Membrane interactions of hemoglobin variants, HbA, HbE, HbF and globin subunits of HbA: effects of aminophospholipids and cholesterol

The interaction of hemoglobin with phospholipid bilayer vesicles (liposomes) has been analyzed in several studies to better understand membrane-protein interactions. However, not much is known on hemoglobin interactions with the aminophospholipids, predominantly localized in the inner leaflet of erythrocytes, e.g., phosphatidylserine (PS), phosphatidylethanolamine (PE) in membranes containing phosphatidylcholine (PC). Effects of cholesterol, largely abundant in erythrocytes, have also not been studied in great details in earlier studies. This work therefore describes the study of the interactions of different hemoglobin variants HbA, HbE and HbF and the globin subunits of HbA with the two aminophospholipids in the presence and absence of cholesterol. Absorption measurements indicate preferential oxidative interaction of HbE and alpha-globin subunit with unilamellar vesicles containing PE and PS compared to normal HbA. Cholesterol was found to stabilize such oxidative interactions in membranes containing both the aminophospholipids. HbE and alpha-globin subunits were also found to induce greater leakage of membrane entrapped carboxyfluorescein (CF) using fluorescence measurements. HbE was found to induce fusion of membrane vesicles containing cholesterol and PE when observed under electron microscope. Taken together, these findings might be helpful in understanding the oxidative stress-related mechanism(s) involved in the premature destruction of erythrocytes in peripheral blood, implicated in the hemoglobin disorder, HbE/beta-thalassemia.     

Membrane interactions of hemoglobin variants, HbA, HbE, HbF and globin subunits of HbA: Effects of aminophospholipids and cholesterol

The interaction of hemoglobin with phospholipid bilayer vesicles (liposomes) has been analyzed in several studies to better understand membrane-protein interactions. However, not much is known on hemoglobin interactions with the aminophospholipids, predominantly localized in the inner leaflet of erythrocytes, e.g., phosphatidylserine (PS), phosphatidylethanolamine (PE) in membranes containing phosphatidylcholine (PC). Effects of cholesterol, largely abundant in erythrocytes, have also not been studied in great details in earlier studies. This work therefore describes the study of the interactions of different hemoglobin variants HbA, HbE and HbF and the globin subunits of HbA with the two aminophospholipids in the presence and absence of cholesterol. Absorption measurements indicate preferential oxidative interaction of HbE and alpha-globin subunit with unilamellar vesicles containing PE and PS compared to normal HbA. Cholesterol was found to stabilize such oxidative interactions in membranes containing both the aminophospholipids. HbE and alpha-globin subunits were also found to induce greater leakage of membrane entrapped carboxyfluorescein (CF) using fluorescence measurements. HbE was found to induce fusion of membrane vesicles containing cholesterol and PE when observed under electron microscope. Taken together, these findings might be helpful in understanding the oxidative stress-related mechanism(s) involved in the premature destruction of erythrocytes in peripheral blood, implicated in the hemoglobin disorder, HbE/beta-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.     

A Study Assessing the Association of Glycated Hemoglobin A1C (HbA1C) Associated Variants with HbA1C,Chronic Kidney Disease and Diabetic Retinopathy in Populations of Asian Ancestry

Glycated hemoglobin A_1C (HbA_1C) level is used as a diagnostic marker for diabetes mellitus and a predictor of diabetes associated complications. Genome-wide association studies have identified genetic variants associated with HbA_1C level. Most of these studies have been conducted in populations of European ancestry. Here we report the findings from a meta-analysis of genome-wide association studies of HbA_1C levels in 6,682 non-diabetic subjects of Chinese, Malay and South Asian ancestries. We also sought to examine the associations between HbA_1C associated SNPs and microvascular complications associated with diabetes mellitus, namely chronic kidney disease and retinopathy. A cluster of 6 SNPs on chromosome 17 showed an association with HbA_1C which achieved genome-wide significance in the Malays but not in Chinese and Asian Indians. No other variants achieved genome-wide significance in the individual studies or in the meta-analysis. When we investigated the reproducibility of the findings that emerged from the European studies, six loci out of fifteen were found to be associated with HbA_1C with effect sizes similar to those reported in the populations of European ancestry and P-value ≤ 0.05. No convincing associations with chronic kidney disease and retinopathy were identified in this study.     

