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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AHSP–/– erythrocytes exhibit abnormal morphology, hemoglobin precipitate...
AHSP–/– erythrocytes exhibit abnormal morphology, hemoglobin precipitates (Heinz bodies), and reduced lifespan. (A) Wright-Giemsa staining (upper panels) shows eosinophilic inclusion bodies (*) in AHSP–/– erythrocytes. Heinz body staining, which detects denatured globin chains (lower panels), is weakly positive in some AHSP+/– erythrocytes (indicated by carets) and is strongly positive in AHSP–/– cells. The boxed area in the top right panel shows an enlargement of an inclusion body. Original magnification, ∞1,000. (B) Erythrocyte survival kinetics determined by biotin labeling. Circulating erythrocytes in 5 animals of each genotype were biotinylated at days –2 and –1. Beginning at day 0, approximately 5 ∝l of blood was removed from the tail vein at the indicated time points, and the fraction of biotin-labeled erythrocytes was quantified by flow cytometry. The half-life of wild-type red blood cells was 22 days, whereas that of the AHSP–/– red blood cells was 12 days. AHSP+/– erythrocytes exhibited normal survival kinetics (not shown). (C) Prussian blue staining for cellular iron in spleen. Increased iron in the AHSP–/– spleen reflects accelerated clearance of erythroid cells by the reticuloendothelial system. Original magnification, ∞200.