Alpha Hemoglobin Stabilizing Protein Research Articles

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

Mammalian hemoglobins (HB) are a kind of heme-binding proteins that play crucial physiological roles in various organisms. The traditional techniques employed for the extraction of HB are expensive and time-consuming, while the yields of mammalian HB in previous reports were quite low. The industrial Pichia pastoris is a highly effective platform for the secretory expression of heterologous proteins. To achieve efficient secretory expression of HB in P. pastoris, multiple strategies were applied, including the selection of a suitable host, the screening of optimal endogenous signal peptides, the knockout of VPS10, VTH1, and PEP5, and the co-expression of Alpha-Hemoglobin Stabilizing Protein (AHSP). In addition, the conditions for producing HB were optimized at shaking-flask level (BMMY medium with 100 mg/L of hemin, 2% methanol, and 24 °C). Based on these conditions, the higher titers of bovine hemoglobin (bHB, 376.9 ± 13.3 mg/L), porcine hemoglobin (pHB, 119.2 ± 7.3 mg/L), and human hemoglobin (hHB, 101.1 ± 6.7 mg/L) were achieved at fermenter level. The engineered P. pastoris strain and comprehensive strategies can also be applied to facilitate the synthesis of other high-value-added hemoproteins or hemoenzymes.

Digital image analysis of erythroblastic islands in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) encompass a diverse group of myeloid neoplasms for which the diagnosis of low-grade subtypes remains challenging. Erythroblastic islands (EBIs) are highly organized units of erythroid proliferation, differentiation, and enucleation. EBI disruption is frequently observed and is believed to be one of the early changes in MDS. In this study, we digitally analyzed bone marrow biopsies dual stained with alpha-hemoglobin stabilizing protein (AHSP) and CD163 to quantitatively study features of EBIs in MDS, among MDS subtypes, as well as those in normal marrows and marrows with other causes of anemia. EBIs in MDS specimens were smaller in size and higher in density compared to both normal and non-MDS anemia specimens. Increased CD163 expression within the EBIs is observed in both MDS and other causes of anemia. A combination of increased EBI density and CD163 expression is seen in association with MDS with high-risk cytogenetics and multiple adverse mutations. As a proof-of-concept study, we show that EBI features can be relatively easily quantified with AHSP/CD163 dual immunohistochemistry and open-source imaging analysis software, highlighting those that are unique to MDS, and which may be prognostically relevant. Further studies of the measurable EBI features may provide valuable and novel tools to aid MDS diagnosis and prognostication in the era of digital pathology.

Keap1-Nrf2 Heterodimer: A Therapeutic Target to Ameliorate Sickle Cell Disease.

Sickle cell disease (SCD) is a monogenic inheritable disease characterized by severe anemia, increased hemolysis, and recurrent, painful vaso-occlusive crises due to the polymerization of hemoglobin S (HbS)-generated oxidative stress. Up until now, only four drugs are approved for SCD in the US. However, each of these drugs affects only a limited array of SCD pathologies. Importantly, curative therapies, such as gene therapy, or hematopoietic stem cell transplantation are not available for every patient because of their high costs, availability of donor matching, and their serious adverse effects. Therefore, there is an unmet medical need for novel therapeutic strategies that target broader SCD sequelae. SCD phenotypic severity can be alleviated by increasing fetal hemoglobin (HbF) expression. This results in the inhibition of HbS polymerization and thus sickling, and a reduction in oxidative stress. The efficacy of HbF is due to its ability to dilute HbS levels below the threshold required for polymerization and to influence HbS polymer stability in RBCs. Nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein-1 (Keap1)-complex signaling is one of the most important cytoprotective signaling controlling oxidative stress. Nrf2 is present in most organs and, after dissociation from Keap1, it accumulates in the cytoplasm, then translocates to the nucleus where it binds to the antioxidant response element (ARE) sequences and increases the expression of various cytoprotective antioxidant genes. Keeping this in mind, various researchers have proposed a role of multiple agents, more importantly tert-Butylhydroquinone (tBHQ), curcumin, etc., (having electrophilic properties) in inhibiting keap1 activity, so that Nrf2 can translocate to the nucleus to activate the gamma globin gene, thus maintaining alpha-hemoglobin-stabilizing protein (AHSP) and HbF levels. This leads to reduced oxidative stress, consequently minimizing SCD-associated complications. In this review, we will discuss the role of the Keap-1-Nrf2 complex in hemoglobinopathies, especially in SCD, and how this complex might represent a better target for more effective treatment options.

