Effect of point mutation on structure-function correlation of hemoglobin variants, HbE and HbD Punjab.

Hemoglobinopathies are examples of autosomal recessive disorders of human hemoglobin. Hemoglobin E (HbE) and Hemoglobin D Punjab (HbD Punjab) are two of the most common hemoglobin variants geographically spread across Asian continent. These two variants differ from normal human hemoglobin (HbA) at a single amino acid residue caused by the point mutation of ß globin gene. The presence of the mutated amino acid residue causes perturbation in the function of both variants. However, the structure-function correlation of these variants has not been established yet. In the present study, we analyzed the conformational changes associated with oxygenation of hemoglobin variants using hydrogen/deuterium exchange-based mass spectrometry of backbone amide hydrogens of α and ß globin chains in the tetrameric hemoglobin molecule. We also performed the functional assay of these variants using oxygen dissociation equilibrium curve. Compared to HbA, both variants showed reduced oxygen affinity, as reported earlier. The functional perturbations exhibited by these variants were correlated well with their structural alterations with respect to the reported changes in the residue level interactions upon oxygenation of normal hemoglobin, monitored through the hydrogen/deuterium exchange kinetics of several peptic peptides originated from the isotopically exchanged oxy and deoxy forms of HbE and HbD Punjab.

Oxidative instability of hemoglobin E (ß26 Glu→Lys) is increased in the presence of free α subunits and reversed by α-hemoglobin stabilizing protein (AHSP): Relevance to HbE/ß-thalassemia.

When adding peroxide (H2O2), ß subunits of hemoglobin (Hb) bear the burden of oxidative changes due in part to the direct oxidation of its Cys93. The presence of unpaired α subunits within red cells and/or co-inheritance of another ß subunit mutant, HbE (ß26 Glu→Lys) have been implicated in the pathogenesis and severity of ß thalassemia. We have found that although both HbA and HbE autoxidize at initially comparable rates, HbE loses heme at a rate almost 2 fold higher than HbA due to unfolding of the protein. Using mass spectrometry and the spin trap, DMPO, we were able to quantify irreversible oxidization of ßCys93 to reflect oxidative instability of ß subunits. In the presence of free α subunits and H2O2, both HbA and HbE showed ßCys93 oxidation which increased with higher H2O2 concentrations. In the presence of Alpha-hemoglobin stabilizing protein (AHSP), which stabilizes the α-subunit in a redox inactive hexacoordinate conformation (thus unable to undergo the redox ferric/ferryl transition), Cys93 oxidation was substantially reduced in both proteins. These experiments establish two important features that may have relevance to the mechanistic understanding of these two inherited hemoglobinopathies, i.e. HbE/ß thalassemia: First, a persistent ferryl/ferryl radical in HbE is more damaging to its own ß subunit (i.e., ßCys93) than HbA. Secondly, in the presence of excess free α-subunit and under the same oxidative conditions, these events are substantially increased for HbE compared to HbA, and may therefore create an oxidative milieu affecting the already unstable HbE.

Blood transfusion from a Hb E trait donor can affect ß-thalassemia diagnosis.

A subject with Hb E (HBB: c.79G > A) trait is asymptomatic and can become a blood donor. However, a blood transfusion from a Hb E trait donor can affect ß-thalassemia (ß-thal) diagnosis. Blood samples from three Thai women were sent to the Associated Medical Sciences (AMS) Clinical Service Center, Chiang Mai, Thailand, for thalassemia diagnosis. Their Hb A2 levels, analyzed by high performance liquid chromatography (HPLC), were higher than 4.0%, thus they were diagnosed to have ß-thal. However, elevated Hb A2 levels in these patients were not certain because the Hb A2 levels analyzed at the initial hospitalization and follow-up were controversial. In addition, there were some cases shown to have controversy between the increased Hb A2 level and red cell indices. The blood transfusion history was confirmed and hemoglobin (Hb) analysis was reanalyzed by capillary electrophoresis (CE). On the CE electrophoregram, Hb A2 levels were observed to be normal and Hb E peaks were present. Therefore, to rule out misdiagnosis and unnecessary genetic counseling, Hb analysis should be performed on the recipient prior to blood transfusions. Moreover, CE has a high efficiency to prevent the misinterpretation of Hb analysis in patients who receive blood transfusions from a donor carrying Hb E.

Genetic heterogeneity of hemoglobin AEBart's disease: a large cohort data from a single referral center in northeast Thailand.

