. This increase can probably be attributed to elevated intracellular glucose concentrations stimulating the pentose phosphate and anaerobic pathways (Rogers et al, ).…”

Therapeutic Strategies to Alter the Oxygen Affinity of Sickle Hemoglobin

“…2,3-DPG is a product of anaerobic glycolysis, which has been found in recent years to be regulated in erythrocytes by oxygen-regulated sequestration and inactivation of glycolytic enzymes by the cytoskeletal protein Band 3. 5–7 Among patients with SCD, P 50 and 2,3-DPG levels vary widely, and more elevated levels appear to decrease solubility of sickle hemoglobin (HbS), 8–10 and to increase red cell sickling under hypoxia, 7,11 although not confirmed by all investigators. 12,13 In vitro manipulation of human sickle blood to reduce 2,3-DPG content in red cells also reduces hypoxia-induced sickling in vitro .…”