Globin Types Research Articles

Cytoglobin has potent superoxide dismutase function

Cytoglobin (Cygb) was discovered as a novel type of globin that is expressed in mammals; however, its functions remain uncertain. While Cygb protects against oxidant stress, the basis for this is unclear, and the effect of Cygb on superoxide metabolism is unknown. From dose-dependent studies of the effect of Cygb on superoxide catabolism, we identify that Cygb has potent superoxide dismutase (SOD) function. Initial assays using cytochrome c showed that Cygb exhibits a high rate of superoxide dismutation on the order of 108 M-1 ⋅ s-1 Spin-trapping studies also demonstrated that the rate of Cygb-mediated superoxide dismutation (1.6 × 108 M-1 ⋅ s-1) was only ∼10-fold less than Cu,Zn-SOD. Stopped-flow experiments confirmed that Cygb rapidly dismutates superoxide with rates within an order of magnitude of Cu,Zn-SOD or Mn-SOD. The SOD function of Cygb was inhibited by cyanide and CO that coordinate to Fe3+-Cygb and Fe2+-Cygb, respectively, suggesting that dismutation involves iron redox cycling, and this was confirmed by spectrophotometric titrations. In control smooth-muscle cells and cells with siRNA-mediated Cygb knockdown subjected to extracellular superoxide stress from xanthine/xanthine oxidase or intracellular superoxide stress triggered by the uncoupler, menadione, Cygb had a prominent role in superoxide metabolism and protected against superoxide-mediated death. Similar experiments in vessels showed higher levels of superoxide in Cygb-/- mice than wild type. Thus, Cygb has potent SOD function and can rapidly dismutate superoxide in cells, conferring protection against oxidant injury. In view of its ubiquitous cellular expression at micromolar concentrations in smooth-muscle and other cells, Cygb can play an important role in cellular superoxide metabolism.

Molecular epidemiological and hematological profile of thalassemia in the Dongguan Region of Guangdong Province, Southern China.

BackgroundThalassemia is a common inherited hematological disease in tropical and subtropical regions. This study aimed to investigate the mutation spectrum of thalassemia in the Dongguan region of southern China and comprehensively analyze hematologic features of thalassemia carriers with various types of globin mutations.MethodsA hematological screening including hematological indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), and mean corpuscular hemoglobin concentration (MCHC) was conducted in 19 442 people from Dongguan region, Guangdong province of China. Then, 4891 suspected thalassemia carriers were further investigated by genetic analysis of combined NGS and gap‐PCR.ResultsTotally, 2319 (11.9%) cases were diagnosed as carriers of thalassemia, of which 1483 cases (7.6%) were α‐thalassemia, 741 cases (3.8%) were β‐thalassemia, and 95 cases (0.5%) were co‐inheritance of α‐ and β‐thalassemia. In α‐thalassemia carriers, the phenotypic severity increases with the number of nonfunctional α‐globin genes. The patients with –SEA/αWSα genotype have less severe clinical phenotypes than those with other Hb H diseases. As for β‐thalassemia, the MCV and MCH in both β0 and β+ carriers are markedly reduced.ConclusionsThis is the first comprehensive molecular epidemiological survey and hematological profiling of thalassemia in Dongguan area. This study will be benefit for genetic counseling in the clinic and may help pediatricians to make a correct diagnosis of different types of thalassemia.

The Globin Gene Family in Arthropods: Evolution and Functional Diversity.

Globins are small heme-proteins that reversibly bind oxygen. Their most prominent roles in vertebrates are the transport and storage of O2 for oxidative energy metabolism, but recent research has suggested alternative, non-respiratory globin functions. In the species-rich and ecologically highly diverse taxon of arthropods, the copper-containing hemocyanin is considered the main respiratory protein. However, recent studies have suggested the presence of globin genes and their proteins in arthropod taxa, including model species like Drosophila. To systematically assess the taxonomic distribution, evolution and diversity of globins in arthropods, we systematically searched transcriptome and genome sequence data and found a conserved, widespread occurrence of three globin classes in arthropods: hemoglobin-like (HbL), globin X (GbX), and globin X-like (GbXL) protein lineages. These globin types were previously identified in protostome and deuterostome animals including vertebrates, suggesting their early ancestry in Metazoa. The HbL genes show multiple, lineage-specific gene duplications in all major arthropod clades. Some HbL genes (e.g., Glob2 and 3 of Drosophila) display particularly fast substitution rates, possibly indicating the evolution of novel functions, e.g., in spermatogenesis. In contrast, arthropod GbX and GbXL globin genes show high evolutionary stability: GbXL is represented by a single-copy gene in all arthropod groups except Brachycera, and representatives of the GbX clade are present in all examined taxa except holometabolan insects. GbX and GbXL both show a brain-specific expression. Most arthropod GbX and GbXL proteins, but also some HbL variants, include sequence motifs indicative of potential N-terminal acylation (i.e., N-myristoylation, 3C-palmitoylation). All arthropods except for the brachyceran Diptera harbor at least one such potentially acylated globin copy, confirming the hypothesis of an essential, conserved globin function associated with the cell membrane. In contrast to other animals, the fourth ancient globin lineage, represented by neuroglobin, appears to be absent in arthropods, and the putative arthropod orthologs of the fifth metazoan globin lineage, androglobin, lack a recognizable globin domain. Thus, the remarkable evolutionary stability of some globin variants is contrasted by occasional dynamic gene multiplication or even loss of otherwise strongly conserved globin lineages in arthropod phylogeny.

