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2.
Br J Haematol ; 196(1): 183-192, 2022 01.
Article in English | MEDLINE | ID: mdl-34378186

ABSTRACT

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.


Subject(s)
Anemia, Sickle Cell/metabolism , Biomarkers , Blood Proteins/metabolism , Erythrocytes/metabolism , Molecular Chaperones/metabolism , Adult , Anemia, Sickle Cell/diagnosis , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/genetics , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Erythrocyte Indices , Female , Hemoglobins/metabolism , Humans , Hydroxyurea/administration & dosage , Hydroxyurea/adverse effects , Hydroxyurea/therapeutic use , Male , Middle Aged , Peptide Fragments/metabolism , Prognosis
4.
Blood Adv ; 2(2): 90-94, 2018 01 23.
Article in English | MEDLINE | ID: mdl-29365316

ABSTRACT

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.


Subject(s)
Blood Substitutes/chemical synthesis , Hemoglobins/chemistry , Nanoparticles/chemistry , Oxygen/chemistry , Silicon Dioxide/chemistry , Adsorption , Drug Design , Hemoglobin, Sickle/chemistry , Humans
6.
Br J Haematol ; 179(1): 142-153, 2017 10.
Article in English | MEDLINE | ID: mdl-28643346

ABSTRACT

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.


Subject(s)
Erythrocytes/metabolism , alpha-Globins/metabolism , beta-Thalassemia/blood , beta-Thalassemia/diagnosis , Adolescent , Adult , Aged , Biomarkers , Case-Control Studies , Female , France , Genotype , Hematologic Tests , Humans , Male , Middle Aged , Mutation , Reproducibility of Results , Sensitivity and Specificity , Young Adult , alpha-Globins/genetics , alpha-Thalassemia/blood , alpha-Thalassemia/genetics , beta-Globins/genetics , beta-Thalassemia/genetics
8.
C R Biol ; 338(2): 95-102, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25543885

ABSTRACT

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.


Subject(s)
Blood Substitutes/pharmacology , Hemoglobins/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Biomarkers , Blood Substitutes/toxicity , Cell Survival/drug effects , Dextrans/pharmacology , Dextrans/toxicity , Down-Regulation/drug effects , Drug Evaluation, Preclinical , E-Selectin/biosynthesis , E-Selectin/genetics , Gene Expression Regulation/drug effects , Heme Oxygenase-1/biosynthesis , Heme Oxygenase-1/genetics , Hemoglobins/analysis , Hemoglobins/chemistry , Hemoglobins/toxicity , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Hydroxyethyl Starch Derivatives/pharmacology , Hydroxyethyl Starch Derivatives/toxicity , Inflammation/chemically induced , Intercellular Adhesion Molecule-1/biosynthesis , Intercellular Adhesion Molecule-1/genetics , Methemoglobin/analysis , Models, Molecular , Nitric Oxide Synthase Type III/biosynthesis , Nitric Oxide Synthase Type III/genetics , Oxidative Stress/drug effects , Plasma Substitutes/pharmacology , Plasma Substitutes/toxicity , Protein Conformation , Real-Time Polymerase Chain Reaction , Vasoconstriction/drug effects
9.
PLoS One ; 9(11): e111395, 2014.
Article in English | MEDLINE | ID: mdl-25369055

ABSTRACT

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.


Subject(s)
Blood Proteins/metabolism , Heme/metabolism , Hemoglobins/metabolism , Molecular Chaperones/metabolism , alpha-Globins/metabolism , Amino Acid Sequence , Blood Proteins/chemistry , Blood Proteins/genetics , Carbon Monoxide/chemistry , Carbon Monoxide/metabolism , Circular Dichroism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Heme/chemistry , Hemoglobins/chemistry , Humans , Kinetics , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Spectrophotometry, Ultraviolet , alpha-Globins/chemistry , alpha-Globins/genetics
10.
Nature ; 514(7521): 242-6, 2014 Oct 09.
Article in English | MEDLINE | ID: mdl-25156257

ABSTRACT

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.


Subject(s)
Erythroblasts/metabolism , Erythropoiesis , HSP70 Heat-Shock Proteins/metabolism , alpha-Globins/metabolism , beta-Thalassemia/blood , beta-Thalassemia/metabolism , Apoptosis , Bone Marrow/metabolism , Caspase 3/metabolism , Cell Nucleus/metabolism , Cell Survival/genetics , Cells, Cultured , Cytoplasm/metabolism , Enzyme Activation , Erythroblasts/cytology , Erythroblasts/pathology , Erythropoiesis/genetics , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Gene Expression Regulation , HSP70 Heat-Shock Proteins/genetics , Humans , Kinetics , Molecular Targeted Therapy , Protein Binding , Protein Refolding , beta-Thalassemia/pathology
11.
Biochim Biophys Acta ; 1840(1): 277-87, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24060751

ABSTRACT

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.