Alpha-Hemoglobin Stabilizing Protein Gene Polymorphism (rs4499252 A/G) and its Association with Beta-Thalassemia Major in Iraqi Patients.

Beta thalassemia (β-thalassemia) major is a genetic disorder of hemoglobin production that results in a diminished rate of synthesis of one or more of the globin chains causing variable degrees of anemia. Alpha-hemoglobin-stabilizing protein (AHSP) is a specific alpha-globin factor that affects the severity of the disease in patients with β-thalassemia. A recent study was conducted to investigate the polymorphism in the AHSP (rs4499252) gene and its association with β-thalassemia in Iraq. Blood samples were obtained from 90 β-thalassemia patients and 60 healthy individuals as a control group in the Wasit Center for Hereditary Anemia from August 2020 to January 2021. After DNA extraction from the whole blood, to determine the genotype of the AHSP gene, the High-Resolution Melt (HRM) Real-Time PCR was used. The results showed a significant increase (P<0.05) in genotype GG (wild type) of the SNP (rs4499252) in β-thalassemia patients, compared to the control group. On the other hand, genotype AA was significantly higher (P≤0.05) in β-thalassemia patients than in the control group, while the genotype GA showed a non-significant difference (P<0.01) between β-thalassemia patients and the healthy controls. The results also showed that the AHSP expression is a biomarker of hemoglobin H disease severity, and the A allele was more frequent in β-thalassemia patients than the G allele in Iraqi patients.

MRNA Analysis of Frameshift Mutations with Stop Codon in the Last Exon: The Case of Hemoglobins Campania [α1 cod95 (-C)] and Sciacca [α1 cod109 (-C)

An insertion or deletion of a nucleotide (nt) in the penultimate or the last exon can result in a frameshift and premature termination codon (PTC), giving rise to an unstable protein variant, showing a dominant phenotype. We described two α-globin mutants created by the deletion of a nucleotide in the penultimate or the last exon of the α1-globin gene: the Hb Campania or α1 cod95 (−C), causing a frameshift resulting in a PTC at codon 102, and the Hb Sciacca or α1 cod109 (−C), causing a frameshift and formation of a PTC at codon 133. The carriers showed α-thalassemia alterations (mild microcytosis with normal Hb A2) and lacked hemoglobin variants. The 3D model indicated the α-chain variants’ instability, due to the severe structural alterations with impairment of the chaperone alpha-hemoglobin stabilizing protein (AHSP) interaction. The qualitative and semiquantitative analyses of the α1mRNA from the reticulocytes of carriers highlighted a reduction in the variant cDNAs that constituted 34% (Hb Campania) and 15% (Hb Sciacca) of the total α1-globin cDNA, respectively. We developed a workflow for the in silico analysis of mechanisms triggering no-go decay, and its results suggested that the reduction in the variant mRNA was likely due to no-go decay caused by the presence of a rare triplet, and, in the case of Hb Sciacca, also by the mRNA’s secondary structure variation. It would be interesting to correlate the phenotype with the quantity of other frameshift mRNA variants, but very few data concerning α- and β-globin variants are available.

Lesional Mechanism of Alpha Thalassemias : Towards a Common Mechanism Alpha Thal / Beta Thal

