Hemoglobin Tetramer Research Articles

An Optimized Lentiviral Vector Efficiently Corrects the Human Sickle Cell Disease Phenotype

Autologous transplantation of hematopoietic stem cells transduced with a lentiviral vector (LV) expressing an anti-sickling HBB variant is a potential treatment for sickle cell disease (SCD). With a clinical trial as our ultimate goal, we generated LV constructs containing an anti-sickling HBB transgene (HBBAS3), a minimal HBB promoter, and different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR). Hematopoietic stem progenitor cells (HSPCs) from SCD patients were transduced with LVs containing either HS2 and HS3 (β-AS3) or HS2, HS3, and HS4 (β-AS3 HS4). The inclusion of the HS4 element drastically reduced vector titer and infectivity in HSPCs, with negligible improvement of transgene expression. Conversely, the LV containing only HS2 and HS3 was able to efficiently transduce SCD bone marrow and Plerixafor-mobilized HSPCs, with anti-sickling HBB representing up to ∼60% of the total HBB-like chains. The expression of the anti-sickling HBB and the reduced incorporation of the βS-chain in hemoglobin tetramers allowed up to 50% reduction in the frequency of RBC sickling under hypoxic conditions. Together, these results demonstrate the ability of a high-titer LV to express elevated levels of a potent anti-sickling HBB transgene ameliorating the SCD cell phenotype.

Analysis of the Quaternary Structure of Hemoglobin Beckman Variant and Molecular Interpretation of Its Functional Abnormality: A Mass-Spectrometry-Based Approach.

Electrostatic attraction between α and β globin chains holds the subunits together in a tetrameric human hemoglobin molecule (α2 β2 ). Compared to normal globin chains, the affinity of a mutant chain to its partner globin might be different in genetic variants of hemoglobin. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous samples, even though the rates of synthesis of both the normal and variant chains are the same. The aforementioned affinities across various globin chains might be assessed by quantification of the different forms of the tetramers present in a variant hemoglobin sample. In the present study, by exploiting mass differences between globin chains, differently populated hemoglobin tetramers present in hemoglobin (Hb) Beckman, a β variant (βA135D), were structurally characterized. The relative populations of dissymmetric tetramers (α2 β2 , α2 ββV , and α2 βV2 ) indicated that both β and βV have different affinities towards the α globin chain. Conformational dynamics analyzed from hydrogen/deuterium exchange kinetics of the three peptide fragments of Hb Beckman in its oxy state displayed molecular insight into its functional abnormality. However, in comparison to normal hemoglobin (α2 β2 ), the point mutation did not show any change in the collision cross-sections of the functionally active conformers of the variant hemoglobin molecules (α2 ββV and α2 βV2 ).

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

AbstractRecently, 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.

Hsp90 chaperones hemoglobin maturation in erythroid and nonerythroid cells

Maturation of adult (α2β2) and fetal hemoglobin (α2γ2) tetramers requires that heme be incorporated into each globin. While hemoglobin alpha (Hb-α) relies on a specific erythroid chaperone (alpha Hb-stabilizing protein, AHSP), the other chaperones that may help mature the partner globins (Hb-γ or Hb-β) in erythroid cells, or may enable nonerythroid cells to express mature Hb, are unknown. We investigated a role for heat-shock protein 90 (hsp90) in Hb maturation in erythroid precursor cells that naturally express Hb-α with either Hb-γ (K562 and HiDEP-1 cells) or Hb-β (HUDEP-2) and in nonerythroid cell lines that either endogenously express Hb-αβ (RAW and A549) or that we transfected to express the globins. We found the following: (i) AHSP and hsp90 associate with distinct globin partners in their immature heme-free states (AHSP with apo-Hbα, and hsp90 with apo-Hbβ or Hb-γ) and that hsp90 does not associate with mature Hb. (ii) Hsp90 stabilizes the apo-globins and helps to drive their heme insertion reactions, as judged by pharmacologic hsp90 inhibition or by coexpression of an ATP-ase defective hsp90. (iii) In nonerythroid cells, heme insertion into all globins became hsp90-dependent, which may explain how mixed Hb tetramers can mature in cells that do not express AHSP. Together, our findings uncover a process in which hsp90 first binds to immature, heme-free Hb-γ or Hb-β, drives their heme insertion process, and then dissociates to allow their heterotetramer formation with Hb-α. Thus, in driving heme insertion, hsp90 works in concert with AHSP to generate functional Hb tetramers during erythropoiesis.

Engineering oxidative stability in human hemoglobin based on the Hb providence (βK82D) mutation and genetic cross-linking.