Evolutionary History of the Globin Gene Family in Annelids.

Animals depend on the sequential oxidation of organic molecules to survive; thus, oxygen-carrying/transporting proteins play a fundamental role in aerobic metabolism. Globins are the most common and widespread group of respiratory proteins. They can be divided into three types: circulating intracellular, noncirculating intracellular, and extracellular, all of which have been reported in annelids. The diversity of oxygen transport proteins has been underestimated across metazoans. We probed 250 annelid transcriptomes in search of globin diversity in order to elucidate the evolutionary history of this gene family within this phylum. We report two new globin types in annelids, namely androglobins and cytoglobins. Although cytoglobins and myoglobins from vertebrates and from invertebrates are referred to by the same name, our data show they are not genuine orthologs. Our phylogenetic analyses show that extracellular globins from annelids are more closely related to extracellular globins from other metazoans than to the intracellular globins of annelids. Broadly, our findings indicate that multiple gene duplication and neo-functionalization events shaped the evolutionary history of the globin family.

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

Globins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure-function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin. It is clear from the literature that E.coli is the most used expression system for expression and purification of recombinant globins. However, the major disadvantage of E. coli is the formation of insoluble aggregates. We experienced that, for more complex multi-domain globins, like the chimeric globin androglobin, it is recommended to switch to a higher eukaryotic expression system.

Functional diversification of sea lamprey globins in evolution and development

Agnathans have a globin repertoire that markedly differs from that of jawed (gnathostome) vertebrates. The sea lamprey (Petromyzon marinus) harbors at least 18 hemoglobin, two myoglobin, two globin X, and one cytoglobin genes. However, agnathan hemoglobins and myoglobins are not orthologous to their cognates in jawed vertebrates. Thus, blood-based O2 transport and muscle-based O2 storage proteins emerged twice in vertebrates from a tissue-globin ancestor. Notably, the sea lamprey displays three switches in hemoglobin expression in its life cycle, analogous to hemoglobin switching in vertebrates. To study the functional changes associated with the evolution and ontogenesis of distinct globin types, we determined O2 binding equilibria, type of quaternary assembly, and nitrite reductase enzymatic activities of one adult (aHb5a) and one embryonic/larval hemoglobin (aHb6), myoglobin (aMb1) and cytoglobin (Cygb) of the sea lamprey. We found clear functional differentiation among globin types expressed at different developmental stages and in different tissues. Cygb and aMb1 have high O2 affinity and nitrite reductase activity, while the two hemoglobins display low O2 affinity and nitrite reductase activity. Cygb and aHb6 but not aHb5a show cooperative O2 binding, correlating with increased stability of dimers, as shown by gel filtration and molecular modeling. The high O2-affinity and the lack of cooperativity confirm the identity of the sea lamprey aMb1 as O2 storage protein of the muscle. The dimeric structure and O2-binding properties of sea lamprey and mammalian Cygb were very similar, suggesting a conservation of function since their divergence around 500million years ago.

Bindings of NO, CO, and O2 to multifunctional globin type dehaloperoxidase follow the 'sliding scale rule'.

Dehaloperoxidase-hemoglobin (DHP), a multifunctional globin protein, not only functions as an oxygen carrier as typical globins such as myoglobin and hemoglobin, but also as a peroxidase, a mono- and dioxygenase, peroxygenase, and an oxidase. Kinetics of DHP binding to NO, CO, and O2 were characterized for wild-type DHP A and B and the H55D and H55V DHP A mutants using stopped-flow methods. All three gaseous ligands bind to DHP significantly more weakly than sperm whale myoglobin (SWMb). Both CO and NO bind to DHP in a one-step process to form a stable six-coordinate complex. Multiple-step NO binding is not observed in DHP, which is similar to observations in SWMb, but in contrast with many heme sensor proteins. The weak affinity of DHP for O2 is mainly due to a fast O2 dissociation rate, in accordance with a longer εN-Fe distance between the heme iron and distal histidine in DHP than that in Mb, and an open-distal pocket that permits ligand escape. Binding affinities in DHP show the same 3-4 orders separation between the pairs NO/CO and CO/O2, consistent with the 'sliding scale rule' hypothesis. Strong gaseous ligand discrimination by DHP is very different from that observed in typical peroxidases, which show poor gaseous ligand selectivity, correlating with a neutral proximal imidazole ligand rather than an imidazolate. The present study provides useful insights into the rationale for DHP to function both as mono-oxygenase and oxidase, and is the first example of a globin peroxidase shown to follow the 'sliding scale rule' hypothesis in gaseous ligand discrimination.

