scholarly journals Amino Acids Responsible for Decreased 2, 3-Biphosphosphoglycerate Binding to Fetal Hemoglobin

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 2916-2920 ◽  
Author(s):  
Kazuhiko Adachi ◽  
Patrick Konitzer ◽  
Jian Pang ◽  
Konda S. Reddy ◽  
Saul Surrey
Keyword(s):  
Expression System ◽  
Oxygen Affinity ◽  
Fetal Hemoglobin ◽  
Oxygen Binding ◽  
Cell Disease ◽  
Binding Properties ◽  
Yeast Expression System ◽  
Normal Human ◽  
Potential Use

Abstract To clarify the role of γN-terminal Gly, γ5 Glu, and γ143 Ser in 2, 3-biphosphosphoglycerate (BPG) binding to fetal hemoglobin (Hb F ), we engineered and produced normal human Hb F and two Hb F variants (Hb F γG1V, γS143H, and Hb F γG1V, γE5P, γS143H) using a yeast expression system and then compared their oxygen-binding properties with those of native human Hb F and adult Hb (Hb A). Oxygen affinity of Hb F γG1V, γS143H in the absence of 2, 3-BPG was slightly higher than that of normal Hb F. The decrease in oxygen affinities for Hb F γG1V, γS143H with increasing 2, 3-BPG concentrations was larger than that of normal Hb F, but significantly less than that of Hb A. In contrast, oxygen affinities of Hb F γG1V, γE5P, γS143H in the absence and presence of 2, 3-BPG were much lower than those of Hb F γG1V, γS143H and were similar to those of Hb A. These results indicate that differences between Pro and Glu at the A2 position in the A helix in Hb A and Hb F, respectively, are critical for reduced binding of 2, 3-BPG to Hb F, even though β5 Pro does not interact directly with 2, 3-BPG in Hb A. Hb F variants such as Hb F γG1V, γE5P, γS143H, which exhibit reduced oxygen affinity, should facilitate design of efficient antisickling fetal Hb variants for potential use in gene therapy for sickle cell disease.

scholarly journals Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations

2008 ◽  
Vol 276 (1658) ◽  
pp. 833-841 ◽  
Author(s):  
Øivind Andersen ◽  
Ola Frang Wetten ◽  
Maria Cristina De Rosa ◽  
Carl Andre ◽  
Cristiana Carelli Alinovi ◽  
...  
Keyword(s):  
Evolutionary Biology ◽  
Quaternary Structure ◽  
Atlantic Cod ◽  
Three Dimensional ◽  
Oxygen Affinity ◽  
Oxygen Binding ◽  
Low Oxygen ◽  
Binding Properties ◽  
Important Species ◽  
The North

A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygen-transporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55β 1 Val and Lys62β 1 Ala are located at crucial positions of the α 1 β 1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water- and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55–Ala62 haplotype, while the temperature-insensitive Met55–Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming.

Vaso-occlusion in sickle cell disease: pathophysiology and novel targeted therapies

Hematology ◽  
2013 ◽  
Vol 2013 (1) ◽  
pp. 362-369 ◽  
Author(s):  
Deepa Manwani ◽  
Paul S. Frenette
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Targeted Therapies ◽  
Therapeutic Intervention ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
The Past ◽  
Symptomatic Management

Abstract Recurrent and unpredictable episodes of vaso-occlusion are the hallmark of sickle cell disease. Symptomatic management and prevention of these events using the fetal hemoglobin–reactivating agent hydroxyurea are currently the mainstay of treatment. Discoveries over the past 2 decades have highlighted the important contributions of various cellular and soluble participants in the vaso-occlusive cascade. The role of these elements and the opportunities for therapeutic intervention are summarized in this review.

Molecular Crowding Limits the Role of Fetal Hemoglobin in Therapy for Sickle Cell Disease

2005 ◽  
Vol 347 (5) ◽  
pp. 1015-1023 ◽  
Author(s):  
Maria Rotter ◽  
Alexey Aprelev ◽  
Kazuhiko Adachi ◽  
Frank A. Ferrone
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Molecular Crowding ◽  
Cell Disease

scholarly journals LC-MS Analysis of Anti-Sickling Compounds in Cord Blood Derived RBCs Demonstrates Modification of Fetal Hemoglobin and Globin Chain Binding Preferences