Subject(s)
Blood Proteins/metabolism , Heme/metabolism , Hemoglobin A/metabolism , Molecular Chaperones/metabolism , Blood Proteins/chemistry , Blood Proteins/genetics , Carbon Monoxide/metabolism , Circular Dichroism , Heme/chemistry , Hemoglobin A/chemistry , Humans , Kinetics , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Mutagenesis, Site-Directed , Oxidation-Reduction , Oxygen/metabolism , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
12.
Am J Mol Biol ; 2(1): 1-10, 2012 Apr 01.
Article in English | MEDLINE | ID: mdl-23847747

ABSTRACT

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.

13.
Hemoglobin ; 35(5-6): 463-8, 2011.
Article in English | MEDLINE | ID: mdl-21950764

ABSTRACT

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.


Subject(s)
Blood Proteins/metabolism , Molecular Chaperones/metabolism , Thalassemia/metabolism , alpha-Globins/metabolism , Animals , Blood Proteins/genetics , Humans , Molecular Chaperones/genetics , Mutation , Phenotype , Protein Binding/physiology , Thalassemia/genetics
14.
Am J Hematol ; 86(2): 199-202, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21264907

ABSTRACT

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.


Subject(s)
Erythrocytes/metabolism , Hemoglobins/analysis , beta-Thalassemia/blood , Adult , Aged, 80 and over , Blood Proteins/genetics , Blood Proteins/metabolism , Carbon Monoxide/metabolism , Chromatography, Affinity , Female , Genotype , Hemoglobins/metabolism , Humans , Kinetics , Male , Middle Aged , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Subunits/blood , Protein Subunits/metabolism , Recombinant Fusion Proteins/metabolism , Severity of Illness Index , Young Adult , alpha-Thalassemia/blood , alpha-Thalassemia/genetics , alpha-Thalassemia/metabolism , beta-Thalassemia/genetics , beta-Thalassemia/metabolism , beta-Thalassemia/physiopathology
15.
J Biol Chem ; 285(23): 17986-92, 2010 Jun 04.
Article in English | MEDLINE | ID: mdl-20371604

ABSTRACT

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.


Subject(s)
Blood Proteins/chemistry , Blood Proteins/genetics , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Mutation , Carbon Monoxide/chemistry , Child , Circular Dichroism , Fluorescence Resonance Energy Transfer , Hemoglobins/chemistry , Humans , Kinetics , Molecular Chaperones/metabolism , Oxygen/chemistry , Phenotype , Protein Structure, Secondary , Recombinant Proteins/chemistry , Thalassemia/genetics
16.
Clin Biochem ; 42(18): 1818-23, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19482015

ABSTRACT

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.


Subject(s)
Blood Proteins/metabolism , Hemoglobins/metabolism , Molecular Chaperones/metabolism , Mutant Proteins/metabolism , Peptide Fragments/metabolism , Blood Proteins/chemistry , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Chaperones/chemistry , Mutant Proteins/chemistry , Protein Stability , Protein Structure, Secondary , Recombinant Fusion Proteins/metabolism , Solubility
17.
Plasmid ; 61(1): 71-7, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18930760

ABSTRACT

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.


Subject(s)
Cloning, Molecular/methods , Genetic Vectors , Hemoglobin A/genetics , Hemoglobin A/isolation & purification , Carbon Monoxide/metabolism , Gene Expression , Glutathione Transferase/genetics , Glutathione Transferase/isolation & purification , Glutathione Transferase/metabolism , Hemoglobin A/metabolism , Humans , Kinetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , alpha-Globins/genetics , beta-Globins/genetics
18.
Hemoglobin ; 32(4): 327-49, 2008.
Article in English | MEDLINE | ID: mdl-18654884

ABSTRACT

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.


Subject(s)
Genetic Variation , Hemoglobins, Abnormal/genetics , alpha-Thalassemia/genetics , Chemical Precipitation , Globins/chemistry , Globins/genetics , Hemoglobins, Abnormal/chemistry , Humans , Protein Binding
19.
Biochim Biophys Acta ; 1784(10): 1448-53, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18342640

ABSTRACT

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.


Subject(s)
Blood Substitutes/therapeutic use , Hemoglobins/therapeutic use , Nanoparticles/therapeutic use , Biological Transport , Blood Substitutes/chemical synthesis , Blood Substitutes/chemistry , Genetic Engineering/methods , Genetic Vectors , Globins/chemistry , Globins/genetics , Globins/therapeutic use , Haptoglobins/chemistry , Haptoglobins/genetics , Haptoglobins/therapeutic use , Hemoglobins/chemistry , Hemoglobins/genetics , Humans , Kinetics , Pharmaceutical Preparations/administration & dosage , Polysaccharides/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/therapeutic use
20.
Gene ; 398(1-2): 35-41, 2007 Aug 15.
Article in English | MEDLINE | ID: mdl-17531404

ABSTRACT

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.


Subject(s)
Hemoglobins/chemistry , Models, Molecular , Software , Amino Acid Sequence , Amino Acids/chemistry , Amino Acids/genetics , Animals , Computer Graphics , Databases, Protein , Hemoglobins/genetics , Humans , Mutation , Protein Conformation , Protein Structure, Tertiary
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