Context and Objectives: Erythropoiesis is a complex process based on the balance between proliferation / maturation and apoptosis, which produces more than 109 red blood cells per day. The lesional mechanism of thalassemia (thal) appears during the synthesis of hemoglobin (Hb) and the classical hypothesis proposes that the clinical severity is proportional to the imbalance of production of alpha / non-alpha-globin chains. However, the severe alpha thal (HbH) phenotypic expression does not adhere to this model since the HbH linked to the association of deletions (alpha0 / alpha +) are less severe than HbH associating deletion and point mutants (alpha0 / alphaCS alpha) while the deficiency is less important, and that there are severe alpha thal linked to homozygosity for an alpha variant without deletion. In order to understand the lesional mechanisms involved, and in light of recent work on beta thal pathophysiology, we have carried out cultures of erythroid progenitors from patients with these two types of « deletional » HbH (alpha0/ -3,7) and « non deletional » HbH (alpha0/ CS) as well as from a homozygous patient of an Agrinio variant (alpha2 : p29 Leu®Pro). We have characterized the amount of alpha globin aggregates in the progenitors of the different patients. In addition, the Agrinio mutation was produced in E. coli to study the consequences of the mutation on interactions between α-HbAgrinio and the alpha-Hemoglobin Stabilizing Protein (AHSP) chaperone.Materials and Methods: Erythroid progenitors were analyzed by multiparametric cytometry (Amnis®) and confocal microscopy to quantify and characterize cytoplasmic aggregates. “Non-thalassemic red cells” (csp) and progenitors from a beta thal major patient were used as negative and positive controls, respectively. Normal α-Hb proteins (α-HbWT) and α-HbAgrinio were produced as glutathione S transferase (GST) fusion proteins associated with their chaperones (GST-AHSP / GST-α -Hb). After cleavage of the GST moiety, the characterization of the AHSP / α-HbAgrinio complex was carried out by fluorescence spectroscopy.Results: The proportion of cells with cellular aggregates analyzed by multiparametric cytometry is proportional to the clinical severity of the patient with an order csp < alpha°/ alpha+ < alpha°/alphaCS < alphaAgrinio alpha / alphaAgrinio alpha < beta thal. The expression in E.coli of α-HbAgrinio is similar to that observed for α-HbWT, but the amount of α-HbAgrinio obtained after purification is greatly decreased indicating a significant instability of α-HbAgrinio. The absorbance spectroscopy shows that the amplitude of the Soret band, characteristic of the heme molecule, is highly decreased for the AHSP / α-HbAgrinio complex compared to that observed for the AHSP / α-HbWT complex. The fluorescence emission spectrum of the AHSP / α-HbAgrinio complex is increased compared to that of the normal complex indicating that the tryptophan AHSP fluorescence signal is less attenuated since fewer heme molecules are present. These results show that α-HbAgrinio is unstable probably due to an alteration of interaction with its chaperone and to a lack of insertion of heme in the globin for this mutant.Conclusion: Recent work has shown that one of the mechanisms responsible for dyserythropoiesis in beta thal major progenitors is the sequestration of the HSP70 chaperone in the cytosol by alpha globin precipitates. HSP70 can therefore no longer protect the nuclear GATA1 factor from cleavage by the activated caspase3 at the beginning of terminal differentiation. This leads to inachieved differentiation and apoptosis. Our results indicate that in HbH and non-deletional alpha thal, a comparable mechanism would be an important element in the pathophysiology unifying the lesional mechanism of two apparently opposite pathologies. Studies in the bacterial model confirm the strong instability of α-HbAgrinio and in vitro studies show that this mutant is partially heminized. DisclosuresNo relevant conflicts of interest to declare.

Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes — these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.

Transient Improvement in Erythropoiesis Is Achieved Via the Chaperone AHSP With Early Administration of Propranolol in Burn Patients.

Burn patients experience erythropoietin resistant anemia in which early commitment and late maturation of erythroblasts are defective. The authors previously showed that propranolol (Prop) treatment restores erythroid committed progenitors, but terminal maturation remains impaired. Hemoglobinization and maturation occur during terminal erythropoiesis and these processes are aided by an erythroblast intrinsic functional protein called alpha-hemoglobin stabilizing protein (AHSP). The authors evaluated the role of AHSP in PBMC- (peripheral blood mono nuclear cell) derived erythroblasts and the implications of Prop in burn patients. Blood samples were collected at three time points from 17 patients receiving standard burn care (SBC) or Prop. Five healthy volunteers provided control plasma (CP). PBMCs were placed in biphasic cultures with 5% autologous plasma (BP) or CP. Erythroblasts were harvested during mid and late maturation stages; the percentage of AHSP+ erythroblasts, AHSP expression, and relative distribution of reticulocytes and polychromatophilic erythroblasts (PolyE) were determined by cytometry. During the second time point (7-10 days postburn), Prop cohort required 35% less transfusions. At mid maturation, PBMCs from Prop-treated patients cultured in BP had 33% more AHSP+ erythroblasts and 40% more AHSP expression compared with SBC. Furthermore, at late maturation, Prop had 50% more reticulocytes and 30% less PolyEs in CP vs BP compared with SBC (11% and 6%, respectively). AHSP is positively associated with late-stage maturation of PBMC-derived erythroblasts in the presence of CP. Albeit transiently, this is more pronounced in Prop than SBC. Early administration of propranolol in burn patients supports erythropoiesis via the chaperone AHSP.

