Loss of α-hemoglobin–stabilizing protein impairs erythropoiesis and exacerbates β-thalassemia
Yi Kong, Suiping Zhou, Anthony J. Kihm, Anne M. Katein, Xiang Yu, David A. Gell, Joel P. Mackay, Kazuhiko Adachi, Linda Foster-Brown, Calvert S. Louden, Andrew J. Gow, Mitchell J. Weiss
Yi Kong, Suiping Zhou, Anthony J. Kihm, Anne M. Katein, Xiang Yu, David A. Gell, Joel P. Mackay, Kazuhiko Adachi, Linda Foster-Brown, Calvert S. Louden, Andrew J. Gow, Mitchell J. Weiss
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Article Hematology

Loss of α-hemoglobin–stabilizing protein impairs erythropoiesis and exacerbates β-thalassemia

Abstract

Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the deleterious effects of free α- and β-Hb subunits, which are unstable and cytotoxic. The α-Hb–stabilizing protein (AHSP) is an erythroid protein that specifically binds α-Hb and prevents its precipitation in vitro, which suggests that it may function to limit free α-Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies. AHSP–/– erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable α-Hb, AHSP–/– erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by α-Hb in solution. Finally, loss of AHSP worsened the phenotype of β-thalassemia, a common inherited anemia characterized by excess free α-Hb. Together, the data support a model in which AHSP binds α-Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in β-thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of β-thalassemia in humans.

Authors

Yi Kong, Suiping Zhou, Anthony J. Kihm, Anne M. Katein, Xiang Yu, David A. Gell, Joel P. Mackay, Kazuhiko Adachi, Linda Foster-Brown, Calvert S. Louden, Andrew J. Gow, Mitchell J. Weiss

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Oxidative stress in AHSP–/– erythrocytes. (A) Relative ROS levels in ery...
Oxidative stress in AHSP–/– erythrocytes. (A) Relative ROS levels in erythrocytes at baseline and with added H2O2. ROS were measured by incubation of cells with DCFH, which is converted by ROS to the fluorescent product 2′,7′-dichlorofluorescein (DCF). (B) Protein oxidation in erythrocyte lysates. Twenty micrograms of hemolysate was treated with DNPH for derivatization of carbonyl groups (by products of protein oxidation). Protein-associated DNP was detected by Western blotting. An identical blot probed with anti-actin (bottom) indicates equal protein loading in each lane. (C) Susceptibility to phenylhydrazine-induced hemolytic anemia. Drug was administered on days –1 and 0. Blood was first sampled on day 0, then hematocrit (top panel) and reticulocyte counts (bottom panel) were assessed daily until recovery. No abnormalities were detected in AHSP+/– mice in the experiments described in panels A–C (not shown).