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 5

Representations of amino acid posttranslational modifications reveal significant changes in both shear stress and circulating microparticles.

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Representations of amino acid posttranslational modifications reveal sig...
MS/MS fragmentation spectra were used to search for posttranslational modifications (PTMs) previously known to be associated with Hb oxidative stress. (A–F) Relative ion current intensity fold changes corresponding to each Hb amino acid–specific PTM from AA and SS mouse shear stress and circulating microparticles (n = 3); measurements were determined by Hb peptide MS1 chromatogram intensities for each PTM. All peptide-spectrum matches were obtained using a strict 1% protein FDR. (G) Ubiquitinated proteins in AA and SS RBC lysates from 3 sets of mice were immunoprecipitated by protein G agarose beads using anti-ubiquitin antibody. (H) Ubiquitin bound Hb complex formation was analyzed using anti-Hbβ antibody. (I) Proteins present in the eluted immunoprecipitate were also resolved by SDS-PAGE and stained with Coomassie blue. Arrows represent Hbβ subunit. Bars represent average mean value; each dot in the bars represents an individual data point; and vertical error bars represent SEM. Student’s t test, 2-tailed, *P < 0.05 (n = 3).