AEBart's disease is a thalassemia intermedia usually characterized by the interaction of α(0)-thalassemia with either deletional or non-deletional α(+)-thalassemia in Hb E heterozygote. Genotypic and phenotypic features are heterogeneous. We studied the hematologic and molecular characteristics of this disease in a cohort of 173 Thai patients encountered at our center in northeast Thailand. Hemoglobin and DNA analyses identified patients with deletional AEBart's disease (n=84), Hb Constant Spring AEBart's disease (n=81), Hb Paksé-AEBart's disease (n=5), AEBart's disease with codon 30 mutation (n=1) and two hitherto un-described forms of AEBart's disease due to interaction of Hb E heterozygote and α(0)-thalassemia with the -α(16.6)kb deletional α(+)-thalassemia (n=1) and Hb Q-Thailand (n=1). Different phenotypic expression of these AEBart's diseases with low Hb, Hct and MCV and increased RDW values with marked reduction in Hb E levels were observed. It was found that all these forms of AEBart's disease showed similar thalassemia intermedia phenotypes but those with non-deletional forms were relatively more anemic. Our data confirm that in such area with high prevalence of hemoglobinopathies such as Southeast Asia, identification of rare thalassemia alleles in a thalassemia intermedia patient should not be ignored. Careful consideration of different phenotypic expression may help in providing presumptive diagnosis of this disease where access to molecular testing is limited. However, molecular diagnostic is useful for predicting the clinical outcome and improving genetic counseling of these complex hemoglobinopathies.

Differential thermal stability and oxidative vulnerability of the hemoglobin variants, HbA2 and HbE.

Apart from few early biophysical studies, the relative thermal instability of HbE has been only shown by clinical investigations. We have compared in vitro thermal stability of HbE with HbA2 and HbA using optical spectroscopy. From absorption measurements in the soret region, synchronous fluorescence spectroscopy and dynamic light scattering experiments, we have found thermal stability of the three hemoglobin variants following the order HbE11.0 in all the three variants. Under oxidative stress conditions in presence of hydrogen peroxide, HbE has been found to be more vulnerable to aggregation compared to HbA and HbA2. Taken together, these studies have shown thermal and oxidative instability of HbE and points towards the role of HbE in the upregulation of redox regulators and chaperone proteins in erythrocyte proteome of patients suffering from HbEbeta thalassemia.

Faster heme loss from hemoglobin E than HbS, in acidic pH: effect of aminophospholipids.

We report studies on loss of heme at or below pH 3.0 from two clinically important hemoglobin variants, HbE and HbS, in the presence and absence of phopholipid membranes. The kinetics of heme loss has been studied at pH 3.0 to simulate the same at a faster rate than at physiological pH, for spectroscopic investigation. Results obtained from the study clearly establish the probable fate of the lost heme to partition into the phospholipid bilayer independent of the pH range. This is also of particular importance to membranes containing the aminophospholipid and cholesterol which are predominantly localized in the inner leaflet of erythrocytes. Absorption measurements indicated such loss of heme when the Soret peak at 415 nm blue-shifted to 380 nm at pH 3.0. The extent of this blue shift decreased from 35 nm to (approx.) 15 nm in the presence of small unilammelar vesicles of both dimyristoyl- and dioleoyl-based phosphatidylcholine and phosphatidylethanolamine, indicating partitioning of the released heme in the membrane bilayer. The kinetics of heme loss was faster from HbE than HbA and HbS, obeying first-order reaction kinetics. Released heme could be involved in the premature destruction of erythrocytes in hemoglobin disorders.

Structural and functional studies indicating altered redox properties of hemoglobin E: implications for production of bioactive nitric oxide.

