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.

HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in ß-thalassaemia.

ß-Thalassaemia major (ß-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing ß-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human ß-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of ß-TM erythroblasts, which may provide a rationale for new targeted therapies of ß-TM.

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.

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.

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.

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.

α-Hemoglobin stabilizing protein: a modulating factor in thalassemias?

α-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 α-hemoglobin (α-Hb) chains against precipitation. α-Hemoglobin stabilizing protein forms a heterodimer complex with α-Hb, then displaying modified oxygen binding kinetics. In the absence of AHSP, α-Hb oxidizes and precipitates within the erythrocyte precursors of bone marrow leading to apoptosis and defective erythropoiesis. Several α-Hb variants with a structural abnormality, frequently located in the contact area between α-Hb and AHSP, exhibit instability and a thalassemia-like syndrome when they are associated with another α-thalassemia (α-thal) determinant. We suggest that this disorder could result from a disturbed interaction between the abnormal α-Hb chains and AHSP. Hb Groene Hart (Pro119>Ser) was one of the first examples in which we observed this abnormality. We later verified this mechanism in a list of several variants, now considered as being nondeletional α-thalassemias. Conversely, it was hypothesized from studies on knock-out mice, that a defect affecting AHSP could cause a thalassemia-like syndrome. This was supported in man by studies showing that a decreased expression of AHSP linked to specific genetic clades could act as a modulating factor in some thalassemia phenotypes. It was also supported by our observation of a family from Southeast Asia, in which a child homozygous for an AHSP mutant (Val56>Gly) displayed, in his first year of life, a moderate thalassemia syndrome. This mutant AHSP was expressed in vitro and demonstrated by biochemical and biophysical studies to display a clear defective interaction with α-Hb, which could support the hypothesis that the reb blood cell (RBC) disorders of the child resulted from this abnormality. It therefore appears that AHSP is a factor with a key role in the formation of Hb tetramers and that structural abnormalities, either on the α-Hb or on the AHSP, may act as a thalassemia modulating factor.

Octamers and nanoparticles as hemoglobin based blood substitutes.

Progress in developing a blood substitute is aided by new biotechnologies and a better understanding of the circulatory system. For Hb based solutions, there is still a debate over the best set of fundamental parameters concerning the oxygen affinity which is correlated with the oxidation rate, the cooperativity, the transporter size, and of course the final source of material. Genetic engineering methods have helped discover novel globins, but not yet the quantity necessary for the high demand of blood transfusions. The expanding database of globin properties has indicated that certain individual parameters are coupled, such as the oxygen affinity and the oxidation rate, indicating that one must accept a compromise of the best parameters. After a general introduction of these basic criteria, we will focus on two strategies concerning the size of the oxygen transporter: Hb octamers, and Hb integrated within a nanoparticle.

Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin.

To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-alpha-[SD]-beta. This vector allows the co-expression of alpha-Hb as a fusion protein with Glutathione S-Transferase (GST-alpha-Hb) and beta-Hb with an additional methionine at the N-terminal extremity (rbeta-Hb). These proteins were solubilized as GST-alpha-Hb/rbeta-Hb complex form and purified in one step by affinity chromatography on immobilized glutathione. The CO binding kinetic studies show that the GST-alpha-Hb/rbeta-Hb complex and recombinant Hb A exhibit the same allosteric behavior as for native Hb A. The GST moiety, which does not modify the function of the complex, can be easily eliminated by cleavage by the PreScission Protease. After cleavage during the rapid purification procedure, over 20mg of recombinant Hb per liter of culture were obtained, more than double the yield of previous co-expression systems. This polycistronic vector system, which offers the additional advantage of a very rapid purification, is especially well suited for the study of abnormal, unstable globins in order to better understand the associated pathology.

Unstable and thalassemic alpha chain hemoglobin variants: a cause of Hb H disease and thalassemia intermedia.

We report an update of the alpha-globin gene point mutations resulting in structural modification associated with an alpha-thalassemia (alpha-thal) phenotype. These variants, barely symptomatic in the heterozygous state, are either unstable due to folding defects and/or defects in binding to alpha-hemoglobin stabilizing protein (AHSP). This is predicted to result in precipitation of the unstable alpha chains or Hb variant, a concomitant decrease in the overall quantity of normal alpha-globin in the red cells and a potential degree of anemia and possibly, hemolysis. Genotype/phenotype correlation and potential genetic risk in combination with common or less common alpha-thal defects are discussed.

