Hemoglobin Tetramer Research Articles

Efficient conversion of hemoglobin to a non-vasoactive oxygen carrier by site-specific cross-linking with azido acyl methyl phosphates followed by bio-orthogonal CuAAC with a bis-alkyne

While cross-linked hemoglobin tetramers are functional acellular oxygen carriers, their ability to scavenge endogenous nitric oxide (NO) by endothelial pore penetration results in adverse cardiovascular effects. Animal studies established that cross-linked human hemoglobins, chemically joined into a double protein, avoid NO scavenging, presumably due to their larger size preventing penetration into endothelial regions that produce NO. In the present report, we utilize azide-containing acyl phosphate reagents to form cross-linked hemoglobins then bio-orthogonally click-couple them with a bis-alkyne (CuAAC). The production of these larger oxygen-carrying hemoglobin conjugates is obtained in high yields through subunit-specific cross-linking between each βLys82 ε-amino group. The methyl phosphate leaving groups provide electrostatically induced β-subunit site-selectivity, producing azido-cross-linked hemoglobin that undergoes highly efficient CuAAC compared with previous cross-linkers. The acyl phosphates also efficiently cross-link both T-state and R-state hemoglobin. The resulting bis- and tris-tetrameric hemoglobin conjugates exhibit oxygen affinity and cooperativity that are comparable to those of the native protein. The hemoglobin derivatives from the process we describe can function as sources of oxygen in biomedical applications, such as in ex-vivo donor organ perfusion.

A Hypovolemic Infusion of Human Methemoglobin in Golden Syrian Hamsters Provides Insight on its effect on the Microcirculation

Introduction: Hemoglobin (HbFe(II)) plays a crucial role in transporting oxygen to tissues for metabolic needs. However, this protein isn't just an oxygen carrier; it also regulates vascular tone and inflammation by its redox coupling with methemoglobin (HbFe(III)). Methemoglobin's oxidized form, Fe (III), possesses the potential to produce water from oxygen as a free energy reaction. It functions as a nitrite reductase, regulating the oxygen level in the blood and controlling vascular tone by generating nitric oxide from nitrite, thereby increasing blood flow when oxygenated hemoglobin decreases. If methemoglobin can't be reduced, it leads to the dissociation of tetrameric hemoglobin into αβ dimers, releasing heme and iron into circulation. Methods: The study involved Golden Syrian Hamsters with dorsal skin fold window chambers, observing acute methemoglobin effects via hypovolemic infusion (10% of the animal’s blood volume) compared to human hemoglobin and a lactated ringer's control. Microhemodynamics, mean arterial pressure, heart rate, blood gases, and blood properties were measured. Results: Methemoglobin did not significantly affect MAP and HR but increased arteriole diameters by 5% and venule diameter by 15% compared to baseline. However, it caused a 50% reduction in functional capillary density compared to baseline, alongside significant cardiac and kidney inflammation. Conclusion: The study concludes that a hypovolemic infusion of methemoglobin induces vasoreactivity but results in poor microcirculatory pressure and inflammation in vital organs. This demonstrates the delicate balance and potential adverse effects associated with alterations in the hemoglobin redox state and highlights the importance of maintaining its balance for vascular and organ health. Project number: R01HL159862 Project number: R01HL162120. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of a rare [Gγ(Aγδβ)0] -thalassemia using tandem mass spectrometry.

Thalassemias are a group of inherited monogenic disorders characterized by defects in the synthesis of one or more of the globin chain subunits of the hemoglobin tetramer. Delta-beta (δβ-) thalassemia has large deletions in the β globin gene cluster involving δ- and β-globin genes, leading to absent or reduced synthesis of both δ- and β-globin chains. Here, we used direct globin-chain analysis using tandem mass spectrometry for the diagnosis of δβ-thalassemia. Two cases from unrelated families were recruited for the study based on clinical and hematological evaluation. Peptides obtained after trypsin digestion of proteins extracted from red blood cell pellets from two affected individuals and their parents were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometric analysis revealed a severe reduction in δ, β, and Aγ globin proteins with increased Gγ globin protein in the affected individuals. The diagnosis of Gγ(Aγδβ)0 -thalassemia in the homozygous state in the affected individuals and in the heterozygous state in the parents was made from our results. The diagnosis was confirmed at the genetic level using multiplex ligation-dependent probe amplification (MLPA). Our findings demonstrate the utility of direct globin protein quantitation using LC-MS/MS to quantify individual globin proteins reflecting changes in globin production. This approach can be utilized for accurate and timely diagnosis of hemoglobinopathies, including rare variants, where existing diagnostic methods provide inconclusive results.

Glutathione Non-Covalent Binding Sites on Hemoglobin and Major Glutathionylation Target betaCys93 Are Conservative among Both Hypoxia-Sensitive and Hypoxia-Tolerant Mammal Species.

