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

Alpha 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.

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

The severity of ß-thalassaemia (ß-thal) intermedia is mainly correlated to the degree of imbalanced α/non α-globin chain synthesis. The phenotypic diversity of ß-thal depends on this imbalance and reflects all possible combinations of α- and ß-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 ß-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 ß-thal and not ß-thal subjects. Significant correlations were observed between the α-Hb pool and biological parameters of ß-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 ß-thal intermedia patients and may be clinically useful for monitoring the evolution of the disequilibrium of globin synthesis in response to treatments.

Interaction of recombinant octameric hemoglobin with endothelial cells.

Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbßG83C. We investigate the potential side effects of rHbßG83C on endothelial cells. The rHbßG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbßG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb ßG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

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 ß-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 ß-Hb, the increase in fluorescence indicates the replacement of AHSP by ß-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.

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

BACKGROUND: 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 ß-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 ß-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.

Interaction of haptoglobin with hemoglobin octamers based on the mutation αAsn78Cys or ßGly83Cys.

Octameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function; in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized. Oxygen binding curves and CO kinetic studies indicate a correct allosteric transition of the tetramers within the octamer. Crystallographic studies of the two rHbs show two disulfide bonds per octamer. Reducing agents may provoke dissociation to tetramers, but the octamers are stable when mixed with fresh human plasma, indicating that the reduction by plasma is slower than the oxidation by the dissolved oxygen, consistent with an enhanced stability. The octameric rHbs were also mixed with a solution of haptoglobin (Hp), which binds the dimers of Hb: there was little interaction for incubation times of 15 min; however, on longer timescales a complex was formed. Dynamic light scattering was used to follow the interaction of Hp with the alpha Asn78Cys octamer during 24 hours; a transition from a simple complex of 15 nm to a final size of 60 nm was observed. The results indicate a specific orientation of the αß dimers may be of importance for the binding to haptoglobin.

Evaluation of the free α-hemoglobin pool in red blood cells: a new test providing a scale of ß-thalassemia severity.

ß-Thalassemias are characterized by an imbalance of globin chains with an excess of α-chains which precipitates in erythroid precursors and red blood cells (RBCs) leading to inefficient erythropoiesis. The severity of the disease correlates with the amount of unpaired α-chains.Our goal was to develop a simple test for evaluation of the free α-hemoglobin pool present in RBC lysates. Alpha-Hemoglobin Stabilizing Protein (AHSP), the chaperone of α-Hb, was used to trap excess a-Hb. A recombinant GST-AHSP fusion protein was bound to an affinity micro-column and then incubated with hemolysates of patients. After washing, the α-Hb was quantified by spectrophotometry in the elution fraction. This assay was applied to 54 patients: 28 without apparent Hb disorder, 20 ß-thalassemic and 6 α-thalassemic. The average value of free α-Hb pool was 93 ± 21 ppm (ng of free α-Hb per mg of Hb subunits)in patients without Hb disorder, while it varies from 119 to 1,756 ppm, in ß-thalassemic patients and correlated with genotype. In contrast,the value of the free α-Hb pool was decreased in α-thalassemic patients (65 ± 26 ppm). This assay may help to characterize ß-thalassemia phenotypes and to follow the evolution of the globin chain imbalance(α/ß+γ ratio) in response to treatment.

Alpha-hemoglobin stabilizing protein (AHSP), a kinetic scheme of the action of a human mutant, AHSPV56G.

A kinetic analysis has been made of the interaction of alpha-Hb chains with a mutant alpha-hemoglobin stabilizing protein, AHSP(V56G), which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent alpha chains until final binding to the partner beta-Hb. Rather than protecting alpha chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with alpha-Hb. For both AHSP(WT) and AHSP(V56G), the binding to alpha-Hb is quite rapid relative to the alpha-beta reaction, as expected because the chaperone binding must be quite competitive to complete the alpha chain folding process before alpha-Hb binds irreversibly to beta-Hb. The main kinetic difference is a dissociation rate of AHSP(V56G).alpha-Hb some four times faster relative to AHSP.alpha-Hb. Considering a role of protein folding, the AHSP(V56G) apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP.alpha-Hb + beta-Hb --> AHSP + alphabeta) can be quite long, especially if there is an excess of AHSP relative to beta-Hb monomers.

The alpha-hemoglobin stabilizing protein and expression of unstable alpha-Hb variants.

OBJECTIVES: To determine the role of the alpha-hemoglobin stabilizing protein (AHSP) in the clinical expression of alpha-hemoglobin (alpha-Hb) variants described as unstable, ten alpha chain variants have been studied with their chaperone. AHSP specifically binds free alpha-Hb to form a soluble heterodimer until it is replaced by the beta-Hb partner. In this way, AHSP prevents the precipitation of free alpha chains which might damage the membrane of erythrocyte. AHSP specifically recognizes the G and H helices of alpha-Hb that are also involved in the alpha1beta1 dimer interface. AHSP may act as a modifier in alpha-thalassemias and lead to the thalassemic phenotypes observed in certain unstable alpha-Hb variants previously considered unstable. The different abnormalities of the alpha chain were located either in the G helix: Hb Bronovo alpha103(G10)His-->Leu, Hb Sallanches alpha104(G11)Cys-->Tyr, Hb Oegstgeest alpha104(G11)Cys-->Ser, Hb Bleuland alpha108(G15)Thr-->Asn, Hb Suan Dok alpha109(G16)Leu-->Arg and as yet undescribed alpha109(G16)Leu-->Gln, in the GH corner: Hb Foggia alpha117(GH5)Phe-->Ser, or in the H helix: Hb Groene Hart alpha119(H2)Pro-->Ser, Hb Diamant alpha119(H2)Pro-->Leu, Hb Utrecht alpha129(H12)Leu-->Pro. DESIGN AND METHODS: These different mutated alpha-Hb were co-expressed with their chaperone AHSP as a fusion protein with glutathione S-transferase (GST) and analyzed by SDS-PAGE. RESULTS: In all cases the proteins were normally synthesized in bacteria as shown by an expression level of mutated GST-alpha-Hbs similar to that observed for normal GST-alpha-Hb. In contrast, the recovered quantities of purified mutated GST-alpha-Hbs associated with AHSP are highly variable. An extreme case is GST-alpha-Hb(Utrecht) which was only found at trace levels. CONCLUSION: One can assume that different mechanisms may be responsible for the amount of abnormal Hb recovered, such as a highly unstable alpha chain or an impaired formation of the complex AHSP/alpha-Hb or a modification of the alphabeta dimer formation.