Polymorphic Variants of Human Rhodanese Exhibit Differences in Thermal Stability and Sulfur Transfer Kinetics

Rhodanese is a component of the mitochondrial H₂S oxidation pathway. Rhodanese catalyzes the transfer of sulfane sulfur from glutathione persulfide (GSSH) to sulfite generating thiosulfate and from thiosulfate to cyanide generating thiocyanate. Two polymorphic variations have been identified in the rhodanese coding sequence in the French Caucasian population. The first, 306A→C, has an allelic frequency of 1% and results in an E102D substitution in the encoded protein. The second polymorphism, 853C→G, has an allelic frequency of 5% and leads to a P285A substitution. In this study, we have examined differences in the stability between wild-type rhodanese and the E102D and P285A variants and in the kinetics of the sulfur transfer reactions. The Asp-102 and Ala-285 variants are more stable than wild-type rhodanese and exhibit k_cat/K_m,CN values that are 17- and 1.6-fold higher, respectively. All three rhodanese forms preferentially catalyze sulfur transfer from GSSH to sulfite, generating thiosulfate and glutathione. The k_cat/K_m,sulfite values for the variants in the sulfur transfer reaction from GSSH to sulfite were 1.6- (Asp-102) and 4-fold (Ala-285) lower than for wild-type rhodanese, whereas the k_cat/K_m,GSSH values were similar for all three enzymes. Thiosulfate-dependent H₂S production in murine liver lysate is low, consistent with a role for rhodanese in sulfide oxidation. Our studies show that polymorphic variations that are distant from the active site differentially modulate the sulfurtransferase activity of human rhodanese to cyanide versus sulfite and might be important in differences in susceptibility to diseases where rhodanese dysfunction has been implicated, e.g. inflammatory bowel diseases.     

Differential Expression and Functionality of TRPA1 Protein Genetic Variants in Conditions of Thermal Stimulation

The role of genetic modifications of the TRPA1 receptor has been well documented in inflammatory and neuropathic pain. We recently reported that the E179K variant of TRPA1 appears to be crucial for the generation of paradoxical heat sensation in pain patients. Here, we describe the consequences of the single amino acid exchange at position 179 in the ankyrin repeat 4 of human TRPA1. TRPA1 wild type Lys-179 protein expressed in HEK cells exhibited intact biochemical properties, inclusive trafficking into the plasma membrane, formation of large protein complexes, and the ability to be activated by cold. Additionally, a strong increase of Lys-179 protein expression was observed in cold (4 °C) and heat (49 °C)-treated cells. In contrast, HEK cells expressing the variant Lys-179 TRPA1 failed to get activated by cold possibly due to the loss of ability to interact with other proteins or other TRPA1 monomers during oligomerization. In conclusion, the detailed understanding of TRPA1 genetic variants might provide a fruitful strategy for future development of pain treatments.     

Genetic Variants of Hemoglobin and Other Blood Proteins in the San Francisco Bay Area

Studies of blood genetics in “normal healthy” persons and patients of different racial groups in the San Francisco Bay Area were carried out from January 1965 to April 1968. They show that diseases due to genetic abnormalities of blood are fairly common in this region. Frequencies for abnormal hemoglobins and erythrocyte enzyme deficiencies and variants were recorded and it was noted that abnormalities in hemoglobin metabolism that may lead to mild or severe clinical and hematological symptoms proved rather common. Accompanying other disease conditions, they may cause difficulties in diagnosis. Several diseases due to or associated with different enzyme abnormalities were encountered.     