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1074-1074
Author(s):  
Benjamin Vieira ◽  
Vu P. Hong ◽  
Kunal Desai ◽  
Martin K. Safo ◽  
David R. Light
Keyword(s):  
Cord Blood ◽  
Binding Affinity ◽  
Oxygen Affinity ◽  
Fetal Hemoglobin ◽  
Oxygen Binding ◽  
Globin Chain ◽  
Employment Equity ◽  
Globin Chains ◽  
N Terminus ◽  
Equity Ownership

Abstract Sickle cell disease (SCD) is a genetic hemoglobinopathy driven largely by a single codon mutation of the β-globin gene resulting in polymerization of hemoglobin S (HbS). Anti-sickling approaches that involve increasing the oxygen affinity of HbS to treat SCD are under development and offer the potential to directly prevent HbS polymerization and its downstream pathophysiology. Two such compounds, 5-hydroxymethylfurfural (5HMF) and voxelotor (GBT440) have entered clinical trials for SCD with promising results and exert their therapeutic effects by modifying the N-terminus of HbS α-globin chains to form a reversible Schiff base. Formation of this N-terminal adduct stabilizes the oxygen-bound R-state (in the R2 conformation) that increases the oxygen affinity of the altered HbS and delaying the polymerization of HbS. In addition, genetic and small molecule therapies designed to increase fetal hemoglobin (HbF) expression hold great potential for the treatment of SCD. Increasing the percentage of HbF in RBCs significantly slows sickling kinetics without affecting oxygen delivery. Combination approaches of high-O2-Hb modification with HbF inducing therapies clinically could result in increased efficacy in the treatment of SCD, but the impact of hemoglobin modifiers on fetal hemoglobin has not been reported. Our present studies investigated the effects of 5HMF and voxelotor in HbF-rich umbilical cord blood derived RBCs. HbF-rich (60-90%) RBCs were isolated from cord blood and incubated with commercially available 5HMF and voxelotor synthesized in-house. The effect of these compounds on hemoglobin oxygen affinity was determined by measuring the p50 of the oxygen saturation curve in whole cells. Sites of modification were determined directly by incubating compounds with the purified RBC lysate, stabilizing the N-terminal adduct by reduction to the amine, and analysis of the resulting modification by LC-MS. Similar to the reported p50 shifts with normal adult hemoglobin (HbA) and HbS, 5HMF and voxelotor increased the oxygen-binding affinity of HbF with an EC50 of 7.9 mM and 560 mM respectively. 1 mM voxelotor lowered cord RBC p50 to 4 mmHg in vitro. LC-MS analysis showed that 5HMF exclusively modified the N-terminus of the α-globin chain, with no modification of b-globin and g-globin chains. Unexpectedly, the α-globin, β-globin and γ-globin chains were all modified by voxelotor following incubation with cord blood. Voxelotor was also shown to modify both α-globin and the β-globin or βS-globin chains on purified HbA or HbS, respectively. These data contrast with published crystallography data demonstrating that voxelotor selectively modifies a single α-globin chain in CO-ligated HbS (Oksenberg et al 2016). Although anti-sickling aromatic aldehydes have similar effects on the oxygen binding affinity of HbA, HbS and HbF, they can vary in their selectivity for modification of the α-globin and beta-like chains of HbF, HbA, and HbS (Abraham et al. 1995). To further investigate our data with voxelotor and increase our understanding of this class of molecules, other hemoglobin modifying aldehyde molecules such as 5-formylsalicyclic acid (5FSA), tucaresol and velaresol (BW12C) will be examined. Disclosures Vieira: Bioverativ a Sanofi Company: Employment. Hong:Bioverativ a Sanofi Company: Employment, Equity Ownership. Desai:Bioverativ a Sanofi Company: Employment, Equity Ownership. Safo:Bioverativ a Sanofi Company: Consultancy; Virginia Commonwealth University: Employment. Light:Bioverativ a Sanofi Company: Employment, Equity Ownership.

Regulation of Blood Oxygen Affinity in the Australian Blackfish Gadopsis Marmoratus: I. Correlations between Oxygen-binding Properties, Habitat and Swimming Behaviour

1982 ◽  
Vol 99 (1) ◽  
pp. 223-243
Author(s):  
G. P. DOBSON ◽  
J. BALDWIN
Keyword(s):  
Whole Blood ◽  
Oxygen Affinity ◽  
Swimming Behaviour ◽  
Oxygen Binding ◽  
Blood Oxygen ◽  
Low Oxygen ◽  
Binding Properties ◽  
Molar Ratios ◽  
Blood Oxygen Affinity ◽  
Low Oxygen Affinity