Previous work suggested that hemoglobin (Hb) tetramer formation slows autoxidation and hemin loss and that the naturally occurring mutant, Hb Providence (HbProv; βK82D), is much more resistant to degradation by H2O2 We have examined systematically the effects of genetic cross-linking of Hb tetramers with and without the HbProv mutation on autoxidation, hemin loss, and reactions with H2O2, using native HbA and various wild-type recombinant Hbs as controls. Genetically cross-linked Hb Presbyterian (βN108K) was also examined as an example of a low oxygen affinity tetramer. Our conclusions are: (a) at low concentrations, all the cross-linked tetramers show smaller rates of autoxidation and hemin loss than HbA, which can dissociate into much less stable dimers and (b) the HbProv βK82D mutation confers more resistance to degradation by H2O2, by markedly inhibiting oxidation of the β93 cysteine side chain, particularly in cross-linked tetramers and even in the presence of the destabilizing Hb Presbyterian mutation. These results show that cross-linking and the βK82D mutation do enhance the resistance of Hb to oxidative degradation, a critical element in the design of a safe and effective oxygen therapeutic.

Immunodetection of Porcine Red Blood Cell Containing Food Ingredients Using a Porcine-Hemoglobin-Specific Monoclonal Antibody.

Monoclonal antibody (mAb) 24C12-E7 has been found to bind to a 12 kDa antigenic protein in the red blood cell (RBC) of porcine blood. The purpose of this study was to determine the identity of this 12 kDa protein and consequently examine its potential as a marker for monitoring porcine RBC-containing ingredients (PRBCIs) in foods. Proteomic techniques identified the 12 kDa antigenic protein to be a monomer of the tetrameric hemoglobin molecule. Further heat-processing of spray-dried PRBCIs diminishes its detectability. Whereas mAb 24C12-E7-based indirect enzyme-linked immunosorbent assay (iELISA) could detect 1% (v/v) or less of PRBCIs in raw and cooked ground meats (beef, pork and chicken), the detection limits were 3 to 30 times higher for spiked cooked beef and pork. The assay is effective for monitoring the presence of PRBCIs in foods to protect the billions of people that avoid consuming blood. In situations where these PRBCIs are present as ingredients in foods that have undergone further heat processing, the assay, however, may not be as sensitive depending on the types of sample matrix, types of PRBCIs and the level of inclusion.

Native mass spectrometry imaging of intact proteins and protein complexes in thin tissue sections

Here, we present native liquid extraction surface analysis (LESA) mass spectrometry imaging of proteins and protein complexes from mouse brain and liver tissue. Intact proteins were detected in characteristically low charge states, indicating that the proteins remain folded. In brain, abundant proteins such as ubiquitin and β thymosin 4 were detected homogeneously across the tissue whereas other proteins, such as neurogranin, were localised in specific anatomical regions. In liver, we demonstrate imaging of a protein complex (tetrameric hemoglobin), as well as fatty acid binding protein. Interestingly, the use of native-like solvents enables extraction of proteins which have not previously been observed in LESA experiments employing denaturing solvents, i.e., native LESA can be applied to extend the range of proteins observed. We also present native LESA ion mobility spectrometry and show that the collision cross sections of proteins extracted from tissue may be determined by travelling wave ion mobility spectrometry. The collision cross section of the 5+ ion of ubiquitin was calculated as 1047Å2, in good agreement with measurements of ubiquitin protein standard solutions. Collision cross sections for the 4+ ions of β-thymosin 4, β-thymosin 10 and two unidentified proteins were also calculated, together with that of a 10+ ion of an unidentified protein of molecular weight 15660Da.

Prothrombotic aspects of sickle cell disease

Sickle cell disease (SCD) is a hematologic disorder caused by a well-characterized point mutation in the β-globin gene. Abnormal polymerization of hemoglobin tetramers results in the formation of sickle red blood cells that leads to vascular occlusions, hemolytic anemia, vascular inflammation and cumulative, multiple organ damage. Ongoing activation of coagulation is another hallmark of SCD. Recent studies strongly suggested that hypercoagulation in SCD is not just a secondary event but contributes directly to the disease pathophysiology. In this article we summarize mechanisms leading to the activation of coagulation, review data indicating direct contribution of coagulation to the pathology of SCD and, we discuss the anticoagulation as a possible treatment strategy to attenuate the disease progression.

Application of isotope exchange based mass spectrometry to understand the mechanism of inhibition of sickle hemoglobin polymerization upon oxygenation.