Intracellular tripeptide glutathione is an important agent of cell survival under hypoxia. Glutathione covalently binds to SH groups of hemoglobin cysteine residues, protecting them from irreversible oxidation, and changes its affinity to oxygen. Reduced glutathione (GSH) can also form a noncovalent complex with hemoglobin. Previously, we showed that hemoglobin tetramer has four noncovalent binding sites of glutathione GSH molecules inside, two of which are released during hemoglobin transition to deoxy form. In this study, we characterized the conserved cysteine residues and residues of noncovalent glutathione binding sites in the sequences of a number of hypoxia-tolerant and hypoxia-sensitive mammals. The solvent accessibility of all HbA and HbB residues in oxy and deoxy forms was analyzed. The alpha subunit of all species considered was shown to have no conserved cysteines, whereas the beta subunit contains Cys93 residue, which is conserved across species and whose glutathionylation changes the affinity of hemoglobin for oxygen 5-6-fold. It was found that the key residues of noncovalent glutathione binding sites in both alpha and beta subunits are absolutely conserved in all species considered, suggesting a common mechanism of hemoglobin redox regulation for both hypoxia-sensitive and hypoxia-tolerant mammals.

Dual α-Globin and Truncated EPO Receptor Knockin Restores Hemoglobin Production in α-Thalassemia-Derived Hematopoietic Stem and Progenitor Cells

Introduction: α-thalassemia major (ATM) is an autosomal recessive disorder where all four copies of the α-globin gene are deleted. A traditionally lethal disease due to severe fetal hypoxia, this condition is now survivable due to the advent of in utero blood transfusions. However, patients still require life-long transfusions postnatally. While allogeneic hematopoietic stem cell transplant (HSCT) can provide a definitive cure, it is limited by the paucity of suitable donors, has a high morbidity and mortality due the prerequisite myeloablative regimens, and carries the risk of immune rejection and graft-versus-host disease. Here, we describe a CRISPR/AAV-mediated genome editing strategy to restore a full-length copy of the α-globin gene at the β-globin locus in ATM patient-derived HSCs to restore normal hemoglobin production to a patient's red blood cells (RBCs). Methods: We identified a Cas9 gRNA that efficiently introduced indels at the HBB locus as well as an AAV6 vector that mediated efficient integration into the start codon of HBB. After designing and testing integration cassettes, we developed two candidate vectors that insert a HBA1 transgene at the HBB locus, with one bicistronic vector that also expresses a truncated form of the erythropoietin receptor (t EPOR) to increase erythropoietic output from edited HSCs. We used this approach to edit CD34 + HSCs derived from the bone marrow of three patients with ATM. Edited cells were cultured for 16 days in RBC differentiation media. Editing rates were quantified by a custom ddPCR assay. To determine the transcriptional response to these editing strategies, bulk RNA sequencing was performed comparing wild-type cells and patient cells that underwent respective editing conditions. Lastly, α-globin protein production was quantified using tetramer and single-globin high protein liquid chromatography (HPLC). Results: While effective editing frequencies in ATM HSCs were achieved by the HBA integration vector (39% edited alleles), we observed that editing rates increased over the course of RBC differentiation in the combined HBA+t EPOR edited condition, reaching 76% edited alleles by day 16. We also found that total cell counts by the end of RBC differentiation more than doubled (2.2-fold) in the HBA+t EPOR condition compared to the HBA alone condition. RNA sequencing of edited ATM-derived HSCs following RBC differentiation revealed an upregulation of HBA1, HBA2, and EPOR transcripts compared to unedited patient cells. These results were supported by HPLC, which demonstrated restoration of hemoglobin tetramers, including fetal hemoglobin (α+γ subunits), adult hemoglobin (α+β subunits), and hemoglobin A2 (α+δ subunits) compared to unedited patient cells. Complementing this with globin single chain analysis found that we had achieved 11% restoration of α-globin compared to wild-type levels. In spite of integration at the HBB locus, neither RNA sequencing nor HPLC showed disruption of β-globin production. α-globin production with a concomitant decrease in the formation of toxic β-globin dimers was confirmed by Western blot, with restoration of the normal balance of α-globin to dimeric β-globin in edited cells to wild-type levels. Conclusions: To our knowledge, these results demonstrate for the first time that gene therapy or genome editing may be used to increase α-globin production in ATM patient-derived RBCs. We found that the editing strategy that paired site-specific α-globin integration with t EPOR expression achieved the highest editing frequencies, greatest production of edited cells, and the most robust production of α-globin protein compared to α-globin integration alone. By increasing erythropoietic output using t EPOR, this approach has the potential to overcome some of the clinical challenges previously seen in HSCT for the hemoglobinopathies, including low editing and engraftment rates. Moreover, as patients with severe anemia such those with ATM have increased levels of circulating erythropoietin due to severe anemia, a t EPOR-based enrichment strategy is likely to be especially potent following transplantation into ATM bone marrow. We believe this could allow clinicians to reduce or eliminate the need for myeloablation, which stand as a major barrier to HSCT safety and efficacy. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for ATM.