Hemoglobin (Hb) E (ß-Glu26Lys) remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms; for example, EE individuals exhibit a mild chronic anemia, and HbE/ß-thalassemia individuals show a range of clinical manifestations, including high morbidity and death, often resulting from cardiac dysfunction. The purpose of this study was to determine and evaluate structural and functional consequences of the HbE mutation that might account for the pathophysiology. Functional studies indicate minimal allosteric consequence to both oxygen and carbon monoxide binding properties of the ferrous derivatives of HbE. In contrast, redox-sensitive reactions are clearly impacted as seen in the following: 1) the ∼2.5 times decrease in the rate at which HbE catalyzes nitrite reduction to nitric oxide (NO) relative to HbA, and 2) the accelerated rate of reduction of aquometHbE by L-cysteine (L-Cys). Sol-gel encapsulation studies imply a shift toward a higher redox potential for both the T and R HbE structures that can explain the origin of the reduced nitrite reductase activity of deoxyHbE and the accelerated rate of reduction of aquometHbE by cysteine. Deoxy- and CO HbE crystal structures (derived from crystals grown at or near physiological pH) show loss of hydrogen bonds in the microenvironment of ßLys-26 and no significant tertiary conformational perturbations at the allosteric transition sites in the R and T states. Together, these data suggest a model in which the HbE mutation, as a consequence of a relative change in redox properties, decreases the overall rate of Hb-mediated production of bioactive NO.

Compound heterozygous Hb Tak/Hb E causes secondary erythrocytosis in a Thai family.

Hb Tak is a rare cause of secondary erythrocytosis. It results from the insertion of two nucleotides (AC) at the termination codon between codon 146 and codon 147 of the beta-globin gene. This insertion causes a frameshift in the terminating codon 147 resulting in an elongated beta chain with an impaired formation of the T-state. We report a Thai family with a compound heterozygosity for Hb Tak [beta147 (+ AC)] and Hb E [beta26(B8)Glu-->Lys] which displayed an asymptomatic erythrocytosis.

A mechanism of ineffective erythropoiesis in ß-thalassemia/Hb E disease.

BACKGROUND: Cells respond to stress stimuli through a number of response pathways, of which one of the most important and well characterized is the unfolded protein response. Despite a large body of work which suggests that stress in erythroblasts may play a pivotal role in the pathogenesis of beta-thalassemia/Hb E disease, this pathway remains uninvestigated. DESIGN AND METHODS: Day 10 erythroblasts from normal controls and beta-thalassemia/Hb E patients were subjected to internal (treatment with tunicamycin) and external (serum and growth factor withdrawal) stress stimuli and the activation of the unfolded protein response pathway was investigated. RESULTS: Normal erythroblasts responded to both internal and external stress by activating the unfolded protein response (UPR) pathway while in contrast, erythroblasts from beta-thalassemia/Hb E patients only showed activation of the unfolded protein response pathway in response to internal stress. This was reflected by a markedly increased induction of apoptosis in serum and growth factor deprived beta-thalassemia/Hb E erythroblasts as compared to control cells. Modulation of the levels of intracellular Ca(2+) in thalassemic erythroblasts restored UPR activation during serum deprivation and significantly reduced the level of serum deprivation induced apoptosis to control levels. CONCLUSIONS: These results suggest the failure of thalassemic erythroblasts to cope with cellular stress caused by an impaired UPR function as a result of high Ca(2+) levels may exacerbate thalassemic cell death during erythropoiesis.

Enhanced oxidative cross-linking of hemoglobin E with spectrin and loss of erythrocyte membrane asymmetry in hemoglobin Ebeta-thalassemia.

Oxidative stress to the erythrocytes is associated with formation of large molecular complexes of hemoglobin and the skeletal protein, spectrin. In this work, such complexes are formed with hemoglobin mixtures isolated from patients suffering from HbEbeta-thalassemia with elevated levels of the HbE and purified erythroid spectrin in the presence of hydrogen peroxide. The complexes are separated on 4% SDS-PAGE and analyzed by densitometry. The results indicate enhanced formation of complexes with higher amounts of HbE, the most common hemoglobin variant prevalent in Southeast Asia. The binding affinity of spectrin with hemoglobin, in the absence of hydrogen peroxide, was found to increase with hemoglobin mixtures enriched with HbE. The presence of ATP was also found to decrease the overall yield of such complexes. Flow cytometric measurements of phosphatidylserine on the red cell surface also showed a lower degree of membrane asymmetry in HbEbeta-thalassemic patients than in normal subjects. The present work shows enhanced formation of high molecular weight cross-linked complexes of hemoglobin derivatives with erythroid spectrin in HbEbeta-thalassemia.

Crystal structures of HbA2 and HbE and modeling of hemoglobin delta 4: interpretation of the thermal stability and the antisickling effect of HbA2 and identification of the ferrocyanide binding site in Hb.

