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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Intercrosses of β-globin+/th-3AHSP+/– double-heterozygous mice. The th-3...
Intercrosses of β-globin+/th-3AHSP+/– double-heterozygous mice. The th-3 mutant β-globin allele represents a targeted deletion of the b1 and b2 adult globin genes; heterozygous animals exhibit β-thalassemia intermedia, while the homozygous state is lethal in utero. (A) Mating strategy. The β-globin and AHSP genes are physically linked on mouse chromosome 7. Double-heterozygous mice used were the F1 progeny of intercrosses of simple AHSP+/– heterozygotes and β-globin+/th-3 heterozygotes, ensuring that the two mutant alleles were on separate chromosomes (trans configuration). The genotype frequencies of live-born offspring resulting from F1 intercrosses are described in Table 2. +, wild-type allele; –, deleted allele. (B) Hematocrits of β-globin–AHSP compound mutant embryos, with genotypes shown below the x axis. For comparison, embryos are grouped according to the presence or absence of thalassemia (β-globin+/th-3 genotype); thalassemic embryos are subdivided into AHSP-null versus AHSP–wild-type or -heterozygous states. Asterisk denotes either + or – alleles for AHSP. Each symbol represents one embryo analyzed: triangles, β-globin+/+ embryos; circles, β-globin+/th-3 with at least one wild-type AHSP allele; diamonds, β-globin+/th-3AHSP–/–. Color coding is used to specify the AHSP genotype within each group: black, +/+; white, +/–; red, –/–. (C) Blood smears of β-globin+/th-3 embryos with +/+ or –/– AHSP genotypes. Most of the erythrocytes are anucleate definitive (fetal liver derived). Nucleated cells: EP, primitive (yolk sac derived); ED, definitive. Among thalassemic embryos, those lacking AHSP exhibited more prominent eosinophilic erythrocyte inclusions (examples marked by double asterisks) and increased circulating ED cells. Original magnification, ×100.