Α-Globin Lentiviral Vectors for Hematopoietic Stem Cell Gene Therapy of α-Thalassemia

α-thalassemia is an inherited blood disorder caused by mutations in α-globin genes (HBA1 and HBA2) resulting in the reduction of α-globin chains, the subunit, along with β-globin chain, constituting adult hemoglobin (α 2β 2). While a reduction of α-globin expression of 40% presents asymptomatic phenotypes, expression levels decreasing to <20% or an α/β-globin mRNA ratio of <0.2, causes severe anima. Treatment for severe cases of α-thalassemia entails lifelong, biweekly blood transfusions with daily chelation therapy. While these therapies enable patients to live into mid- to late-adulthood, they continue to engender serious clinical manifestations, such as iron toxicity. To answer this clinical need, our laboratory has developed a stem cell gene therapy in which functional copies of α-globin gene integrate into the genome of patient's hematopoietic and progenitor stem cells (HSPCs) by lentiviral vectors (LVs) to normalize the globin chain imbalance and restore hemoglobin function in derived red blood cells (RBCs). The design of our α-globin LVs (AGLVs) is based on GLOBE β-globin LV utilized in a clinical trial for β-thalassemia (NCT02453477), which has achieved transfusion reduction and independence in patients with transfusion-dependence β-thalassemia major. We have constructed a series of short proviral length AGLVs for optimized titer production, HSPC infectivity, and lineage-specific α-globin gene expression. Twelve AGLVs varying in gene and regulatory element compositions were constructed and assessed for raw titer yields and characterized for gene transfer efficiency, mRNA expression and hemoglobin production in a α-globin knockout (KO) human erythroid cell line. Successfully, all AGLVs confer high raw titers ~ 1e7 TU/mL and enabled the production of adult hemoglobin (HbA). We identified two optimal AGLVs for further characterization in human primary HSPCs: 1) A2-UV for yielding highest raw titers and gene transfer efficiency, and 2) A2-Globe for producing the most α-globin mRNA. Both A2-UV and A2-Globe LVsharbor HBA2, are regulated by the β-globin promoter and enhanced by the core or large β-LCR enhancer region, respectively. To assess candidate AGLVs in human HSPCs, AGLVs were tagged at the transcription level to enable identification and quantification of vector-derived α-globin mRNA proceeding HSPCs transduction anderythroid differentiation. A2-UV demonstrated optimum CD34 + infectivity and gene transfer, and A2-Globe expressed a high ~30% of α-globin mRNA per total β-globin per vector copy number (VCN), achieving levels of one endogenous α-globin gene (~25% per total β-globin). Current studies examined A2-UV and A2-Globe in α-thalassemia major patient HSPCs - lacking all four α-globin genes - harboring a lethal homozygous 20kb deletion of HBA1 and HBA2, known as SEA –/– mutation. Patient HSPCs were transduced and differentiated into RBCs. A high CD34+ infectivity and gene transfer was obtained by both candidate vectors, without utilization of transduction enhancers. Furthermore, a correction of α/β-globin mRNA ratio of ~0.7 was obtained while retaining a low VCN (<3).RBCs were then analyzed at the protein level . Chromatograms of hemoglobin tetramers revealed that HbA represented 98% of all hemoglobin, compared to 0% in non-transduced patient RBCs. Single globin chain chromatograms successfully demonstrated α/β-globin chain ratio of ~0.4 or higher and successfully demonstrated that a low average of copies per cell (~3) restored 60% of adult hemoglobin when normalized to healthy donor RBCs. These data demonstrate α/β-globin correction and hemoglobin restoration in α-thalassemia patient cells with the most severe mutation. Three assays were conducted on patient cells, all with similar and highly encouraging results for disease amelioration in severe cases of α-thalassemia. Overall, this project is paving the way for the first development of a curative gene therapy approach for α-thalassemia.