Stage Specific Expression Pattern of Alpha-Hemoglobin-Stabilizing-Protein (AHSP) Portrayed in Erythroblast Chronology.

During erythropoiesis, the molecular chaperone alpha-hemoglobin-stabilizing protein (AHSP) sequesters free alpha-hemoglobin (αHb) and prevents precipitation of excess αHb. While AHSP is linked to hereditary anemia, the pattern of expression during specific erythroblast stages is poorly understood. We investigated gene and protein expressions of AHSP throughout progressive maturation stages of erythroblasts in biphasic cultures of blood and bone marrow samples from healthy donors. Differentiating erythroblasts were periodically subjected to flow cytometry, Amnis imaging and RT-qPCR analyses. We made parallel in vivo validations from naive murine bone marrow cells. Percentages of AHSP+ erythroblasts, protein expressions and AHSP gene expressions are negligible on culture day 6 (CFU-Es) and progressively increases from culture days 8–12 (peaks on day 12) and declines on day 14. Notably, sub-cellular location of AHSP is both in the cytoplasm and nucleus in the early erythroblasts while in the late stages of maturation AHSP is found predominantly in the nucleus, being expelled with it during enucleation. As both human bone marrow and peripheral blood mononuclear cells (PBMC) derived erythroblasts demonstrated similar expression patterns, sampling of erythroblasts from day 11 cultures could portray erythroblast chronology and provide optimum representative stage specific expression patterns. PBMCs may be suitable for comparison studies of AHSP expression in pathologic erythropoiesi

48 Burn Injury Impedes Late Erythropoiesis via Decreased Alpha Hemoglobin Stabilizing Protein and siglec1

Abstract Introduction Transfusions are of no benefit to burn patients with chronic anemia. We are elucidating the mechanisms of erythropoietin resistant anemia in burn injury using a mouse model in addition to a contrived system with human PBMCs. We have found that Alpha Hemoglobin Stabilizing Protein (AHSP), an erythroblast specific protein essential to effective hemoglobinization, is decreased in PBMC derived erythroblasts (EB) from burn patients (male + female). Hemoglobinization begins at the polychromatic EB (PolyE) stage and intensifies at the orthochromatic EB (OrthoE) stage, culminating in ejection of the nucleus and the cell becoming a reticulocyte (Reti). EB needs the support of a central island macrophage (EBI MØ) until they progress to maturation. We have seen in bone marrow from mice (male) following burn injury that Siglec1, an essential adhesion molecule is decreased in EBI MØ. It is these MØ that provide iron for the assembly of the hemoglobin unit in late erythropoiesis. EB maturation defects seen in burn injury could be due to the intrinsic changes in EB phenotype or the phenotype of supporting EBI MØ or a combination of the two. Our goal was to investigate in the animal model using male and female mice, how myeloid/erythroid intrinsic changes after burn impact the progression of late erythropoiesis from an EB and EBI MØ perspectives. Methods With IACUC approval, 12 male and female mice were anesthetized and subjected to sham burn or scald burn (15% TBSA) upon dorsal immersion in a 100oC water bath for 8 seconds. Animals were sacrificed on post burn day 7 (PBD 7). Total bone marrow cells (TBM) were harvested from bilateral femurs. Cells were characterized using CD71, Ter119, and Syto16 antibodies. EBI MØ were identified using a combination of F4/80, ERHR3, Vcam1, Ly6G and Siglec 1 antibodies. Geometric mean of fluorescence intensity (MFI) and cell counts were obtained by flow cytometric analysis and presented as x103 per million TBM cells. Peripheral blood was harvested via cardiac puncture and hemoglobin concentrations (g/dL) were obtained using a hematology analyzer. Results Among the erythroblasts, EEBs were 2.4 fold higher with a 2.4 fold lower AHSP expression in PBD 7 mice compared to sham as shown in left, top and bottom figures. Similarly, more EEBs were associated with EBIMØ with a lower Siglec1 expression as shown in right, top and bottom figures. Blood Hbg concentrations were lower in burn (sham=15.5±0.7 vs PBD 7= 13.1±0.5; p&amp;lt; 0.01). Conclusions The combined influence of Siglec1 and AHSP expression interferes with effective hemoglobinization impeding late maturation of erythroblasts following burn injury. Whether there is a synergistic action between the two deficiencies is being studied. Applicability of Research to Practice Underpinning the mechanism of erythropoiesis will lead to better options to ineffective transfusions in burn patients with anemia.