1. The regulation of whole blood oxygen affinity in the freshwater blackfish Gadopsis marmoratus Richardson has been examined, and correlations made between oxygen-binding properties and the habitat and swimming behaviour of the fish. 2. Blackfish whole blood has a low oxygen affinity relative to other fish bloods reported in the literature. This is not due to a low oxygen affinity of the stripped haemoglobins, but arises from interactions between haemoglobin and intraerythrocytic modulators. 3. The presence of high concentrations of ATP, and to a lesser extent GTP, in the erythrocyte, together with the effect of these nucleoside triphosphates on the oxygen affinity of haemoglobin solutions at physiological NTP: Hb4 molar ratios, demonstrates that this class of compounds is a major regulator of oxygen affinity in blackfish blood. 4. The oxygen affinities of whole blood and haemoglobin solutions are sensitive to pH, with haemoglobin solutions displaying a relatively large alkaline Bohr coefficient of - 1.05 over the physiologically relevant pH range of 6.5–7.0. 5. Although increasing Pco2, lowers the oxygen affinity of whole blood, it does so only through the effect on pH, as pH-buffered haemoglobin solutions show no oxygen-linked CO2 binding. This lack of oxygen-linked CO2 binding has not been reported for any other naturally occurring vertebrate haemoglobins. 6. Muscle morphology and biochemistry, and behavioural observations, indicate that the blackfish uses anaerobic energy metabolism during rapid swimming and in recovery. 7. It is concluded that the oxygen-binding properties of blackfish blood reflect adaptations for maintaining adequate tissue oxygenation for animals at rest and during slow sustained swimming in waters of high oxygen tensions.

Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1839-1842 ◽  
Author(s):  
Vijay G. Sankaran ◽  
Tobias F. Menne ◽  
Jian Xu ◽  
Thomas E. Akie ◽  
Guillaume Lettre ◽  
...  
Keyword(s):  
Association Studies ◽  
Globin Gene ◽  
Genetic Association Studies ◽  
Fetal Hemoglobin ◽  
Sequence Variants ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Direct Role ◽  
Globin Gene Regulation

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the β-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the β-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in β-hemoglobin disorders.

Magnetic resonance investigations of blood: From hemoglobin to lymphocyte migration

1996 ◽  
Vol 54 ◽  
pp. 880-881
Author(s):  
C. Ho
Keyword(s):  
T Cells ◽  
Magnetic Resonance ◽  
Expression System ◽  
Oxygen Affinity ◽  
Hill Coefficient ◽  
Oxygen Binding ◽  
Lymphocyte Migration ◽  
Magnetic Resonance Imaging Mri ◽  
Function Relationship ◽  
The Hill

We have applied various nuclear magnetic resonance (NMR) techniques, from high-resolution solution-state NMR spectroscopy to magnetic resonance imaging MRI), to investigate blood components such as hemoglobin and T-cells. We are interested in the structure-function relationship in human normal adult hemoglobin (Hb A) and in tracking the movement of T-cells in live rats. In this presentation, we shall give a brief summary of our approach and recent experimental results.We have recently developed an expression system to produce authentic Hb A in Escherichia coli. With our Hb A expression plasmid (pHE2) (Fig. 1), we are in a position to produce any desired hemoglobin in E. coli. We are carrying out a study to learn the rules governing oxygen affinity and cooperativity of the oxygen binding process in hemoglobin. We have constructed several recombinant hemoglobins which exhibit varying degrees of oxygen affinity and cooperativity (as measured by the Hill coefficient).

Temperature acclimation and oxygen-binding properties of blood and multiple haemoglobins of rainbow trout

1976 ◽  
Vol 65 (2) ◽  
pp. 333-345 ◽  
Author(s):  
R. E. Weber ◽  
S. C. Wood ◽  
J. P. Lomholt
Keyword(s):  
Rainbow Trout ◽  
Whole Blood ◽  
Relative Concentration ◽  
Oxygen Affinity ◽  
Temperature Acclimation ◽  
Organic Phosphate ◽  
Red Cells ◽  
Oxygen Binding ◽  
Binding Properties

Acclimation of rainbow trout to 5, 15 and 22 degrees C for periods exceeding 4 months had no significant effect on the oxygen affinity of whole blood or on the concentration of ATP, which is the main organic phosphate in red cells. Slight differences were, however, found in the oxygenation properties of the haemolysates, which correlate with changes in the relative concentration of the multiple haemoglobins. The oxygen-binding properties of the main haemoglobin components account for the observed differences in the haemolysates. The possible thermoacclimatory significance of changes in haemoglobin multiplicity and co-factor concentrations is discussed.