Manipulating hemoglobin oxygenation using silica nanoparticles: a novel prospect for artificial oxygen carriers.

Recently, nanoparticles have attracted much attention as new scaffolds for hemoglobin-based oxygen carriers (HBOCs). Indeed, the development of bionanotechnology paves the way for the rational design of blood substitutes, providing that the interaction between the nanoparticles and hemoglobin at a molecular scale and its effect on the oxygenation properties of hemoglobin are finely controlled. Here, we show that human hemoglobin has a high affinity for silica nanoparticles, leading to the adsorption of hemoglobin tetramers on the surface. The adsorption process results in a remarkable retaining of the oxygenation properties of human adult hemoglobin and sickle cell hemoglobin, associated with an increase of the oxygen affinity. The cooperative oxygen binding exhibited by adsorbed hemoglobin and the comparison with the oxygenation properties of diaspirin cross-linked hemoglobin confirmed the preservation of the tetrameric structure of hemoglobin loaded on silica nanoparticles. Our results show that silica nanoparticles can act as an effector for human native and mutant hemoglobin. Manipulating hemoglobin oxygenation using nanoparticles opens the way to the design of novel HBOCs.

Exploiting a list of protein sequences.

We describe a software program to help exploit a database of aligned protein sequences. In addition to the classical lists of sequences, a graphical representation is used to get a better overview of the information. As natural parameters, the type of amino acid and sequence position are used. Various plots or 3D representations are then updated. Examples are shown based on globin sequences from various species and on the abnormal human hemoglobins. The software should be of interest to protein engineers who need to know what variants are already known.

High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone.

The alpha-subunits of human hemoglobin (Hb) have been more difficult to express than beta-chains owing to the high instability of alpha-chains. Here, we describe the production in Escherichia coli of a soluble recombinant alpha-Hb with human alpha-hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX-alpha-AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express alpha-hemoglobin and human AHSP. While the GST-alpha-Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX-alpha-AHSP permits the co-expression of soluble GST-alpha-Hb and GST-AHSP. The alpha-Hb, produced at a high yield of 12 to 20 mg per liter of culture, was then purified as a complex with its chaperone. Biochemical and biophysical properties of recombinant AHSP/recombinant alpha-Hb complex were similar to those of recombinant AHSP/native alpha-Hb complex as assessed by UV/visible and CO or O(2) binding properties. This co-expression technique can be use to study the interaction between a molecular chaperone and its target protein and, more generally, this system would be particularly interesting for the study of partner proteins when one or both proteins are individually unstable.

Recombinant hemoglobin betaG83C-F41Y.

We have engineered a stable octameric hemoglobin (Hb) of molecular mass 129 kDa, a dimer of recombinant hemoglobin (rHb betaG83C-F41Y) tetramers joined by disulfide bonds at the beta83 position. One of the major problems with oxygen carriers based on acellular hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb betaG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinetics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb betaG83C-F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 M(-1) x h(-1) (observed time coefficient of 1 h at 1 mM glutathione) at 25 degrees C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 microM Hb and 25 degrees C. In addition, the octameric rHb betaG83C-F41Y was resistant to potential reducing agents present in fresh plasma.

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.

Stable octameric structure of recombinant hemoglobin alpha(2)beta(2)83 Gly-->Cys.

We have engineered a recombinant hemoglobin (rHb betaG83C) based on the variant Hb Ta-Li, which oligomerizes through intertetramer disulfide bonds. Size exclusion chromatography and electrospray ionization mass spectrometry show that the rHb betaG83C assembles into an oligomeric structure the size of a dimer of tetramers. The oligomer has carbon monoxide-binding properties similar to those of natural human hemoglobin. Unlike HbA, the oligomer does not participate in dimer exchange. The CO kinetics, auto-oxidation rate, and gel filtration experiments on the oligomeric betaG83C did not show the usual concentration dependence, implying that it does not dissociate easily into smaller species. The octamer could be dissociated by the use of reducing agents. The action of reduced glutathione on oligomeric betaG83C exhibited biphasic kinetics for the loss of the octameric form, with a time constant for the rapid phase of about 2 h at 1 mM glutathione. However, the size of oligomer betaG83C was not modified after incubation with fresh plasma.