Incidence of Sickle Cell Disease and Other Hemoglobin Variants in 10,095 Lebanese Neonates

Hemoglobinopathies are highly prevalent diseases and impose a public health burden. Early diagnosis and treatment can ameliorate the course of these diseases and improve survival. Despite purported high incidence of hemoglobinopathies in Lebanon, there are no nationwide screening programs. In this study, newborn screening utilizing high pressure liquid chromatography was executed in all public hospitals across Lebanon between 2010 and 2013. All newborns with an abnormal hemoglobin (Hb) were offered genetic counseling and all those with disease were enrolled in comprehensive hemoglobinopathy clinics. Among newborns, 2.1% were found to have an abnormal Hb variant with sickle Hb being the most common while 0.1% were found to have sickle cell disease (SCD). The majority of those with SCD had non-Lebanese origins. The most common causes of hospitalizations in infants with SCD were acute splenic sequestration and pain crises. No bacteremia or other life threatening infections were noted. At a median follow up 14 months (follow up range 7 to 34 months), all children with disease are alive and compliant with treatment. Systematic screening for SCD and other Hb variants was shown to be feasible, cost effective, and of accurate predictive value. This program was also clinically effective because it led to the identification of babies with disease and to providing them with free early multidisciplinary care. Conclusively, a newborn screening program should be implemented across Lebanon to detect hemoglobinopathies and initiate early therapeutic and preventive strategies and genetic counseling.     

Ethnic Variation in the Correlation Between Fasting Glucose Concentration and Glycated Hemoglobin (HbA1C)

ObjectiveWe aimed to determine the relationship between fasting serum glucose (FSG) concentration and glycated hemoglobin-Alc (HbAlc) in the 3 ethnicities in Singapore after adjustment for demographic and therapeutic variables.MethodsFasting serum glucose (FSG), HbAlc, and serum creatinine levels were simultaneously sampled from 575 patients with diabetes (389 Chinese, 97 Indians, 89 Malays) in this cross-sectional study between January and May 2008, and the results were subjected to multivariate linear regression analysis.ResultsWe found a significant interaction between FSG and ethnicity on HbAlc. The correlation between FSG and HbAlc among Chinese subjects was 0.25 (95% confidence interval [CI]:0.2-0.3) relative to the Malays (0.38, 95% CI: 0.30-0.45) after adjustment for age; gender; serum creatinine concentrations; body mass index (BMI); duration of diabetes; use of sulfonylureas, metformin, and insulin; and hemoglobin (Hb) and red cell indices (P = .005). Hence, for a given FSG, the predicted HbAlc will be higher in Malays compared to Chinese subjects.We did not observe a statistically significant difference between Indians and Malays with respect to the correlation between FSG and HbAlc.ConclusionWe showed a higher correlation between HbAlc and FSG in Malay subjects relative to the Chinese in this cohort. The ethnic variation in the HbAlc-FSG relationship may be related to differences in percentage contribution by the FSG to overall HbAlc among ethnic groups. Future studies using continuous glucose monitoring (CGM) to elucidate the relative contributions by FSG and postprandial glucose (PPG) to the daily blood glucose profile and the overall HbA1c by ethnicity are required. (Endocr Pract. 2013;19:812-817)     

Peroxidase Activity and Involvement in the Oxidative Stress Response of Roseobacter denitrificans Truncated Hemoglobin

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm^-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (k_cat/K_m) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pK_a value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.     

Storage Stability and Thermal Stability of Hordeumin,an Anthocyanin Pigment from Barley

Hordeumin stored at –40 to –80^oC in 1% HCI–methanol suffered neither from color reduction nor discoloration. After heating at 80°C for 60 min, hordeumin showed a pigment retention rate of 100%. This characteristic is because the pigment is a composite high-molecular weight compound consisting of anthocyanins and polyphenols, It was determined, however, that discoloration and browning occurred more rapidly than color reduction during storage and heating of the pigment.     

