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 prevents α-Hb–mediated production of ROS and oxidation of Hb A in s...
AHSP prevents α-Hb–mediated production of ROS and oxidation of Hb A in solution. (A) α-Hb or β-Hb was preincubated with AHSP at the indicated molar ratios for 30 minutes at 4°C, then was mixed with H2O2 and TMPD, a ROS indicator dye. The rate of TMPD oxidation, shown on the y axis, was measured by spectrophotometry. Graph shows the average value of three experiments. (B) Effect of AHSP on H2O2-induced heme loss from α-Hb in the experiment in A, as measured by reduced absorbance at 412 nm. The molar ratio of α-Hb to AHSP was 1:1 in the sample on the right. (C) Effect of AHSP or control protein SBTI on α-Hb–mediated oxidation of Hb A. Oxidation of Hb A after 30 minutes at 37°C was measured by spectrophotometry. Where indicated, α-Hb was added to Hb A at a molar ratio of 1:8. AHSP or SBTI was preincubated with α-Hb (1:1 molar ratio) for 30 minutes at 4°C before it was added to Hb A.