Structural Effect of Gγ and Aγ Globin Chains in Fetal Hemoglobin Tetramer

Introduction. Hemoglobin (Hb) is a tetrameric oxigen and carbon dioxide transportation protein; it is composed of four polypeptide chains, two of alpha type, which can be ζ, α 2 or α 1; and two beta chains, which include ε, Gγ, Aγ, δ or β (Antonini and Brunori, 1970).The synthesis of these globin chains depends on the stage of human development; in embryonic stages the hemoglobins Gower I (ζ 2ε 2), Gower II (α 2ε 2) and Portland (ζ 2γ 2) are synthesized; subsequently, from the third week of gestation begins the synthesis of fetal hemoglobin (HbF) formed by two alpha chains and two gamma chains (α 2γ 2); finally during the last trimester the second hemoglobin switching takes place consisting on decreased synthesis of gamma globin chains and the beginning of beta and delta globin chain synthesis that will be forming adult hemoglobin (α 2β 2) and hemoglobin A 2 (α 2δ 2), which will remain constant throughout extrauterine life (Wilber, 2011). One of the molecular mechanisms that has allowed the evolutionary success of humans has been gene duplication, although it results from a random process it favors DNA variation that increases the adaptive capacity of a population (Magadum, 2013). The high homology between duplicated genes is clear, however, it allows differences to arise because of genetic conversion, globin genes HBG2, HBG1, for instance ,which can be observed in their functional products; Gγ and Aγ globin chains (Chen, 2007), both formed by 146 residues, with their distinct difference a position 136 where there is a change of glycine in Gγ by alanine in Aγ and maintain a ratio of 3:1 ( Gγ: Aγ) during fetal stages; while in extra uterine life this ratio is reversed to 2:3 ( Gγ: Aγ), the heterogeneity of γ globins available for HbF synthesis allows the formation of three variants of the same molecule α 2Gγ 2; 2α A/Gγ and 2α Aγ 2; these γ globin chainsdifferences potential implication on HbF interaction with oxygen has not been elucidated as well as whether the presence of a specific type occurs to a greater or lesser extent in different pathologies(Alter 1979). Objective . To evaluate the structural effect of the type of γ globin chain in three models of Fetal Hemoglobin. Methodology . Modeling of the three HbF possible structures: α 2Gγ 2; 2α A/Gγ and 2α Aγ 2 was performed on UCSF Chimera using the crystallography structures of a HbA (1a3n) and Hb Bart (1i3d) hemoglobins as atomic position templates of α and Aγglobin chains. HbF models as well as HbA and Bart hemoglobin (as reference) where then uploaded to CHARMM-GUIplatform to establish system conditions for molecular dynamic simulation such as force field (c36m), water box size (90 Å) and temperature (310 K), molecular dynamics simulation was run for 100 ns on GROMCS (by triplicate), results were analyzed by Root Mean Square Deviation (RMDS) and Root Mean Square Fluctuation (RMSF) of γ globin chains. Results.Three different HbF tetrameric structures where created based on γ globin chains available α 2Gγ 2; 2α A/Gγ and 2α Aγ 2, RMSD shows fluctuation in atomic positions through time among the three HbF models (figure 1: a, b, c), it is shown that the HbF behavior most similar to HbA (figure 1d) is the 2α A/Gγ (figure 1b)which could indicate that this particular type of HbF would be the more abundant during healthy extrauterine life since HbA responds to a normoxic environment that can become hypoxic at medular level when some disease are present, leukemia for instance. By comparing Bart hemoglobin to HbF models is clear that Aγreduces residues movement, limiting tetramer flexibility thus restraining allosteric changes needed for gases exchange, which can be seen when analyzing HbF γ globin chains at 100-146 residues region where there is no fluctuation in the Aγ 2chains opposite to what is shown by Gγ 2 (Figure 2a, b and c). To evaluate these RMSD results, cluster analysis was performed for each replica in the three HbF structures, resulting in values of α 2Gγ 2: 0.13, 2α A/Gγ and 2α Aγ 2: 0.12 with differences below 0.20 among replicates showing the validity of the results. Conclusion and perspectives . There are structural changes in HbF depending on the presence of Gγ or Aγ globin chains. Further studies are required to identify the type of tetramer present in healthy individuals as in patients with different blood malignancies where HbF has been reported as a therapeutic target or risk factor to understand its effect and association with opposite outcomes.

Assessing the Impact of the Covid-19 Pandemic on Outcomes of Patient Admitted with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is an autosomal recessive hemoglobinopathy which affects millions of people worldwide. It is caused by a substitution of the glutamic acid (Glu) amino acid with valine (Val) at the sixth position of the β-globin gene, resulting in a mutated hemoglobin tetramer, HbS, in the erythrocytes of affected individuals. This morphology results in sickle-shaped red blood cells and hemolysis when deoxygenated and leads to vaso-occlusion with associated ischemia. SCD is characterized by repeated severe acute pain episodes, acute chest syndrome, and can be complicated by stroke, nephropathy, avascular necrosis, and more. Many studies have shown disparities in outcomes in racial/ethnic minorities as well as in those with lower socioeconomic status. These factors have also been linked to increased risk of COVID-19 infection and infection related mortality. In this study, we aim to explore the baseline characteristics of patients with SCD, prevalence of hospitalizations, and compare inpatient outcomes during the COVID-19 pandemic with those prior to the pandemic. Methods: Nationwide Inpatient Sample (NIS) was queried to determine adult hospitalized patients with a primary diagnosis of SCD using ICD-10 codes. They were stratified into two cohort groups based on year of admission, comparing admissions during 2016-2019 with admission during the year 2020. Primary outcomes assessed were inpatient mortality, length of stay (LOS), and total hospitalization charges. Secondary outcomes included respiratory failure, need for mechanical ventilation, cerebrovascular accident (CVA), myocardial infarction (MI), acute kidney injury (AKI), pulmonary embolism (PE), and need for blood transfusions. Multivariate linear and logistic regression analysis was performed to adjust for confounding variables. Results: A total of 456, 750 patients were hospitalized with SCD. 377, 285 were admitted between 2016-2019 and 79, 465 were admitted during 2020. Age distribution and sex were similar between the two groups. Patients admitted in the year 2020 were older and had more comorbidities when compared to patients admitted during prior years. Distribution of race, insurance type, hospital type, hospital size, hospital locations, and household income was not significantly different between the two groups. In 2020 patients admitted with SCD had greater odds of developing acute respiratory failure and PE, though the overall prevalence of these secondary outcomes were very low (3.6% and 0.80% respectively). Despite these outcomes, there was no significant difference in inpatient mortality (0.31% vs. 0.23%) for SCD patients admitted during 2020 when compared to prior years. In addition, the length of hospital stay was not significantly different in these two groups as well (5.1 vs. 4.9 days). Finally, patients admitted in 2020 incurred significantly higher hospital costs than SCD during pre-pandemic years ($42, 307 vs. $36, 087). Conclusion: SCD is the most common hemoglobinopathy in the United States. The African American population is most commonly affected by this disease. In addition, it is well known that this racial minority suffers inequities in accessing and receiving health care, which is associated with worse overall outcomes. During the COVID-19 pandemic, reduced access to hospital services combined with fear of exposure to the virus, led to a significant drop in overall access to healthcare and treatment. In this study, we found that those with SCD admitted during the COVID-19 pandemic overall had more comorbidities and more commonly had respiratory failure and pulmonary embolism, yet there was no change in inpatient mortality and LOS for these patients. Although disparities existed during the pandemic, it is fortunate that SCD itself did not serve as an additional risk factor for inpatient mortality and hospitalization duration.