Hemoglobin A(2) (alpha(2)delta(2)) is an important hemoglobin variant which is a minor component (2-3%) in the circulating red blood cells, and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is beta-chain production failure, HbA(2) acts as the predominant oxygen deliverer. HbA(2) has two more important features. (1) It is more resistant to thermal denaturation than HbA, and (2) it inhibits the polymerization of deoxy sickle hemoglobin (HbS). Hemoglobin E (E26K(beta)), formed as a result of the splice site mutation on exon 1 of the beta-globin gene, is another important hemoglobin variant which is known to be unstable at high temperatures. Both heterozygous HbE (HbAE) and homozygous HbE (HbEE) are benign disorders, but when HbE combines with beta-thalassemia, it causes E/beta-thalassemia which has severe clinical consequences. In this paper, we present the crystal structures of HbA(2) and HbE at 2.20 and 1.74 A resolution, respectively, in their R2 states, which have been used here to provide the probable explanations of the thermal stability and instability of HbA(2) and HbE. Using the coordinates of R2 state HbA(2), we modeled the structure of T state HbA(2) which allowed us to address the structural basis of the antisickling property of HbA(2). Using the coordinates of the delta-chain of HbA(2) (R2 state), we also modeled the structure of hemoglobin homotetramer delta(4) that occurs in the case of rare HbH disease. From the differences in intersubunit contacts among beta(4), gamma(4), and delta(4), we formed a hypothesis regarding the possible tetramerization pathway of delta(4). The crystal structure of a ferrocyanide-bound HbA(2) at 1.88 A resolution is also presented here, which throws light on the location and the mode of binding of ferrocyanide anion with hemoglobin, predominantly using the residues involved in DPG binding. The pH dependence of ferrocyanide binding with hemoglobin has also been investigated.

Attenuation of oxidative stress-induced changes in thalassemic erythrocytes by vitamin E.

The oxidative stress status of the transfusion-dependent Ebeta- and beta-thalassemia patients were studied before and after treatment with vitamin E for a period of four weeks. The level of cellular vitamin antioxidants viz. ascorbic acid and vitamin E in the thalassemia patients were found to be considerably lower compared to normal subjects. The activities of enzymatic antioxidants viz. catalase, glutathione peroxidase and glutathione reductase were found to be drastically reduced in untreated Ebeta- and beta-thalassemic patients when compared to normal subjects. However, the activity of superoxide dis-mutase was found to be increased in both types of untreated thalassemic patients when compared to normal individuals. An increase in superoxide dismutase and a decrease in catalase activity reflects the presence of a severe oxidative stress situation in the erythrocytes of the untreated transfusion dependent Ebeta- and beta-thalassemia patients. Changes in erythrocyte membrane protein pattern in untreated Ebeta- and beta-thalassemia patients when compared to normal erythrocyte further confirm the presence of continued oxidative stress in the ailing thalassemic erythrocytes. All these changes in the antioxidant status as well as the changes in the erythrocyte membrane proteins are ameliorated to considerable extent when the transfusion-dependent Ebeta- and beta-thalassemia patients were treated with vitamin E at a dose of 10 mg/kg/day for a period of four weeks. The patients during the treatment period did not exhibit any side effects and gained in body weight indicating a healthy status. The present study reveals that the lipophilic antioxidant vitamin E could be useful in the management of transfusion-dependant Ebeta- and beta-thalassemia patients.

Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E, isolated from the blood samples of beta-thalassemic patients.

Hemoglobin A(2) (alpha(2)delta(2)), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is a failure of beta-chain production, HbA(2) acts as the predominant oxygen delivery mechanism. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of beta globin gene, when combines with beta-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA(2) and HbE are reported here. HbA(2) and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from beta-thalassemia minor and E beta-thalassemia. X-ray diffraction data of HbA(2) and HbE were collected upto 2.1 and 1.73 A, respectively. HbA(2) crystallized in space group P2(1) with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and beta=99.80 degrees whereas HbE crystallized in space group P2(1)2(1)2(1) with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R(2) state.

Effect of HbE and HbH on HbA1C level by ionic exchange HPLC comparing to immunoturbidimetry.

