Hemoglobin oxidation–dependent reactions promote interactions with band 3 and oxidative changes in sickle cell–derived microparticles
Sirsendu Jana, … , Gregory M. Vercellotti, Abdu I. Alayash
Sirsendu Jana, … , Gregory M. Vercellotti, Abdu I. Alayash
Published November 2, 2018
Citation Information: JCI Insight. 2018;3(21):e120451. https://doi.org/10.1172/jci.insight.120451.
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Research Article Hematology Vascular biology

Hemoglobin oxidation–dependent reactions promote interactions with band 3 and oxidative changes in sickle cell–derived microparticles

Abstract

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes–sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent β-globin posttranslational modifications, including the irreversible oxidation of βCys93 and the ubiquitination of βLys96 and βLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, βCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.

Authors

Sirsendu Jana, Michael Brad Strader, Fantao Meng, Wayne Hicks, Tigist Kassa, Ivan Tarandovskiy, Silvia De Paoli, Jan Simak, Michael R. Heaven, John D. Belcher, Gregory M. Vercellotti, Abdu I. Alayash

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Figure 10

Proposed erythrocyte internal oxidative changes that trigger membrane alternations and microparticle formation.

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Proposed erythrocyte internal oxidative changes that trigger membrane al...
Oxidative reactions within RBCs include the following pathways: (1) Ferrous Hb (Fe2+) undergoes spontaneous oxidation (autoxidation) to form ferric Hb (Fe3+), and in the presence of H2O2 ferryl Hb (Fe4+) is formed when antioxidative mechanisms are compromised. Ferryl Hb undergoes oxidative changes including irreversible oxidation of βCys93 and ubiquitination of some lysine residues. Ferryl Hb can oxidatively target band 3, leading to complex formation and microparticle formation (2) or denaturation and proteasome degradation (3). Some ferryl Hbs revert back to ferric by a process of autoreduction (4). Hydroxyurea acts as an antioxidant by forming SNO-Hb at βCys93 by a process of S-nitrosylation, thereby minimizing the consequences of these processes (adapted with modification from ref. 23).