Alpha-hemoglobin-stabilizing protein (AHSP): a modulatory factor in β-thalassemia.

Hemoglobin (Hb) is an iron-containing metalloprotein that transports oxygen molecules from the lungs to the rest of the human body. Among the different variants of Hb, HbA1 is the most common and is composed of two alpha (αHb) and two beta globin chains (βHb) constructing a heterotetrameric protein complex (α2β2). Due to the higher number of AHSP genes, there is a tendency to produce approximately twice as much of α subunit as β subunit. Therefore, there is a chance of presenting excess α subunit leftover in human blood plasma; excess subunits subsequently bind with each other and aggregates β-thalassemia occurs due to lack of or reduced numbers of βHb subunit. Alpha-hemoglobin-stabilizing protein (AHSP) is a scavenger protein which acts as a molecular chaperon by reversibly binding with free αHb forming a complex (AHSP-αHb) that prevents aggregation and precipitation preventing deleterious effects towards developing serious human diseases including β-thalassemia. Clinical severity worsens if mutations in AHSP gene co-occur in patients with β-thalassemia. Considering the mechanism of action of AHSP and its contribution to ameliorating β-thalassemia severity, it could potentially be used as a modulatory agent in the treatment of β-thalassemia.

Association of alpha hemoglobin-stabilizing protein (AHSP) gene mutation and disease severity among HbE-beta thalassemia patients.

In this study, we aimed to investigate the pattern and association of genetic mutations occurring within the alpha hemoglobin-stabilizing protein (AHSP) gene among HbE beta thalassemia patients with varying phenotypic expressions. Fifty-four diagnosed cases of HbE beta thalassemia (transfusion dependent and independent) were included in the study. Among them, 38 patients with similar genotypes (IVS 1-5, alpha gene deletion and triplication, Xmn polymorphism) were selected for further analysis. AHSP gene sequencing was done for these 38 samples to study associated mutations in AHSP gene. HbE beta thalassemia patients with similar genotypes but different phenotypic expressions were found to have mutations in the AHSP gene. There were five mutations found most prevalent among the samples analyzed for AHSP gene sequencing. Among these, two mutations were from intron 1 region of AHSP and three mutations were found in exon 3. The most prevalent mutation was found at the Oct binding site at intron 1 of AHSP. The mutations in exon 3 were more prevalent among the TDT groups. A mutation in exon 3 changing the amino acid (33rd) from serine to phenylalanine was found to be associated with only TDT group. This study documents that among the HbE beta thalassemia patients with varying severity, an exon mutation in AHSP is significantly prevalent only among the TDT group. Further understanding of the mechanism will shed light upon the impact of AHSP in modifying the disease severity in thalassemia.

T4 Mechanistic Insights on Late Stage Erythropoiesis in Burn Patients Treated with Propranolol

Burn patients suffer from Epo resistant anemia in which the Epo-independent stages of early commitment and late maturation of erythroblasts are defective. We have previously shown in mice and patients, that propranolol (prop) treatment after burn injury restored erythroid committed progenitors, while terminal maturation was still impaired. During terminal erythropoiesis, hemoglobinization is aided by alpha hemoglobin stabilizing protein (AHSP), which is an erythroblast intrinsic functional protein predominantly expressed in nucleated erythroblasts. Effective hemoglobinization results in maturation of polychromatic erythroblasts (PolyE) to reticulocytes. Here we demonstrate in PBMC derived ex vivo erythroblast cultures, AHSP is positively associated with late stage maturation in the presence of allogeneic control plasma (CP); and more so in combination with prop than with standard burn care (SBC) in burn patients. After IRB approval, blood samples were collected from 6 burn patients (age=39±16 yeas; %TBSA=32±7) at three time points on post burn days 2–4, 7–10 and 27–33. Control plasma was obtained from healthy volunteers. Fifteen PBMC samples were placed in growth factor cocktail (SCF, GMCSF, IL-3, Cyclosporine) for 0–5 days to nurture erythroid progenitors. On day 5, each sample was split into two and supplemented with either autologous burn plasma (BP) or CP along with SCF, EPO, and holotransferrin from days 5–15. We harvested cells on culture days 9, 11 and 15; subjected them to FACS and AMNIS to determine the expression (mean fluorescence intensity) and percentage of AHSP+ erythroblasts on days 9 and 11 and the percentage of Reticulocytes (Sytox- CD235a+ CD71+/- Syto16-) and PolyE (Sytox- CD235a+ CD71+/- Syto16+ SSChigh) on day 15. See figure. Overall, CP supplementation in PBMC cultures produced more reticulocytes with a proportional decrease in PolyE, indicating enhanced late stage maturation. Nonetheless, the decrease in PolyE and increase in reticulocytes are more striking in prop treated patients compared to SBC. This is consistent with about 20% increase in AHSP+ cells and AHSP expression in developing erythroblasts in prop compared to SBC. Together, CP imparts a positive influence on reticulocyte maturation by augmenting AHSP expression in PBMC derived erythroblasts. Plasma resuscitation in addition to prop treatment is a promising alternate strategy to reduce transfusions in burn patients.

