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#25369055   2014/11/05 Save this To Up

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.

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#24060751   2013/12/10 Save this To Up

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

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).

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#22287545   2012/07/03 Save this To Up

Insights into hemoglobin assembly through in vivo mutagenesis of α-hemoglobin stabilizing protein.

α-Hemoglobin stabilizing protein (AHSP) is believed to facilitate adult Hemoglobin A assembly and protect against toxic free α-globin subunits. Recombinant AHSP binds multiple forms of free α-globin to stabilize their structures and inhibit precipitation. However, AHSP also stimulates autooxidation of αO(2) subunit and its rapid conversion to a partially unfolded bishistidyl hemichrome structure. To investigate these biochemical properties, we altered the evolutionarily conserved AHSP proline 30 in recombinantly expressed proteins and introduced identical mutations into the endogenous murine Ahsp gene. In vitro, the P30W AHSP variant bound oxygenated α chains with 30-fold increased affinity. Both P30W and P30A mutant proteins also caused decreased rates of αO(2) autooxidation as compared with wild-type AHSP. Despite these abnormalities, mice harboring P30A or P30W Ahsp mutations exhibited no detectable defects in erythropoiesis at steady state or during induced stresses. Further biochemical studies revealed that the AHSP P30A and P30W substitutions had minimal effects on AHSP interactions with ferric α subunits. Together, our findings indicate that the ability of AHSP to stabilize nascent α chain folding intermediates prior to hemin reduction and incorporation into adult Hemoglobin A is physiologically more important than AHSP interactions with ferrous αO(2) subunits.

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#21529291   2011/05/02 Save this To Up

Characterization of a monoclonal antibody to erythroid related factor.

We describe here a novel IgG monoclonal antibody to erythroid-related factor (ERAF), also known as alpha hemoglobin stabilizing protein (AHSP) and eryththroid differentiation related factor (EDRF). Our antibody named PCE 5 is an IgG(1) kappa chain and is to the peptide sequence MVTVVE ranked highly in our active site analysis and binds with high affinity to ERAF.

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#21264907   2011/01/25 Save this To Up

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), 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 β-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 β-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 β-thalassemia phenotypes and to follow the evolution of the globin chain imbalance(α/β+γ ratio) in response to treatment.

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#20920461   2010/12/08 Save this To Up

Modulation of expression and polymerization of hemoglobin Polytaur, a potential blood substitute.

Chemically or genetically modified hemoglobins are a therapeutic class indicated for the treatment of a variety of hypo-oxygenation pathologies, severe trauma-related hemorrhages or elective surgery when blood transfusions are refused or not available. Recombinant heterologous hemoglobins offer the possibility of a potentially unlimited production and genetically optimized properties in terms of oxygen affinity, NO reactivity and resistance to autoxidation. Hemoglobin Polytaur is an autopolymerizing human-bovine hybrid mutant, previously obtained as a 500kDa polymer, shown to reduce the infarct volume from focal cerebral ischemia in in vivo animal models. In this work, hemoglobin Polytaur polymerization, carried out under conditions to minimize heme oxidation and modification, resulted in a 180kDa cyclic homogeneous trimer of hemoglobin tetramers. This novel oligomer was characterized by electrophoresis, MALDI-TOF mass spectrometry and gel filtration. The size and the oxygen binding properties were shown to be ideally suited for its use as a blood substitute. Co-expression with the human α hemoglobin-stabilizing protein (AHSP), a chaperone that assists hemoglobin folding in vivo, resulted in an unexpected decrease in yield and in unusual spectroscopic and functional properties, suggesting the formation of strong protein-protein interactions that reduce the expression, hinder the tetramer assembly and prevent purification.

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#20815047   2010/09/27 Save this To Up

Analysis of alpha hemoglobin stabilizing protein overexpression in murine β-thalassemia.

Excess free alpha-globin is cytotoxic and contributes to the pathophysiology of b-thalassemia. Alpha hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free alpha-globin to promote its folding and inhibit its ability to produce damaging reactive oxygen species. Reduced AHSP levels correlate with increased severity of b-thalassemia in some human cohorts, but causal mechanistic relationships are not established for these associations. We used transgenic and lentiviral gene transfer methods to investigate whether supraphysiologic AHSP levels could mitigate the severity of b-thalassemia intermedia by providing an increased sink for the excess pool of alpha-globin chains. We tested wild-type AHSP and two mutant versions with amino acid substitutions that confer 3- or 13-fold higher affinity for alpha-globin. Erythroid overexpression of these AHSP proteins up to 11-fold beyond endogenous levels had no major effects on hematologic parameters in b-thalassemic animals. Our results demonstrate that endogenous AHSP is not limiting for a-globin detoxification in a murine model of b-thalassemia.

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#20371604   2010/05/31 Save this To Up

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.

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#19482015   2009/11/27 Save this To Up

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

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.

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#17052927   2006/11/19 Save this To Up

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. In the absence of AHSP, alpha-Hb oxidizes and precipitates within the erythrocyte precursors of the bone marrow leading to apoptosis and defective erythropoiesis. In vitro the binding of AHSP to ferrous alpha-Hb accelerates oxidation of the heme iron in alpha-Hb, but the complex is more resistant to protein unfolding. AHSP could act as a modulating factor in beta-thalassemia. Recent studies showed more severe thalassemic syndromes in patients with decreased levels of AHSP and in one patient who carried a structurally abnormal AHSP. Some alpha-Hb variants with structural abnormality located in the contact area between alpha-Hb and AHSP exhibit an instability and a thalassemic like syndrome. We suggest that this could result from a disturbed interaction between alpha-Hb variants and AHSP. To study this interaction, we constructed the pGEX-alpha-AHSP vector that co-expressed human alpha-Hb and AHSP. Using this approach, we investigated the alpha42 (C7), alpha104 (G11) and alpha119 (H2) sites, where variants with some thalassemic features have been described. Results obtained with recombinant Groene Hart alpha-Hb and Diamant alpha-Hb, in which proline 119 is replaced by a serine and a leucine, respectively, showed clearly an impaired interaction with AHSP. In contrast, the alpha mutants at the sites 42 and 104 exhibit a normal interaction with AHSP. The CO rebinding kinetics of the AHSP/alpha-Hb(42mutant) complexes were similar to those previously obtained with the AHSP/alpha-Hb(WT) complex, which shows a modified rate that is intermediate to the classical Hb allosteric states.

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