Impaired binding of AHSP to α chain variants: Hb Groene Hart illustrates a mechanism leading to unstable hemoglobins with α thalassemic like syndrome

Abstract

Alpha hemoglobin stabilizing protein (AHSP) is a small protein of 102 residues induced by GATA-1, Oct-1- and EKLF. It is synthesized at a high level in the red blood cell precursors and acts as a chaperone protecting the alpha hemoglobin (α-Hb) chains against precipitation. AHSP and α-Hb form a heterodimer complex. In the absence of AHSP, α-Hb oxidizes and precipitates within the erythrocyte precursors of the bone marrow leading to apoptosis and defective erythropoiesis. In vitro the binding of AHSP to ferrous α-Hb accelerates oxidation of the heme iron in α-Hb, but the complex is more resistant to protein unfolding. AHSP could act as a modulating factor in beta-thalassemia. Recent studies showed more severe thalassemic syndromes in patients with decreased levels of AHSP and in one patient who carried a structurally abnormal AHSP. Some α-Hb variants with structural abnormality located in the contact area between α-Hb and AHSP exhibit an instability and a thalassemic like syndrome. We suggest that this could result from a disturbed interaction between α-Hb variants and AHSP. To study this interaction, we constructed the pGEX-α-AHSP vector that co-expressed human α-Hb and AHSP. Using this approach, we investigated the α42 (C7), α104 (G11) and α119 (H2) sites, where variants with some thalassemic features have been described. Results obtained with recombinant Groene Hart α-Hb and Diamant α-Hb, in which proline 119 is replaced by a serine and a leucine, respectively, showed clearly an impaired interaction with AHSP. In contrast, the α mutants at the sites 42 and 104 exhibit a normal interaction with AHSP. The CO rebinding kinetics of the AHSP/α-Hb 42mutant complexes were similar to those previously obtained with the AHSP/α-Hb WT complex, which shows a modified rate that is intermediate to the classical Hb allosteric states.