Fetal Hemoglobin Expression Is Differentially Affected by Inhibition of the Proposed Dred Complex Constituents, LSD1 and DNMT1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3263-3263 ◽  
Author(s):  
Tara L Arvedson ◽  
Lynn Tran ◽  
Sandra L Ross ◽  
Sean Yoder ◽  
Alexandra Hertz ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Beta Thalassemia ◽  
Cell Disease ◽  
Hematopoietic Progenitor ◽  
Gamma Globin ◽  
Hbf Induction ◽  
Effect Of Treatment ◽  
Globin Promoter

Abstract Abstract 3263 Introduction Sickle cell disease and beta thalassemia are disorders caused by mutations in adult hemoglobin (HbA) or defects in HbA expression. A potential therapeutic solution is reactivation of fetal hemoglobin (HbF) expression. Although HbF, comprising two alpha and two gamma globin chains, is the primary form of hemoglobin expressed in utero, gamma globin expression is silenced in adults. One proposed mechanism of gamma globin silencing involves binding of the direct repeat erythroid definitive (DRED) repressor complex to sequences in the gamma globin promoter. The DRED complex is reported to include the orphan nuclear hormone receptors TR2 and TR4, lysine specific demethylase (LSD1) and DNA methyltransferase (DNMT1). As both LSD1 and DNMT1 are epigenetic modifiers, gamma globin repression is proposed to be mediated by LSD1- and DNMT1-induced epigenetic changes. To investigate the role of DNMT1 and LSD1 in HbF silencing, HbF expression was evaluated in an erythroid differentiation model where hematopoietic progenitor cells were treated with either DNMT1 or LSD1 small molecule inhibitors or siRNA. Methods Human hematopoietic progenitor cells from healthy donors were induced to become erythroid using a two step protocol including erythropoietin, SCF, IL-3 and hydrocortisone for days 1–7 and erythropoietin and SCF for days 8–14. Cultures were treated with a range of concentrations of either tranylcypromine or S2101 (LSD1 inhibitors) or 5-azacytidine (DNMT1 inhibitor) and compared to HbF-inducing, positive control small molecules pomalidomide and lenalidomide. Cultures were also treated with LSD1 siRNAs and compared to controls. The effect of treatment on gamma, beta and alpha globin transcription was determined by qRT-PCR. The effect of treatment on HbA and HbF levels was determined by ELISA, HPLC, flow cytometry and imaging. Differentiation was characterized by morphology and flow-based detection of CD34 and glycophorin. Effects on viability were characterized by ViCell and flow cytometry. Results Treatment with a concentration range of 5-azacytidine increased the rate of red blood cell differentiation as measured by daily changes in CD34 and glycophorin and hemoglobinization. Quantitative ELISA demonstrated that HbF expression increased two-fold. In contrast, LSD1 inhibition reduced both the rate of proliferation and differentiation of erythroid progenitors. Consistent with impaired differentiation, both beta globin transcription and HbA expression were reduced by up to 84% (qRT-PCR) and 65% (quantitative ELISA), respectively. No increase in gamma globin transcription or HbF expression was observed in response to LSD1 inhibition. Control cultures differentiated as expected: after 14 days of treatment the majority of vehicle-, lenalidomide- or pomalidomide-treated cells were glycophorin-positive and enucleation was readily apparent. Both lenalidomide and pomalidomide treatment induced a two-fold increase in HbF expression, as previously reported. Conclusions Although both LSD1 and DNMT1 are reported to be components of the DRED complex and are proposed to be jointly responsible for epigenetically modifying the gamma globin promoter to silence HbF expression, inhibition of the two proteins had different outcomes on HbF expression. DNMT1 inhibition upregulated HbF expression to a similar extent as pomalidomide (currently in Phase 1 clinical trials for HbF induction), whereas LSD1 inhibition impaired erythroid differentiation and hemoglobinization. These results suggest that the mechanism of gamma globin silencing and the proposed role of the DRED complex require further evaluation. Furthermore, this work also suggests that LSD1 inhibition is not a therapeutic strategy for HbF induction in patients with sickle cell disease or beta thalassemia. Disclosures: Arvedson: Amgen: Employment. Tran:Amgen: Employment. Ross:Amgen: Employment. Yoder:Amgen: Employment. Hertz:Amgen: Employment. Hale:Amgen: Employment. Eschelbach:Amgen: Employment. Dineen:Amgen: Employment. Matyas:Amgen: Employment. Hartley:Amgen: Employment. Morgenstern:Amgen: Employment. Winters:Amgen: Employment. Cindy:Amgen: Employment. Molineux:Amgen: Employment. Coxon:Amgen: Employment.

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