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.

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.

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

The 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.

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) HbβG83C. We investigate the potential side effects of rHbβG83C on endothelial cells.

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.

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

β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-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 β-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.

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.

Interaction of haptoglobin with hemoglobin octamers based on the mutation Asn78Cys or Gly83Cys.

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.

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.

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

β-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), 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.

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.

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.

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.

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.

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.

Reversible hexacoordination of alpha-hemoglobin-stabilizing protein (AHSP)/alpha-hemoglobin Versus pressure. Evidence for protection of the alpha-chains by their chaperone.

Using high hydrostatic pressure or hydrogen peroxide as perturbing agents, we demonstrate a protective effect of the chaperone AHSP for the alpha-chains of Hb. High pressure induces an irreversible aggregation of the ferrous deoxy alpha-chains, whereas the AHSP/alpha-Hb complex shows reversible hexacoordination of the alpha-Hb without protein aggregation. Upon pressure release, the relaxation kinetics of the transition from the hexacoordinated to pentacoordinated form of alpha-Hb in the presence of AHSP exhibit a biphasic shape.

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

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 (alpha-Hb) chains against precipitation. AHSP and alpha-Hb form a heterodimer complex.

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.

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.

Neuroglobin ligand binding kinetics.

Neuroglobin, cytoglobin, and hemoglobins from Drosophila melanogaster and Arabidopsis thaliana were studied for their ligand binding properties versus temperature. These globins have a common feature of being hexacoordinated (via the distal histidine) under deoxy conditions, displaying and enhanced amplitude for the alpha absorption band at 560 nm. External ligands can bind, but the transition from the hexacoordinated form to the ligand (L) bound species is slow, as expected for a replacement reaction Fe-His <--> Fe <--> Fe-L.