Resolving Hidden Solution Conformations of Hemoglobin Using IMS-IMS on a Cyclic Instrument.

Ion mobility spectrometry-mass spectrometry (IMS-MS) experiments on a cyclic IMS instrument were used to examine heterogeneous distributions of structures found in the 15+ to 18+ charge states of the hemoglobin tetramer (Hb). The resolving power of IMS measurements is known to increase with increasing drift-region length. This effect is not significant for Hb charge states as peaks were shown to broaden with increasing drift-region length. This observation suggests that multiple structures with similar cross sections may be present. To examine this hypothesis, selections of drift time distributions were isolated and subsequently reinjected into the mobility region for additional separation. These IMS-IMS experiments demonstrate that selected regions separate further upon additional passes around the drift cell, consistent with the idea that initial resolving power was limited due to the presence of many closely related conformations. Additional variable temperature electrospray ionization (vT-ESI) experiments were conducted to study how changing the solution temperature affects solution conformations. Some features in these IMS-IMS studies were observed to change similarly with solution temperature compared to features in the single IMS distribution. Other features changed differently in the selected mobility data, indicating that solution structures that were obscured upon IMS analysis because of the complex heterogeneity of the original distribution are discernible after reducing the number of conformers that are analyzed by further IMS analysis. These results illustrate that the combination of vT-ESI with IMS-IMS is useful for resolving and exploring conformer distributions and stabilities in systems that exhibit a large degree of structural heterogeneity.

Crystal structural investigations of heme protein derivatives resulting from reactions of aryl- and alkylhydroxylamines with human hemoglobin

Phenylhydroxylamine (PhNHOH) and nitrosobenzene (PhNO) interact with human tetrameric hemoglobin (Hb) to form the nitrosobenzene adduct Hb(PhNO). These interactions also frequently lead to methemoglobin formation in red blood cells. We utilize UV–vis spectroscopy and X-ray crystallography to identify the primary and secondary products that form when PhNHOH and related alkylhydroxylamines (RNHOH; R = Me, t-Bu) react with human ferric Hb. We show that with MeNHOH, the primary product is Hb[α-FeIII(H2O)][β-FeII(MeNO)], in which nitrosomethane is bound to the β subunit but not the α subunit. Attempts to isolate a nitrosochloramphenicol (CAMNO) adduct resulted in our isolation of a Hb[α-FeII][β-FeII-cySOx]{CAMNO} product (cySOx = oxidized cysteine) in which CAMNO was located outside of the protein in the solvent region between the β2 and α2 subunits of the same tetramer. We also observed that the βcys93 residue had been oxidized. In the case of t-BuNHOH, we demonstrate that the isolated product is the β-hemichrome Hb[α-FeIII(H2O)][β-FeIII(His)2]{t-BuNHOH}, in which the β heme has slipped ∼4.4 Å towards the solvent exterior to accommodate the bis-His heme coordination. When PhNHOH is used, a similar β-hemichrome Hb[α-FeIII(H2O)][β-FeIII(His)2-cySOx]{PhNHOH} was obtained. Our results reveal, for the first time, the X-ray structural determination of a β-hemichrome in a human Hb derivative. Our UV–vis and X-ray crystal structural result reveal that although Hb(PhNO) and Hb(RNO) complexes may form as primary products, attempted isolation of these products by crystallization may result in the structural determination of their secondary products which may contain β-hemichromes en route to further protein degradation.

Renal glomerular and tubular responses to glutaraldehyde- polymerized human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and volume replacement therapeutics, however, their molecular and cellular effects on the vasculature and different organ systems are not fully defined. Using a guinea pig transfusion model, we examined the renal glomerular and tubular responses to PolyHeme, a highly characterized glutaraldehyde-polymerized human hemoglobin with low tetrameric hemoglobin content. PolyHeme-infused animals showed no major changes in glomerular histology or loss of specific markers of glomerular podocytes (Wilms tumor 1 protein, podocin, and podocalyxin) or endothelial cells (ETS-related gene and claudin-5) after 4, 24, and 72 h. Relative to sham controls, PolyHeme-infused animals also showed similar expression and subcellular distribution of N-cadherin and E-cadherin, two key epithelial junctional proteins of proximal and distal tubules, respectively. In terms of heme catabolism and iron-handling responses, PolyHeme induced a moderate but transient expression of heme oxygenase-1 in proximal tubular epithelium and tubulointerstitial macrophages that was accompanied by increased iron deposition in tubular epithelium. Contrary to previous findings with other modified or acellular hemoglobins, the present data show that PolyHeme does not disrupt the junctional integrity of the renal glomerulus and tubular epithelium, and triggers moderate activation of heme catabolic and iron sequestration systems likely as part of a renal adaptive response.

