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
View: Text | PDF
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

×

Figure 9

Hydroxyurea reduces ferryl heme and shields βCy93 by a process of S-nitrosylation.

Options: View larger image (or click on image) Download as PowerPoint
Hydroxyurea reduces ferryl heme and shields βCy93 by a process of S-nitr...
Effects of HU on the levels of ferryl Hb (made with the addition of 150 μM H2O2). (A) Overlaid sulfHb spectra (absorption at 620 nm) of HbS (60 μM) treated (dashed line) and non-treated with HU (240 μM) (solid line). (B) Amounts of sulfHb produced at various ratios of H2O2 to Hb; nontreated HbS (solid black) and HU-treated (pattern). (C) MS/MS fragmentation spectrum representing the y and b ions matched to the modified tryptic peptide GTFATSELHCDKLHVDPENFR (residues 83–104) (residues 82–104) from Hb incubated with HU; this peptide contains the diglycine signature modification (at βLys96) associated with ubiquitination and S-nitrosylation (at βCys93). 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 HbS vs. corresponding HbS + HU (n = 3).