Insights into Hemoglobin Assembly through in Vivo Mutagenesis of α-Hemoglobin Stabilizing Protein

Abstract

Background:The ␣-hemoglobin-stabilizing protein (AHSP) facilitates hemoglobin assembly. Results: AHSP mutations that enhance binding affinity for ␣-globin or slow its rate of autooxidation in vitro do not affect normal or stressed erythropoiesis in mice. Conclusion: AHSP exhibits robust molecular chaperone activity in vivo even when its biochemical interactions with reduced ␣-globin are perturbed. Significance: Our findings support new models for AHSP activities in vivo.

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

“…Time courses for these reactions are provided in Fig. 2 of Khandros et al (36). c The time course for met-␣ dissociation from AHSP P30A indicated two phases, and fitting to a two-exponential expression gave a fast phase with an amplitude of ϳ33% and k AHSP of Ϸ0.04 s Ϫ1 and a slow phase with an amplitude of ϳ67% and k AHSP of Ϸ0.002 s…”

“…2B in Ref. 36), suggesting multiple conformations for the mutant AHSP P30A ⅐ met-␣ complex. However, even the rate of the most rapid phase is 5-10-fold smaller than k AHSP for the corresponding reduced ␣ complex.…”

α-Hemoglobin Stabilizing Protein (AHSP) Markedly Decreases the Redox Potential and Reactivity of α-Subunits of Human HbA with Hydrogen Peroxide

“…To further investigate our findings, we altered the evolutionarily conserved AHSP proline 30 in recombinant AHSP to generate AHSP(P30A) and AHSP(P30W) mutant proteins (17,40). Although these mutations do not detectably perturb erythropoiesis in mouse models, AHSP(P30W) has been shown to bind oxygenated ␣-subunits with a 30-fold increased affinity in vitro, and both variants cause decreased rates of autoxidation and increased rates of hemin loss relative to wild-type AHSP (17,40).…”

“…Although these mutations do not detectably perturb erythropoiesis in mouse models, AHSP(P30W) has been shown to bind oxygenated ␣-subunits with a 30-fold increased affinity in vitro, and both variants cause decreased rates of autoxidation and increased rates of hemin loss relative to wild-type AHSP (17,40). In conducting experiments analogous to those presented in Fig.…”

Hemoglobin Variants: Biochemical Properties and Clinical Correlates

“…Asn) (Lacan et al 2004) bind b globin normally but show impaired interaction with AHSP and may be mildly destabilizing (Fig. 2D, violet spheres) (see also Yu et al 2009;Khandros et al 2012;Mollan et al 2012). Antitermination mutations can also destabilize a globin in part by impairing its binding to AHSP (Turbpaiboon et al 2006).…”