What Is the Relevance of Murburn Concept in Thalassemia and Respiratory Diseases?

Murburn concept is a novel perspective for understanding cellular function, deeming cells as simple chemical engines (SCE) that are powered by redox reactions initiated by effective charge separation (ECS). The 1-electron active diffusible reactive (oxygen) species, or DR(O)S, equilibriums involved in these processes are also crucial for homeostasis, coherently networking cells, and rendering electromechanical functions of sensing and responding to stimuli. This perspective presents the true physiological function of oxygen, which is to enable ECS and the generation of DR(O)S. Therefore, DR(O)S must now to be seen as the quintessential elixir of life, although they might have undesired effects (i.e., the traditionally perceived oxidative stress) when present in the wrong amounts, places and times. We also elaborated that tetrameric hemoglobin (Hb) is actually an ATP-synthesizing murzyme (an enzyme working via murburn concept) and postulated that several post-translational modifications (such as glycation) on Hb could result from murburn activity. Murburn perspective has also enabled the establishment of a facile rationale explaining the sustenance of erythrocytes for 3–4 months, despite their lacking nucleus or mitochondria (to coordinate their various functions and mass-produce ATP, respectively). Although thalassemia has its roots in genetic causation, the new awareness of the mechanistic roles of oxygen-hemoglobin-erythrocyte trio significantly impacts our approaches to interpreting research data and devising therapies for this malady. These insights are also relevant in other clinical manifestations that involve respiratory distress (such as asthma, lung cancer, COVID-19 and pneumonia) and mitochondrial diseases. Herein, these contexts and developments are briefly discussed.

Endothelial beta-globin is differentially expressed across mammalian species

Globin proteins interact with endothelial nitric oxide synthase at the myoendothelial junctions of resistance arteries to regulate nitric oxide signaling between endothelium and smooth muscle. Human resistance arteries express both alpha and beta globin to form heterotetrameric hemoglobin (α2β2). To better understand the role of hemoglobin in human resistance arteries, we need to identify an animal model in which both alpha and beta globin are expressed in the resistance vasculature. To identify potential candidates, we used microscopy to survey a range of mammalian laboratory animals for hemoglobin in the artery wall. We isolated arteries (100-200 μm in diameter) from mesenteric tissue collected from human patients undergoing clinically-indicated surgery and from the following laboratory animals: mouse ( Mus musculus, C57BL/6 strain), rat ( Rattus norvegicus, CD strain), African thicket rat ( Grammomys surdaster), guinea pig ( Cavia porcellus), New Zealand white rabbit ( Oryctolagus cuniculus), Beagle dog ( Canis lupus familiaris), and cynomolgus macaque ( Macaca fascicularis) after they underwent euthanasia on approved animal study protocols. We leveraged the autofluorescence of heterotetrameric hemoglobin under multiphoton excitation to visualize hemoglobin in the wall of intact, unstained, paraformaldehyde-fixed artery segments. Autofluorescent puncta representing tetrameric hemoglobin were observed in the wall of resistance arteries from the human patients, non-human primate, and canines, but not from rabbit nor any of the rodent species. The ability of beta globin to restrict endothelial NO signaling was confirmed in canine arteries ex vivo by testing a beta globin mimetic peptide that inhibited vasoconstriction to phenylephrine. In conclusion, primates and canines appear to have tetrameric hemoglobin in the resistance artery wall, whereas rodents and rabbits do not. This study highlights differences in vascular globin expression between humans and rodents and extends the recent discovery of hemoglobin in resistance arteries of humans to two additional mammalian species that may serve as models for studying the unique activities of tetrameric hemoglobin in the resistance vasculature. Funding: Intramural Research program, NIAID, NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Molecular evolution of the hemoglobin gene family across vertebrates

Adaptation to various altitudes and oxygen levels is a major aspect of vertebrate evolution. Hemoglobin is an erythrocyte protein belonging to the globin superfamily, and the α-, β-globin genes of jawed vertebrates encode tetrameric ((α2β2) hemoglobin, which contributes to aerobic metabolism by delivering oxygen from the respiratory exchange surfaces into cells. However, there are various gaps in knowledge regarding hemoglobin gene evolution, including patterns in cartilaginous fish and the roles of gene conversion in various taxa. Hence, we evaluated the evolutionary history of the vertebrate hemoglobin gene family by analyses of 97 species representing all classes of vertebrates. By genome-wide analyses, we extracted 879 hemoglobin sequences. Members of the hemoglobin gene family were conserved in birds and reptiles but variable in mammals, amphibians, and teleosts. Gene motifs, structures, and synteny were relatively well-conserved among vertebrates. Our results revealed that purifying selection contributed substantially to the evolution of all vertebrate hemoglobin genes, with mean dN/dS (ω) values ranging from 0.057 in teleosts to 0.359 in reptiles. In general, after the fish-specific genome duplication, the teleost hemoglobin genes showed variation in rates of evolution, and the β-globin genes showed relatively high ω values after a gene transposition event in amniotes. We also observed that the frequency of gene conversion was high in amniotes, with fewer hemoglobin genes and higher rates of evolution. Collectively, our findings provide detail insight into complex evolutionary processes shaping the vertebrate hemoglobin gene family, involving gene duplication, gene loss, purifying selection, and gene conversion.