BACKGROUND: The hemoglobin (Hb)A1C level is widely used to monitor diabetes mellitus patients. The N-terminal amino acid valine of its beta chain is glycated. The assay of HbA1C is based on differences in the charge, chemical and structural properties of the protein. METHODS: There are fully automated instruments available in clinical chemistry laboratory to assay HbA1C level. The effect of hemoglobinopathies was studied between an ionic exchange high-pressure liquid chromatography (HPLC) (Bio-Rad Laboratories, USA) and immunoturbidimetry (BM/Hitachi 912 with Roche HBA1CII, Germany-Japan) assay. The influence of high level HbF relative to the HbA1C level by ionic exchange HPLC is known. The effect of HbE and HbH to the HbA1C level by ionic exchange HPLC comparing to immunoturbimetry was examined. The evaluation was performed on 34 normal controls (A2A), 17 beta thalassemia traits (A2 upward arrow A), 36 HbE heterozygotes (EA), 37 HbE homozygotes (EE), 36 beta thalassemia/HbE (EF/EFA), 11 EABart's diseases (EABart's), 34 Hb H diseases (A2/CSAH) and 13 cord blood samples (FA). CONCLUSIONS: Hemoglobinopathies can impact on the assay of HbA1C level such as HbE and HbH to ionic exchange HPLC. Although not studied as yet, this effect may influence the other methods such as affinity chromatography.

Renal function in adult beta-thalassemia/Hb E disease.

Beta-Thalassemia hemoglobin E (beta-thal/Hb E) is the commonest form of hemoglobinopathy in Thailand. Shortened red cell life span, rapid iron turnover and tissue deposition of excess iron are major factors responsible for functional and physiological abnormalities found in various forms of thalassemia. Increased deposition of iron had been found in renal parenchyma of thalassemic patients, but no systematic study of the effect of the deposits on renal functions has been available. The purpose of this study is to describe the functional abnormalities of the kidney in patients with beta-thal/Hb E and provide evidence that increased oxidative stress might be one of the factors responsible for the damage. Urine and serum samples from 95 patients with beta-thal/Hb E were studied comparing with 27 age-matched healthy controls. No difference in the creatinine clearance was observed. beta-thal/Hb E patients excreted significantly more urinary protein (0.8+/-0.5 vs. 0.3+/-0.1 g/day, p < 0.001). Aminoaciduria was found in 16 % of the patients. Analysis of urinary protein by SDS-PAGE electrophoresis and silver staining revealed abnormal pattern of protein with increased small molecular weight (<45 kD) bands. Morning urine analysis showed significant lower urine osmolality (578.3+/-164.6 vs. 762.4+/-169.9 mosm/kg, p < 0.001) in patients. Patients excreted more NAG (N-acetyl beta-D-glucosaminidase, 26.3+/-41.3 vs. 8.4+/-3.9 U/g Cr, p < 0.0001) and beta2-microglobulin, 124.3+/-167 vs. 71+/-65.5 microg/g Cr, p = 0.001. Plasma and urine MDA (malonyldialdehyde) levels were both raised (p < 0.0001). Nine patients were selected for renal acidification study. All were found to be normal, but showed poor response to DDAVP challenge (urine osmolality 533+/-71). This is the first report of renal tubular defects found associated with beta-thal/Hb E disease. The mechanism leading to the damage is not known but it might be related to increased oxidative stress secondary to tissue deposition of iron, as indicated by the raised levels of serum and urine MDA. It is not known whether these functional defects would have any long-term effects on the patients. Further studies are warranted and means of prevention of these defects should urgently be sought.

Use of a polybrene capillary coating in capillary electrophoresis for rapid analysis of hemoglobin variants with on-line detection via an ion trap storage/reflectron time-of-flight mass spectrometer.

A polybrene capillary coating in capillary electrophoresis (CE) has been used for rapid analysis of hemoglobin variant digests. The use of the polybrene capillary coating has allowed sufficient separation to resolve the large number of digest products formed upon tryptic digestion of the whole protein, so that prior separation of the hemoglobin alpha and beta chains is not required. The resolution of the digest peaks obtained by CE is sufficient so that even single amino acid substitutions can easily be detected using UV absorption detection. The digest is further analyzed by capillary electrophoresis separation with on-line detection using electrospray ionization interfaced to the ion trap storage/reflectron time of flight device (CE/ESI-IT/reTOF), where a comparison of the total ion electropherograms and mass spectra of the mutant and normal hemoglobins can detect the presence of a mutation site. The CE separation and mass analysis can be accomplished in typically 10-15 min. The unique capability of the CE/ESI-IT/reTOF system for detection of fast separations with narrow peaks that may be under 1 s fwhm is demonstrated. The speed of this system is essential for resolution of the large number of peaks that are separated in a short time duration using CE separations.