Alpha-haemoglobin-stabilising protein

The α-hemoglobin-stabilizing protein (AHSP), also known as EDRF (erythroid differentiation related factor) and ERAF (erythroid associated factor) Mammalian hemoglobin A (HbA) is a tetramer of two α- and two β-globin chains. It is crucial to erythrocyte formation that the two globin chains be produced at balanced levels since any disruptions resulting in an excess of either chain have significant, deleterious effects on red cell survival, a situation most clearly illustrated by important human diseases, such as thalassemias [122]. In addition, free α-Hb (α-globin plus heme, or holo-α-globin) is a potent oxidant, catalyzing the production of reactive oxygen species (ROSs), which damage erythroid precursors and mature erythrocytes [123].

Using a screen for genes induced by the essential erythroid transcription factor GATA-1, Kihm and coworkers [124] identified the AHSP protein (102 aa) that stabilizes free α-hemoglobin. AHSP mRNA is expressed exclusively in hematopoietic tissues including bone marrow, spleen and fetal liver. AHSP is an abundant, erythroid-specific protein that forms a stable heterodimeric complex with free α-hemoglobin. Although AHSP is able to interact with multiple forms of α-globin, including apo-, ferrous and ferric states bound to a variety of ligands, it does not interact with β-hemoglobin or HbA (α2β2). Moreover, AHSP specifically protects free α-hemoglobin from precipitation both in vitro and in live cells, and reduces oxidant-induced precipitation of α-globin in solution [124, 125]. Finally, AHSP promotes refolding of denatured α-globin and HbA assembly from newly synthesized α-globin in vitro [126].

Gene ablation studies in mice demonstrated that AHSP is required for normal erythropoiesis. AHSP (−/−) homozygous knockout mice exhibit mild hemolytic anemia, shortened erythrocyte lifespan and high levels of reactive oxygen species, consistent with the presence of unstable α-globin [124]. AHSP-null erythrocytes are short-lived, contain Hb precipitates, and exhibit signs of oxidative damage. Predictably, loss of AHSP exacerbates β-thalassemia in mice and results in increased α-globin precipitation, suggesting that altered AHSP expression or function could modify thalassemia phenotypes in humans [124, 127]. Recently, the first mutant of AHSP, AHSP(V56G), was discovered associated with clinical symptoms of mild thalassemia [128]. The kinetics analysis showed that the AHSP(V56G) apparently does not bind long enough (0.5 vs. 2 s for the WT) to stabilize α-globin. Biophysical characterization demonstrated that monomeric AHSP in solution forms a moderate affinity complex with α-globin with 1:1 stoichiometry and with an association constant of 1 × 107 M−1 [129]. A far-UV circular dichroism spectrum showed that slightly elongated AHSP is primarily α-helical in conformation, probably with an extended C-terminal region. The crystal structure of AHSP bound to Fe(II)-αHb revealed that AHSP binds α-globin at the α1β1 dimer interface, opposite the heme binding pocket, where it is easily displaced by attachment of β-globin [125]. The structure of AHSP bound to ferrous αHb is thought to represent a transitional complex through which αHb is converted to a non-reactive, hexacoordinate ferric form. The crystal structure of this ferric α-Hb-AHSP complex at 2.4 Å resolution revealed a striking bis-histidine configuration, in which both the proximal and the distal histidines of α-Hb coordinate the heme iron atom. To attain this unusual conformation, segments of α-Hb undergo drastic structural rearrangements, including the repositioning of several α-helices. Moreover, conversion to the ferric bis-histidine configuration strongly and specifically inhibits redox chemistry catalysis and heme loss from α-Hb. The observed structural changes, which impair the chemical reactivity of heme iron, explain how AHSP stabilizes α-Hb and prevents its damaging effects in cells [130]. Taken together, X-ray crystallography, NMR spectroscopy, and mutagenesis data indicate the importance of an evolutionarily conserved proline, Pro-30, in loop 1 of AHSP. In complex with αHb, AHSP Pro-30 adopts a cis-peptidyl conformation and establishes contact with the N-terminus of helix G in α-Hb. As described above, complex formation suppresses the heme-catalyzed evolution of reactive oxygen species by converting α-Hb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidine coordination). Mutations that stabilize the cis-peptidyl conformation of free AHSP also enhance the conversion of α-Hb. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of α-Hb [131].

Since AHSP and β-Hb have overlapping binding sites on αHb, the addition of β-Hb to either Fe(II)- or Fe(III) αHb.AHSP displaces AHSP to generate tetrameric (α2β2) HbA species. These findings suggest a biochemical pathway through which AHSP might participate in normal Hb synthesis and modulate the severity of thalassemias. Indeed, some thalassemia phenotypes could be explained by a decreased expression of the Ahsp gene [132], or by an α-Hb variant displaying an impaired interaction with the AHSP or partner β chain [133], thereby hampering formation of a α1β1 dimer [134, 135]. Recently, in a murine model of β(IVS-2-654)-thalassemia, the expression of wt AHSP was able to improve the anemia phenotype and partially relieve this thalassemia syndrome [48], suggesting a possible role for AHSP in the treatments of thalassemia.