Relation of Immunoglobulin Level and White Blood Cell Count with Frequency of Infection in Splenectomized and Non-Splenectomized B Thalassemia Major Patients

Background: Beta-thalassemia major is an autosomal recessive condition caused by absent (β0) synthesis of the β globin chains of the hemoglobin tetramer. Objectives: Evaluation of immunoglobulin level and white blood cell count in splenectomized and non non splenectomized patient as well as comparison of the levels of Ig and WBC with frequency of infection. Materials and Methods: This cross sectional study included a total of 60 patients with beta-thalassemia major and 20 age and sex matched apparently healthy individual as a control, blood taken from them for an evaluation of immunoglobulin level and white blood cell count. Results: There is no significant difference in immunoglobulin level between splenectomized and non splenectomized B-thalassemia major patients and control groups. Also, there is significant increase in mean Frequency of infection / year for patients with thalassemia major in comparison to control, Specifically, splenectomized patients are having significant increase in frequency of infection in comparison to non splenectomizd. There is a significant difference in mean of White blood cell count, Neutrophil, Lymphocytes in patients with splenectomy, non-splenectomy and control group. Conclusion: Immunoglobulin levels are within normal range in thalassemia major patients whether splenectomized or non splenectomized. Leukocytes count mainly neutrophils and lymphocytes are higher in splenectomised patients. Frequency of infection is higher in splenectomed patients and there is a positive correlation with IgA and IgG.

Modulation of Allosteric Control and Evolution of Hemoglobin.

Allostery arises when a ligand-induced change in shape of a binding site of a protein is coupled to a tertiary/quaternary conformational change with a consequent modulation of functional properties. The two-state allosteric model of Monod, Wyman and Changeux [J. Mol. Biol. 1965; 12, 88-118] is an elegant and effective theory to account for protein regulation and control. Tetrameric hemoglobin (Hb), the oxygen transporter of all vertebrates, has been for decades the ideal system to test for the validity of the MWC theory. The small ligands affecting Hb's behavior (organic phosphates, protons, bicarbonate) are produced by the red blood cell during metabolism. By binding to specific sites, these messengers make Hb sensing the environment and reacting consequently. HbI and HbIV from trout and human HbA are classical cooperative models, being similar yet different. They share many fundamental features, starting with the globin fold and the quaternary assembly, and reversible cooperative O2 binding. Nevertheless, they differ in ligand affinity, binding of allosteric effectors, and stability of the quaternary assembly. Here, we recollect essential functional properties and correlate them to the tertiary and quaternary structures available in the protein databank to infer on the molecular basis of the evolution of oxygen transporters.

Water dynamics around T0 vs R4 of hemoglobin from local hydrophobicity analysis.

The local hydration around tetrameric hemoglobin (Hb) in its T0 and R4 conformational substates is analyzed based on molecular dynamics simulations. Analysis of the local hydrophobicity (LH) for all residues at the α1β2 and α2β1 interfaces, responsible for the quaternary T → R transition, which is encoded in the Monod-Wyman-Changeux model, as well as comparison with earlier computations of the solvent accessible surface area, makes clear that the two quantities measure different aspects of hydration. Local hydrophobicity quantifies the presence and structure of water molecules at the interface, whereas "buried surface" reports on the available space for solvent. For simulations with Hb frozen in its T0 and R4 states, the correlation coefficient between LH and buried surface is 0.36 and 0.44, respectively, but it increases considerably if the 95% confidence interval is used. The LH with Hb frozen and flexible changes little for most residues at the interfaces but is significantly altered for a few select ones: Thr41α, Tyr42α, Tyr140α, Trp37β, Glu101β (for T0) and Thr38α, Tyr42α, Tyr140α (for R4). The number of water molecules at the interface is found to increase by ∼25% for T0 → R4, which is consistent with earlier measurements. Since hydration is found to be essential to protein function, it is clear that hydration also plays an essential role in allostery.

Thalassemia: A Review of the Challenges to the Families and Caregivers.

Thalassemias area group of congenital hemoglobin (Hb) disorders distinguished by dwindling or total curtailment of production of one or more globin chains of hemoglobin tetramers, ensuing in unrestrained destruction of red blood cells (RBC) that causes severe anemia. The severity of the disease often remains immensely variable. Children with thalassemia suffer from the disease's consequences and treatment complications. The disease also causes a negative impact on family members, who suffer mentally, socially, financially, and even physically. In this review, we highlight the challenges experienced by the family and caregivers; for instance, repeated blood transfusion as the dominant origin of tissue casualty, morbidity, and fatal clinical outcomes. Treatment modalities regarding thalassemias were not successful until the inception of bone marrow transplantation and gene therapy.

