Hemoglobin oxidation–dependent reactions promote interactions with band 3 and oxidative changes in sickle cell–derived microparticles
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
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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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 6

Hemoglobin oxidation promotes complex formation with band 3.

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Hemoglobin oxidation promotes complex formation with band 3. Co-IP of ba...
Co-IP of band 3 was done and probed with anti-Hb antibody. (A) The absorbance spectra for the ferrous, ferric, and ferryl Hbs used in experiments. (B) Band 3-Hb complex formation in RBC ghost membrane was analyzed following in vitro incubation with different Hb oxidation states using a co-IP assay and immunoblotted using anti–band 3 and anti-Hb antibodies (lower panel). Upper panel: 90-kDa bands show equal band 3 loading; lower panel: Hb binding developed with anti-Hb antibody. Upper panel: 120-kDa bands indicate high molecular complex with band 3. (C) Eluted proteins from band 3 immunoprecipitates from RBC ghost membranes treated with or without Hb of different oxidation states were resolved by SDS-PAGE, followed by staining with Coomassie blue. The gel bands were further analyzed by LC-MS. Higher levels of membrane protein complexes were found in ferryl Hb–treated RBC ghost membranes (some of the major complexed proteins with Hb α or β subunits include [1] spectrin α; [2] spectrin β, spectrin α, Hbβ, band 3 anion transport protein, Hbα, ankyrin 1; [3] spectrin β isoform A, Hbβ, band 3 anion transport protein, Hbα, ankyrin 1; [4] band 3 anion transport protein, ankyrin 1; [5] erythrocyte membrane protein band 4.2, Hbβ subunit, band 3 anion transport protein, Hbα subunit; [6] Hbβ, and band 3 anion transport protein). All proteins were searched using Mascot and listed in order of Mascot score. (D) Plot representing relative band intensities measured from a specific area of Coomassie-stained gel (120-kDa region, denoted by the red box in C). The values in the graph represent average mean value of 3 independent observations, and each dot in the bars represents individual data points. Vertical error bars represent SEM. Student’s t test, 2-tailed, *P < 0.05 vs. control (n = 3).