Role of Alpha Hemoglobin Stabilizing Protein Expression in Beta Thalassemia Patients

AbstractBackground: Beta (b)-Thalassemia is an autosomal reces-sive inherited disease causing variable degrees of anemia. The molecular defects are owing to point mutations or small deletions leading to diminution or absence of b-globin chain synthesis. The unmatched a-Hb is destructive to itself and other cellular components, resulting in ineffective erythropoi-esis and hemolysis. Alpha-Hemoglobin Stabilizing Protein (AHSP) is a scavenger protein of erythroid that fastly and reversibly attach the a subunit of hemoglobin molecule preventing its precipitation and subsequent hemolysis.Aim of Work: Is to assess AHSP gene expression in thalassemia major and intermedia and its relation to clinical features, laboratory investigations and to determine its role on the severity of thalassemia.Subjects and Methods: This case-control study is consisted of 120 studied subjects categorized into 40 thalassemia major patients, 40 thalassemia intermedia patients and 40 age healthy individuals as a control group. All subjects underwent the following; history, clinical picture, complete blood picture and Hb electrophoresis, serum ferritin and measurement of AHSP, alpha globin and beta globin genes expression by real time PCR.Results: AHSP gene in thal. intermedia group was higher than both thal. major (p<0.001). Beside thal. intermedia group had higher a globin/AHSP ratio versus thal. major group (p=0.011). b globin/AHSP ratio was not significant between both thal. groups. AHSP in thal.major group correlated with a globin and b globin expression and negatively correlated with organomegally, ferritin and no. of blood transfusion. Whereas in thal. intermedia group, was positively correlated with Hb, HCT, MCV, MCH, a globin gene expression, b globin gene expression and b globin/a globin genes ratio.Conclusion: AHSP expression was higher in thalassemia intermedia group than thalassemia major group. Consequently, AHSP can be considered genetic modifier in b thalassemia.

The effect of histone deacetylase inhibitors on AHSP expression.

Alpha-hemoglobin stabilizing protein (AHSP) is a molecular chaperone that can reduce the damage caused by excess free α-globin to erythroid cells in patients with impaired β-globin chain synthesis. We assessed the effect of sodium phenylbutyrate and sodium valproate, two histone deacetylase inhibitors (HDIs) that are being studied for the treatment of hemoglobinopathies, on the expression of AHSP, BCL11A (all isoforms), γ-globin genes (HBG1/2), and some related transcription factors including GATA1, NFE2, EKLF, KLF4, and STAT3. For this purpose, the K562 cell line was cultured for 2, 4, and 6 days in the presence and absence of sodium phenylbutyrate and sodium valproate. Relative real-time qRT-PCR analysis of mRNA levels was performed to determine the effects of the two compounds on gene expression. Expression of all target mRNAs increased significantly (p < 0.05), except for the expression of BCL11A, which was down-regulated (p < 0.05) in the cells treated with both compounds relative to the levels measured for untreated cells. The findings indicated that sodium valproate had a more considerable effect than sodium phenylbutyrate (p < 0.0005) on BCL11A repression and the up-regulation of other studied genes. γ-Globin and AHSP gene expression continuously increased during the culture period in the treated cells, with the highest gene expression observed for 1 mM sodium valproate after 6 days. Both compounds repressed the expression of BCL11A (-XL, -L, -S) and up-regulated GATA1, NFE2, EKLF, KLF4, STAT3, AHSP, and γ-globin genes expression. Moreover, sodium valproate showed a stronger effect on repressing BCL11A and escalating the expression of other target genes. The findings of this in vitro experiment could be considered in selecting drugs for clinical use in patients with β-hemoglobinopathies.

A molecular study on the role of alpha-hemoglobin-stabilizing protein in hemoglobin H disease.