Sickle hemoglobin (HbS) polymerization initiates in the deoxy state with the binding of hydrophobic patch formed by the isopropyl group of βVal6 residue of a hemoglobin tetramer with the hydrophobic pocket of another tetramer, whose hydrophobic patch binds to the hydrophobic groove of a third molecule. Subsequent elongation of a single stranded polymer followed by the formation of a double strand and finally combination of seven such pairs of double strands results in a fourteen stranded fibrous polymer. Precipitation of this fiber inside the erythrocytes results in sickling of red blood cells. Surprisingly, the polymerization does not occur in the oxy state of HbS. Due to the unavailability of crystal structure of oxy form of HbS, the molecular basis of inhibition of polymerization in the oxy state is unknown to date. In the present study, we have attempted to understand the molecular mechanism of inhibition of polymerization by exploiting the exchange of backbone amide hydrogens of HbS with deuterated solvent. Hydrogen/deuterium exchange kinetics of peptide amide hydrogens of both oxy and deoxy form of HbS were monitored through ESI mass spectrometry. Upon oxygenation changes in the conformational flexibility across different regions of α and β globin chains in the tetrameric HbS molecule were investigated. It was observed that oxygenation led to perturbation in the conformation of several residues around the hydrophobic patch, groove of a tetramer and axial, lateral contacts across the double strands that are involved in HbS polymerization.

Alteration of the α1β2/α2β1 subunit interface contributes to the increased hemoglobin-oxygen affinity of high-altitude deer mice.

BackgroundDeer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant.ResultsHighland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules.ConclusionsThe analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.

"Inside-Out" PEGylation of Bovine β-Cross-Linked Hemoglobin.

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to β-cross-linked (βXL-Hb) hemoglobin (Hb) tetramers through the Cys β93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three βXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P50 =9.18Torr), but the oxygen affinity was not significantly altered by PEGylation (P50 =9.67Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect.

Fine Sampling of the R→T Quaternary-Structure Transition of a Tetrameric Hemoglobin.

Although the end points of the functional transitions of tetrameric hemoglobins (Hbs) have been well characterized, atomic-resolution data on R-T intermediate states are extremely limited. Herein, the X-ray structures of two independent tetramers of the fully ligated carbomonoxy form of Trematomus newnesi hemoglobin (Hb1Tn) within the same crystal are described. These structures show peculiar features in the heme pocket, EF corner, and tertiary/quaternary structure. Distal histidine side chains have a propensity to swing out of the heme pocket and thus allow compression of the EF corner. In this rotameric state, the distal His group does not interact with the CO ligand, consistent with FTIR spectra recorded in solution. At the quaternary-structure level, one tetramer is an intermediate R-T state, whereas the other assumes a T-like structure. Altogether, the structures of these tetramers provide the best available atomic-level picture of the R→T transition of vertebrate Hbs.

Beneficial effects of novel cross-linked hemoglobin YQ23 on hemorrhagic shock in rats and pigs

BackgroundTo overcome the problems of previously reported hemoglobin-based oxygen carriers, we developed a stabilized nonpolymeric cross-linked tetrameric hemoglobin solution (YQ23). The aims of this study were to investigate the oxygen carrying and releasing properties of this novel hemoglobin-based oxygen carrier and to determine whether it has beneficial effects for hemorrhagic shock. MethodsUsing a hemorrhagic shock model in Sprague–Dawley rats and mini-pigs, we tested the effects of infusing 0.1, 0.3, and 0.5 g/kg YQ23 on animal survival, tissue oxygen delivery (DO2) and consumption (VO2), hemodynamics parameters, and liver, renal, and cardiac function. ResultsYQ23 infusion increased the survival rate of rats and pigs with severe hemorrhagic shock in a dose-dependent manner. Moreover, it improved the hemodynamic parameters, cardiac output, DO2 and VO2, and the mitochondrial respiratory function of vital organs. Among the three doses of YQ23, 0.5 gHb/kg YQ23 achieved a similar beneficial effect as whole blood. ConclusionsThis study indicated that the novel cross-linked tetrameric hemoglobin YQ23 has good oxygen carrying and releasing properties and exhibits beneficial effects on hemorrhagic shock in rats and pigs by improving the oxygen carrying and delivery function of blood, which maintains organ function.

Hb Alesha [β67(E11)Val→Met (GTG>ATG); HBB: c.202G > A] Found in a Chinese Girl

Mutations that cause destabilization of the hemoglobin (Hb) tetramer are a rare cause of hemolytic anemia. In contrast to the hemolytic anemia caused by enzyme deficiencies, a dominant mode of inheritance characterizes the unstable Hbs. Hb Alesha [β67(E11)Val→Met; HBB: c.202G>A] is caused by a G>A mutation at codon 67 of the β-globin gene, resulting in a valine to methionine substitution at helix E11. This replacement disrupts the apolar bonds between valine and the heme group, producing an unstable Hb and severe hemolysis. We report this rare hemoglobinopathy in a Chinese girl with severe hemolytic anemia, splenomegaly and frequent requirement for red blood cell (RBC) transfusions.