Development of a β-Globin Gene Replacement Strategy As a β-Thalassemia Therapy

Background: β-thalassemia is a genetic disorder with high unmet need worldwide. Over 300 genetic mutations that result in the reduction, or loss, of β-globin (HBB) expression have been characterized to date. Patients with thalassemic mutations in both HBB alleles often experience severe anemia resulting from ineffective erythropoiesis due to the loss of adult hemoglobin (HbA) tetramers (α2β2) and imbalanced excess of α-globin chains. One challenge is creating a genome-editing strategy that replaces the diversity of disease-causing mutations in the HBB gene while restoring physiological HbA expression. Therefore, a gene-editing platform using homology-directed repair (HDR) that could replace the entire HBB gene in situ and under the control of normal regulatory elements would be an ideal strategy. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were edited using high-fidelity Cas9, HBB guide RNA, and different AAV6 DNA donor templates and then differentiated into red blood cells in vitro. We evaluated HBB expression in 39 AAV6 donors engineered with heterologous introns and different polyadenylation signals first using a flow cytometry screening assay and later HPLC. Candidates that met or exceeded wild-type HBB expression were moved forward for further comparison. Next, we edited HSPCs with the 8 highest expressing AAV6 donors, performed in vitro differentiation, and evaluated HDR frequencies by droplet digital polymerase chain reaction (ddPCR) and HbA formation by HPLC. The candidate that demonstrated the highest conversion of donor knock-in to HbA was further engineered to boost expression and reduce construct size. Two final candidates were moved forward to further characterize their ability to rescue loss of HbA in CD34+ HSPCs isolated from patients with sickle cell disease (SCD) at the protein and RNA level. Additionally, we performed DNA analysis on the gene-edited cells using ddPCR and a targeted PCR-free long-range sequencing approach. Results: Adding heterologous introns to the HBB coding sequence significantly increased HBB expression relative to the no-intron construct containing only coding DNA. The results from our flow cytometry screen and HPLC analysis were positively correlated, and the top DNA donors with heterologous introns resulted in physiologic HbA expression. Using this gene-replacement strategy, we achieved HDR frequencies of up to 40% in CD34+ HSPCs. Further optimization resulted in a smaller DNA template, maintenance of HbA conversion rates, and a lower rate of unintended recombinations. Finally, using HSPCs from patients with SCD as a therapeutically relevant model, we found, by HPLC and RNA sequencing, the optimized DNA donor can successfully replace the entire nonfunctional HBB gene, thereby restoring physiologic HbA expression. Future experiments are investigating in vitro gene correction in cells isolated from patients with β-thalassemia and additional studies evaluating in vivo stem cell repopulation capacity of gene-edited HSPCs. Conclusion: We developed an HBB AAV6 donor engineered with heterologous introns that demonstrates efficient HBB gene replacement and HbA rescue in the erythroid progeny of CD34+ HSPCs isolated from patients with SCD, offering a potential differentiated approach for treating β-thalassemia.

Ex Vivo CRISPR-Based Sickle Cell Modeling

Autologous hematopoietic stem cell transplantation combined with genome editing holds promise for curing patients with hemoglobinopathies such as sickle cell anemia (SCA). Hence, experimental models to assess the impact of genome editing modalities (i.e. nucleases, base editors, and prime editors) are important tools that could complement the development of safe curative approaches for hemoglobinopathies. The culture and ex vivo engineering of red blood cells (RBC) can help characterize genetic variants, model diseases, and may eventually spur the development of applications in transfusion medicine. In the last decade, improvements in the in vitro production of RBCs have enabled efficient erythroid progenitor proliferation and high enucleation levels from several sources of hematopoietic stem and progenitor cells (HSPCs). Despite these advances, there remains a need for refining the terminal step of in vitro human erythropoiesis - i.e., the terminal maturation of reticulocytes into erythrocytes - so that the RBCs produced ex vivo resemble mature circulating blood cells. Completing their maturation will also be required in some instances to recapitulate specific RBC phenotypes. Here we describe the near-complete erythroid differentiation of cultured RBCs (cRBCs) from adult HSPCs in feeder-cell-free and animal-protein-free conditions. The approach improves post-enucleation cell integrity and survival, and enables subsequent storage of cRBCs for up to 42 days in classical nutritive solution conditions, without any specialized equipment. We next used a non-viral, selection-free CRISPR-Cas9-based genome editing to efficiently insert the HBB-βs mutation at the endogenous locus in HSPCs. From this process, mature RBCs with crescent shape, a hallmark of SCA, where readily obtained. The sickle phenotype was recapitulated at high frequencies - comparable to the levels of biallelic HBB-βs modification obtained. Importantly, the sickle shape was observed exclusively in RBCs that underwent the complete cRBC culture protocol - consistent with the deformability properties acquired during the late-stages of RBC maturation. Moreover, a β-globin variant forming an in-frame, 9bp deletion (HBBΔ9), was frequently observed following the targeting of HBB. Contrary to usual editing by-products creating β-globin knockout alleles, this variant was expressed in erythroid cells and formed a hemoglobin tetramer, suggesting that its potential impact should be further studied in the context of clinical trials relying on this editing strategy. We foresee that this system, combining efficient genome editing of HSPCs and production of mature cRBCs, will support the functional study of various erythroid traits and could be exploited to chaperone the development of gene-modified HSPC transplants for hemoglobinopathies.