The clinical course of hemoglobin H (HbH) disease is remarkably variable. It is not completely clear how genetic and environmental factors interplay to modify clinical severity in affected individuals. Previous studies suggested that altered structure or function of alpha-hemoglobin-stabilizing protein (AHSP) could modify the clinical phenotypes of thalassemias. The present study attempted to explore the potential role of AHSP in the pathophysiology of HbH disease in 95 Chinese and Thai/Sino-Thai patients with deletional and non-deletional form of this disease. We identified six polymorphic sites in AHSP which were subgrouped into major haplotype clades. No association between AHSP genotypes or haplotypes and clinical phenotypes was observed. Instead, multiple linear regression analysis indicated that expression of AHSP correlated negatively with age (P<0.001) and hemoglobin (P=0.007), but positively with reticulocyte count (P=0.003) and severity score (P=0.003). Subgroup analysis showed that AHSP expression was higher in the non-deletional form than in the deletional form (P<0.001). Moreover, specific types of non-deletional HbH disease with production of mutant alpha-globin chains that do not bind to AHSP (Hb Constant Spring and Hb Pakse) showed the highest AHSP expression. The present findings demonstrate that AHSP expression is a biomarker of HbH disease severity and infer an important role of AHSP in modulating the pathophysiology of this disease. Pharmacological or genetic means to alter AHSP expression may be a novel approach for amelioration of disease severity in HbH disease.

Endothelial Cell-Expressed α Hemoglobin and Its Molecular Chaperone Ahsp Modulate Arterial Vascular Reactivity

We are studying a novel mechanism by which prototypical erythrocyte proteins also act in endothelial cells to regulate vascular tone. Previously, we reported that the alpha subunit of hemoglobin (αHb) is expressed in the myoendothelial junction of endothelial cells in resistance arterioles where it binds endothelial nitric oxide (NO) synthase (eNOS) and degrades NO, thereby stimulating vasoconstriction. To extend this observation, we examined arterioles of mice lacking 2 out of 4 α globin genes [HbA1 knockout *α2/*α2]. Compared to wild type (wt) controls, the thoracodorsal artery (TDA, a resistance arteriole) from mutant animals exhibited abnormal morphology with thinned internal elastic lamina, excessive dilation, and reduced contractility after treatment with the vasoconstrictor phenylephrine (EC50 8.2e-6M-1 n=6 in wt vs 2.3e-5 M-1 n=6 in mutant; p<0.0001).Wild type TDAs expressed αHb (Fig. 1A), but not βHb mRNA and protein. However, free αHb is unstable and not expected to exist as an isolated monomer. Alpha hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free αHb, stabilizes its structure and facilitates assembly of HbA tetramers (α2β2) in red blood cells. Immunohistochemistry showed that AHSP and αHb co-localize in endothelial cells lining the TDA. In the TDA of Ahsp knockout (KO) mice, αHb immunostaining was disorganized and reduced in intensity (Fig. 1A), and Western blotting showed reduced αHb protein compared to wt controls. Moreover, TDAs from Ahsp KO mice exhibited abnormal thinning of the internal elastic lamina, excessive dilation (lumen diameter 7500 μM2 in Ahsp KO vs 4100 μM2 in controls; n=5 mice, p=0.0037) and reduced constriction after phenylephrine treatment (EC50 9.6e-6 M-1 in wt n=7 vs 5.6e-6 M-1 in mutant n=6; p<0.0001) (Fig. 1B-C), similar to what we observed in HbA1-/- mice.To examine physical and functional interactions between AHSP, αHb and eNOS, we expressed fluorescent-tagged proteins in cultured human coronary ECs. αHb-GFP alone was expressed at relatively low level (mean fluorescent intensity (MFI) = 631) (Fig. 1D). Coexpressed mCherry-AHSP colocalized with αHb-GFP and increased its expression level (MFI 5312) (Fig. 1D). Similarly, coexpressed mCherry-eNOS colocalized with αHb-GFP and enhanced its expression (MFI 1519) (Fig. 1D). Purified αHb co-immunoprecipitated with AHSP or eNOS, but not both, suggesting mutually exclusive interactions.Overall, our studies provide genetic evidence that αHb expressed in arteriolar endothelial cells regulates blood vessel tone in vivo. Moreover, biochemical studies show that AHSP stabilizes endothelial-expressed αHb and facilitates its assembly with eNOS. Thus, AHSP acts as a molecular chaperone for αHb in erythrocytes and endothelial cells to promote the formation of HbA (α2b2) for O2 transport, and αHb-eNOS for NO degradation. We hypothesize that the αHb-AHSP-eNOS axis functions to fine-tune systemic blood pressure and/or regional blood flow to specific vascular beds. Defining the magnitude of these effects in mice and humans during specific circulatory stresses and in α thalassemia is an important and interesting topic for future investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vascular effects of alpha hemoglobin stabilizing protein deletion

