Alpha haemoglobin‐stabilising protein concentration in the red blood cells of patients with sickle cell anaemia with and without hydroxycarbamide treatment

International audienceAlpha haemoglobin-stabilising protein (AHSP) is a key chaperone synthesised in red blood cell (RBC) precursors. Many studies have reported AHSP as a potential biomarker of various diseases. AHSP gene expression has been studied in detail, but little is known about AHSP protein levels in RBCs. We investigated the AHSP concentration of RBC lysates from control subjects (n = 10) and patients with sickle cell anaemia (SCA) with (n = 10) and without (n = 12) hydroxycarbamide (HC) treatment, to evaluate the clinical relevance of AHSP in SCA. We developed a sandwich enzyme-linked immunosorbent assay method, with which we were able, for the first time, to determine the mean AHSP concentration in control RBC lysates (0·82 µg/ml). The AHSP concentration (2·23 µg/ml) was significantly higher in untreated patients with the SS genotype than in controls. The AHSP concentration decreased significantly on HC treatment (1·50 µg/ml) but remained significantly higher than that in controls. A strong positive correlation was observed between the AHSP concentration and the α-haemoglobin pool with the three groups of subjects pooled into a single group. Our present findings indicate that AHSP concentration can be considered a candidate biomarker for monitoring HC responses in patients with SCA and suggest a role for AHSP in various RBC diseases

Dynamics of α-Hb chain binding to its chaperone AHSP depends on heme coordination and redox state

International audienceBackground: AHSP is an erythroid molecular chaperone of the α-hemoglobin chains (α-Hb). Upon AHSP binding, native ferric α-Hb undergoes an unprecedented structural rearrangement at the heme site giving rise to a 6th coordination bond with His(E7).Methods: Recombinant AHSP, WT α-Hb:AHSP and α-Hb(HE7Q):AHSP complexes were expressed in Escherichia coli. Thermal denaturation curves were measured by circular dichroism for the isolated α-Hb and bound to AHSP. Kinetics of ligand binding and redox reactions of α-Hb bound to AHSP as well as α-Hb release from the α-Hb:AHSP complex were measured by time-resolved absorption spectroscopy.Results: AHSP binding to α-Hb is kinetically controlled to prevail over direct binding with β-chains and is also thermodynamically controlled by the α-Hb redox state and not the liganded state of the ferrous α-Hb. The dramatic instability of isolated ferric α-Hb is greatly decreased upon AHSP binding. Removing the bis-histidyl hexacoordination in α-HbH58(E7)Q:AHSP complex reduces the stabilizing effect of AHSP binding. Once the ferric α-Hb is bound to AHSP, the globin can be more easily reduced by several chemical and enzymatic systems compared to α-Hb within the Hb-tetramer.Conclusion: α-Hb reduction could trigger its release from AHSP toward its final Hb β-chain partner producing functional ferrous Hb-tetramers. This work indicates a preferred kinetic pathway for Hb-synthesis.General significance: The cellular redox balance in Hb-synthesis should be considered as important as the relative proportional synthesis of both Hb-subunits and their heme cofactor. The in vivo role of AHSP is discussed in the context of the molecular disorders observed in thalassemia

Role of α-globin H helix in the building of tetrameric human hemoglobin: interaction with α-hemoglobin stabilizing protein (AHSP) and heme molecule.

Alpha-Hemoglobin Stabilizing Protein (AHSP) binds to α-hemoglobin (α-Hb) or α-globin and maintains it in a soluble state until its association with the β-Hb chain partner to form Hb tetramers. AHSP specifically recognizes the G and H helices of α-Hb. To investigate the degree of interaction of the various regions of the α-globin H helix with AHSP, this interface was studied by stepwise elimination of regions of the α-globin H helix: five truncated α-Hbs α-Hb1-138, α-Hb1-134, α-Hb1-126, α-Hb1-123, α-Hb1-117 were co-expressed with AHSP as two glutathione-S-transferase (GST) fusion proteins. SDS-PAGE and Western Blot analysis revealed that the level of expression of each truncated α-Hb was similar to that of the wild type α-Hb except the shortest protein α-Hb1-117 which displayed a decreased expression. While truncated GST-α-Hb1-138 and GST-α-Hb1-134 were normally soluble; the shorter globins GST-α-Hb1-126 and GST-α-Hb1-117 were obtained in very low quantities, and the truncated GST-α-Hb1-123 provided the least material. Absorbance and fluorescence studies of complexes showed that the truncated α-Hb1-134 and shorter forms led to modified absorption spectra together with an increased fluorescence emission. This attests that shortening the H helix leads to a lower affinity of the α-globin for the heme. Upon addition of β-Hb, the increase in fluorescence indicates the replacement of AHSP by β-Hb. The CO binding kinetics of different truncated AHSPWT/α-Hb complexes showed that these Hbs were not functionally normal in terms of the allosteric transition. The N-terminal part of the H helix is primordial for interaction with AHSP and C-terminal part for interaction with heme, both features being required for stability of α-globin chain