A Large Cohort of Hemoglobin Variants in Thailand: Molecular Epidemiological Study and Diagnostic Consideration

Background

Hemoglobin (Hb) variants are structurally inherited changes of globin chains. Accurate diagnoses of these variants are important for planning of appropriate management and genetic counseling. Since no epidemiological study has been conducted before, we have investigated frequencies, molecular and hematological features of Hb variants found in a large cohort of Thai subjects.

Materials and Methods

Study was conducted on 26,013 unrelated subjects, inhabiting in all geographical parts of Thailand over a period of 11 years from January 2002-December 2012. Hb analysis was done on high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). Mutations causing Hb variants were identified using PCR and related techniques.

Results

Among 26,013 subjects investigated, 636 (2.4%) were found to carry Hb variants. Of these 636 subjects, 142 (22.4%) carried α-chain variants with 13 different mutations. The remaining included 451 (70.9%) cases with 16 β-chain variants, 37 (5.8%) cases with Hb Lepore (δβ-hybrid Hb) and 6 (0.9%) cases with a single δ-chain variant. The most common α-globin chain variant was the Hb Q-Thailand (α^{74GAC-CAC, Asp-His}) which was found in 101 cases (15.8%). For β-globin chain variants, Hb Hope (β^{136GGT-GAT, Gly-Asp}) and Hb Tak (β^{146+AC, Ter-Thr}) are the two most common ones, found in 121 (19.0%) and 90 (14.2%) cases, respectively. Seven Hb variants have never been found in Thai population. Hb analysis profiles on HPLC or CE of these variants were illustrated to guide presumptive diagnostics.

Conclusions

Hb variants are common and heterogeneous in Thai population. With varieties of thalassemias and hemoglobinopathies in the population, interactions between them leading to complex syndromes are common and render their diagnoses difficult in routine practices. Knowledge of the spectrum, molecular basis, genotype-phenotype correlation and diagnostic features should prove useful for prevention and control of the diseases in the region.     

糖化血红蛋白（HbA1c）检测技术与标准化研究进展

糖尿病是世界性疾病,更是严重的公共卫生问题。世界卫生组织（World Health Organization, WHO）将糖化血红蛋白A_1c（glycated hemoglobin A_1c,HbA_1c）确定为糖尿病诊断标准,这对于糖尿病的诊断、监测和治疗具有重要临床意义。近年来,国内外开展了大量有关HbA_1c实验室检测方法与标准化的相关技术研究工作,形成了一系列检测方法和标准体系,取得了一定成果。介绍了具有代表性的HbA_1c实验室检测技术及国内外HbA_1c标准化研究进程,并对当前存在的技术难题进行了分析和展望,以期有助于临床实验室选择合适的检测方法,并推进我国HbA_1c标准化工作的发展。     

Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability,Nucleotide Hydrolysis,and Helicase Activity

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg²⁺ ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.     

Comparative Cytotoxicity,Oxidative Stress,and Cytokine Secretion Induced by Two Cyanotoxin Variants,Microcystin LR and RR,in Human Intestinal Caco‐2 Cells

While MC‐LR and MC‐RR share significant structural similarity, MC‐RR is less cytotoxic than MC‐LR. In the current study, we have compared the effects of MC‐LR and MC‐RR in Caco‐2 cells by evaluating cytotoxicity, oxidative stress (reactive oxygen species production), and the cellular proinflammatory response (IL‐6 and IL‐8 production). Following treatment with 100 µM microcystins (MC), cytotoxicity was two‐fold greater with MC‐LR as compared to MC‐RR after 24 h exposure. Whereas the reactive oxygen species production and IL‐6 secretion were similar following a 24‐h treatment with either MC, 100 µM MC‐LR induced a five‐fold greater IL‐8 secretion when compared to MC‐RR. Our study has demonstrated that, although both MC‐LR and MC‐RR induced some cytotoxicity in human intestinal cells, a major difference in IL‐8 production was observed between the two variants. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:253‐258, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21482