Hemoglobin H is a tetramer made up of 2 alpha globin subunits and 2 beta globin subunits. The alpha globin subunit requires chaperone activity by alpha hemoglobin stabilizing protein (AHSP) in order to properly associate with hemoglobin beta. In addition to erythrocytes, alpha globin is expressed in endothelial cells (EC), specifically in the small projections of the EC that make contact with the neighboring smooth muscle cells (SMC), known as myoendothelial junctions (MEJ). Immuno‐transmission electron microscopy indicates that AHSP is expressed in EC and the MEJ but not SMC of C57Bl/6 resistance arteries. There is no expression of vascular AHSP in the global AHSP knockout mice. Correlating with its role as a chaperone protein for alpha globin, 32% of AHSP is found in the EC while 60% is found in the MEJ. The percent distribution of alpha globin beads in the EC and MEJ is 35% and 60%, respectively. In AHSP −/−mice, we found that alpha globin “un‐polarizes” from the MEJ and is more equivalently spread throughout the EC and MEJ (47% and 41.5%). To determine if AHSP is regulating alpha globin distribution, we immunoprecipitated eNOS and blotted for alpha globin. In a concentration‐dependent manner, AHSP competed off eNOS for binding to alpha globin. This correlated with data from AHSP−/− mice where an increase in alpha globin‐eNOS interaction was found via PLA signal. We hypothesized that the increase in alpha globin/eNOS interaction may then alter available nitric oxide. Indeed, using radiotelemetery blood pressure measurements, we found that mean arterial pressure in AHSP−/− mice was significantly increased. These results hint that deletion of AHSP may strengthen the interaction between alpha globin and eNOS resulting in altered blood pressure stasis.Support or Funding InformationAmerican Heart AssociationNational Institute of Health

Alpha hemoglobin stabilizing protein: Its causal relationship with the severity of beta thalassemia.

Thalassemia major is characterized by anemia, iron overload and cellular damage. The severity of symptoms correlates with the alpha/non-alpha globin imbalance and is proportional to the magnitude of alpha chain excess. Alpha hemoglobin stabilizing protein (AHSP), the erythroid specific alpha globin chaperone, stabilizes free alpha chains, and prevents the formation of reactive oxygen radicals. Though AHSP expression has been linked to the severity of beta thalassemia, its role as a probable genetic modifier of disease severity, has still not been unequivocally established. In the present study, the level of the chaperone has been seen to vary in regularly transfused beta thalassemia patients, being underexpressed in 64% of cases, upregulated in 16% and comparable to controls in 20% of the cases. This discrepancy may be attributed to the degree of DNA damage, % HbF, and the number of nucleated RBCs in the peripheral blood of these patients. Results reveal that a decrease in the free alpha chain pool, and hence the repertoire of unbound iron, due to elevated HbF and/or the presence of nucleated RBCs in the peripheral blood results in the upregulation of the AHSP gene.

Rôle du chaperon moléculaire de l’alpha-hémoglobine dans la formation de l’hémoglobine et l’expression clinique de certaines hémoglobinopathies

Alpha-hemoglobin stabilizing protein (AHSP), described as a chaperone of alpha-hemoglobin (α-Hb), is synthesized at a high concentration in the erythroid precursors. AHSP specifically recognizes the G and H helices of α-Hb and forms a stable complex with free α-Hb until its association with the partner β-subunits. Unlike the free β-Hb which are soluble and form homologous tetramers, freshly synthesized α-Hb chains are highly unstable molecular species which precipitate and generate reactive oxygen species within the erythrocyte precursors of the bone marrow leading to apoptosis and ineffective erythropoiesis. AHSP protects the free α-Hb chains in maintaining it in the soluble state. In this review, we report data from the literature and our laboratory concerning the key role of AHSP in the biosynthesis of Hb and its possible involvement in some disorders of the red blood cell as well as the hemoglobinopathies and we discuss its use as a prognostic tool in thalassemia syndromes.