Hydroxycarbamide decreases the free alpha‐hemoglobin pool in red blood cells of adult patients with sickle cell anemia

International audienceNo abstract availabl

Detection and follow-up of a soluble alpha-haemoglobin pool in the red cells of stored blood units.

International audienceNo abstract availabl

Elevated soluble α-hemoglobin pool in sickle cell anemia

International audienceNo abstract availabl

Red blood cells free α-haemoglobin pool: a biomarker to monitor the β-thalassemia intermedia variability. The ALPHAPOOL study

International audienceThe severity of β-thalassaemia (β-thal) intermedia is mainly correlated to the degree of imbalanced α/non α-globin chain synthesis. The phenotypic diversity of β-thal depends on this imbalance and reflects all possible combinations of α- and β-globin genotypes, levels of fetal haemoglobin (HbF) and co-inheritance of other modulating factors. This study aimed to demonstrate the validity of a new surrogate of α/non α-globin biosynthetic ratio by measuring the soluble α-Hb pool in lysed red blood cells. Our results confirm that the α-Hb pool measurement allows a good discrimination between β-thal intermedia patients, controls and α-thal patients (P < 0·003). Receiver operator characteristic analyses revealed an area under the curve of 0·978 for the α-Hb pool measurement at a threshold of 120 ng free α-Hb/mg of total Hb/ml of haemolysate (ppm) with a sensitivity and specificity of 86% and 100%, respectively, to discriminate between β-thal and not β-thal subjects. Significant correlations were observed between the α-Hb pool and biological parameters of β-thal, the most significant association being observed with red cell hexokinase activity. This study indicates that the α-Hb pool could be a new marker for assistance in diagnostic orientation of β-thal intermedia patients and may be clinically useful for monitoring the evolution of the disequilibrium of globin synthesis in response to treatments

Ligand binding studies of the various truncated GST-AHSP^WT/GST-α-Hb complexes (A) and after addition of β-Hb in the presence of IHP (B).

<p>The CO recombination kinetics reveal whether the protein complexes display the known rates for the classical R and T states, or the reference intermediate rate for the AHSP^WT/α-Hb^WT complex. The truncated forms of α-Hbs complexed with AHSP show less of the I state, indicating a modified interaction. Usually, the addition of β-Hb in the presence of IHP provokes a replacement of the AHSP by the β-Hb and leads to formation of Hb dimers and tetramers which display the usual slow T-state kinetics; however, the truncated forms displayed little of this allosteric form. Experimental conditions were 50 mM Bis-Tris buffer at pH 7.0, 100 mM CO at 25°C in absence or in presence of IHP at a final concentration of 1 mM. The concentration of different complexes on heme basis varies between 3.4 and 10.6 µM depending on the truncation.</p

Conformational Stability of truncated AHSP^WT/α-Hb complexes.

<p>(A) Thermal unfolding curve measured by CD experiments. Fraction unfolded (fu) is versus temperature for AHSP^WT/native α-Hb (♦), AHSP^WT/α-Hb^WT (•), AHSP^WT/α-Hb1-138, (▴) and AHSP^WT/α-Hb1-134 (▪) complexes. The solid lines are a smooth curve (sigmoidal representation) to help estimate Tm. Protein concentration was around 20 µM (on a heme basis) in 2.5 mM Na₂HPO₄, 37.5 mM NaCl buffer at pH 7.4 in the presence of sodium dithionite at 1 mM. The change in ellipticity was recorded at 222.6 nm from 20 to 80°C with a heat rate of 1°C.min⁻¹. (B) Hydrodynamic diameter for the complexes of AHSP^WT with native α-Hb, α-Hb^WT, α-Hb1-138 and α-Hb1-134. Protein concentration was around 20 µM (on a heme basis) in PBS.</p

Absorption spectra of truncated AHSP^WT/α-Hbs complexes.

<p>The spectra are represented in black for AHSP^WT/α-Hb^WT complex, in red for of AHSP/α-Hb1-138 complex, in black dashed line for AHSP/α-Hb1-134 complex and in grey for AHSP/α-Hb 1-126 complex. The measurements were performed in PBS pH 7.4 at 25°C.</p