Liquid extraction surface analysis for native mass spectrometry: Protein complexes and ligand binding

Native liquid extraction surface analysis (LESA) mass spectrometry enables the direct sampling of protein complexes from a solid surface. We have previously demonstrated native LESA mass spectrometry of holomyoglobin (∼17kDa) from glass slides and tetrameric haemoglobin (∼64kDa) from dried blood spots and thin tissue sections. Here, we further explore the capabilities of this emerging technique by investigating a range of proteins which exist in various oligomeric states in vivo. Tetrameric avidin (∼64kDa), octameric (∼190kDa) and hexadecameric (∼380kDa) CS2 hydrolase, and tetradecameric GroEL (∼800kDa) were all detected by native LESA mass spectrometry. Moreover, trimeric AmtB, a membrane protein, could also be observed by native LESA mass spectrometry. The suitability of LESA mass spectrometry for probing protein-ligand binding was also investigated. Non-covalent complexes of the ligand biotin with the proteins avidin, haemoglobin and bovine serum albumin were detected. The results indicate that non-specific binding is minimal and that native LESA mass spectrometry is a promising tool for the investigation of biologically significant ligand binding.

Enhanced Nitrite Reductase Activity and Its Correlation with Oxygen Affinity in Hemoglobin Bis-Tetramers.

The vasoactivity of circulating cross-linked hemoglobin is consistent with the acellular protein penetrating the endothelial lining of blood vessels where hemoglobin can bind nitric oxide, the signal for relaxation of the muscles that surround blood vessels. In an important contrast, derivatives of bis-tetramers that are produced from hemoglobin by chemical coupling do not cause vasoconstriction in animal models. Presumably, they are unable to enter the endothelia where hemoglobin tetramers bind to nitric oxide. In addition, hemoglobin bis-tetramers can produce nitric oxide in circulation through their intrinsic nitrite reductase activity. Examination of this activity for hemoglobin-derived bis-tetramers that are acetylated at lysyl amino groups in their α subunits reveals enhanced activity (k = 2.21 M(-1) s(-1)) compared to that of nonacetylated bis-tetramers (k = 0.70 M(-1) s(-1)). Plots of nitrite reductase activities as a function of the corresponding oxygen affinities of certain allosteric-state-stabilized derivatives reveal a significant correlation, providing a basis for interpretation of the correlated functions.

Thermogravimetric analysis coupled with chemometrics as a powerful predictive tool for ß-thalassemia screening

β-Thalassemia is a hemoglobin genetic disorder characterized by the absence or reduced β-globin chain synthesis, one of the constituents of the adult hemoglobin tetramer. In this study the possibility of using thermogravimetric analysis (TGA) followed by chemometrics as a new approach for β-thalassemia detection is proposed. Blood samples from patients with β-thalassemia were analyzed by the TG7 thermobalance and the resulting curves were compared to those typical of healthy individuals. Principal Component Analysis (PCA) was used to evaluate the correlation between the hematological parameters and the thermogravimetric results. The thermogravimetric profiles of blood samples from β-thalassemia patients were clearly distinct from those of healthy individuals as result of the different quantities of water content and corpuscular fraction. The hematological overview showed significant decreases in the values of red blood cell indices and an increase in red cell distribution width value in thalassemia subjects when compared with those of healthy subjects. The implementation of a predictive model based on Partial Least Square Discriminant Analysis (PLS-DA) for β-thalassemia diagnosis, was performed and validated. This model permitted the discrimination of anemic patients and healthy individuals and was able to detect thalassemia in clinically heterogeneous patients as in the presence of δβ-thalassemia and β-thalassemia combined with Hb Lepore. TGA and Chemometrics are capable of predicting ß-thalassemia syndromes using only a few microliters of blood without any pretreatment and with an hour of analysis time. A fast, rapid and cost-effective diagnostic tool for the β-thalassemia screening is proposed.

Molecular oxygen migration through the xenon docking sites of human hemoglobin in the R-state

A nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to tetrameric human hemoglobin and its isolated α and β chains in buffer solutions equilibrated with 1atm of air and up to 25atm of xenon. Xenon binding to the isolated α chains and to the α subunits within tetrameric hemoglobin was found to cause a decrease in the efficiency of O2 escape by a factor of ~1.30 and 3.3, respectively. A kinetic model for O2 dissociation, rebinding, and migration through two alternative pathways in the hemoglobin subunits was introduced and discussed. It was shown that, in the isolated α chains and α subunits within tetrameric hemoglobin, nearly one- and two-third escaping molecules of O2 leave the protein via xenon docking sites, respectively. The present experimental data support the idea that O2 molecule escapes from the β subunits mainly through the His(E7) gate, and show unambiguously that, in the α subunits, in addition to the direct E7 channel, there is at least one alternative escape route leading to the exterior via the xenon docking sites.