The Full Globin Repertoire of Turtles Provides Insights into Vertebrate Globin Evolution and Functions

Globins are small heme proteins that play an important role in oxygen supply, but may also have other functions. Globins offer a unique opportunity to study the functional evolution of genes and proteins. We have characterized the globin repertoire of two different turtle species: the Chinese softshell turtle (Pelodiscus sinensis) and the western painted turtle (Chrysemys picta bellii). In the genomes of both species, we have identified eight distinct globin types: hemoglobin (Hb), myoglobin, neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Therefore, along with the coelacanth, turtles are so far the only known vertebrates with a full globin repertoire. This fact allows for the first time a comparative analysis of the expression of all eight globins in a single species. Phylogenetic analysis showed an early divergence of neuroglobin and globin X before the radiation of vertebrates. Among the other globins, cytoglobin diverged first, and there is a close relationship between myoglobin and globin E; the position of globin Y is not resolved. The globin E gene was selectively lost in the green anole, and the genes coding for globin X and globin Y were deleted in chicken. Quantitative real-time reverse transcription polymerase chain reaction experiments revealed that myoglobin, neuroglobin, and globin E are highly expressed with tissue-specific patterns, which are in line with their roles in the oxidative metabolism of the striated muscles, the brain, and the retina, respectively. Histochemical analyses showed high levels of globin E in the pigment epithelium of the eye. Globin E probably has a myoglobin-like role in transporting O2 across the pigment epithelium to supply in the metabolically highly active retina.

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell (RBC) transfusion. When ES cell-derived erythroid cells are generated using embryoid bodies, these cells predominantly express embryonic type ε-globin, with lesser fetal type γ-globin and small amounts of adult type β-globin; however, no β-globin expression is detected in iPS cell-derived erythroid cells. Recently, the ES cell-derived sac (ES sac) was reported to express hemangioblast markers and could generate functional platelets (Takayama, Blood. 2008). We previously demonstrated that erythroid cells were also efficiently generated via the ES sac (2013 ASH). We extend this work to evaluate globin expression in ES sac-derived erythroid cells.We generated ES sacs from human H1 ES or iPS cells using VEGF for 15 days, as previously described. The spherical cells within ES sacs were harvested and cultured on OP9 feeder cells for 2 days, and the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002). The globin types expressed in erythroid cells were evaluated by RT-qPCR and hemoglobin electrophoresis.When hematopoietic cell-stimulating cytokines (SCF, FLT3L, TPO, IL3, EPO, and BMP4) were added in ES sac cultures on day 9-15, we observed 1.4-fold greater amounts of GPA+ erythroid cells (p<0.05) and 1.3-fold lower ε-globin expression in ES sac-derived erythroid cells (p<0.05), suggesting that cytokine stimulation might induce more hematopoietic/stem progenitor cells (HSPC) which can be differentiated to γ- or β-globin-expressing erythroid cells. Thus, we hypothesized that the ES sac contains both primitive and definitive erythroid progenitor cells capable of ε-globin-expression or γ- or β-globin-expression upon differentiation; respectively, and that these progenitors are selectable based upon surface markers of erythroid progenitor cells or HSPCs.To investigate whether primitive erythropoiesis is switched to definitive erythropoiesis during ES sac maturation, we evaluated spherical cells within the ES sac on day 9, 12, 15, and 18 after ES sac culture. A high percentage of GPA+ erythroid cells (29.2±3.7%) were observed on as early as day9. At that time point, almost no CD34+CD45+ HSPCs were present; however, the number increased upon further ES sac maturation until day 15 (6.8±1.6%). Cells further differentiated in erythroid culture had lower ε-globin expression and higher β-globin expression (up to 13.8±1.5%) when harvested from the ES sac at later time points. These data suggest that more matured ES sacs favor less primitive erythropoiesis and more definitive erythropoiesis.On day 15, the ES sacs contained a high percentage of GPA+(CD34-) erythroid cells (68.7±4.0%) and relatively lower amounts of CD34+(GPA-) HSPCs (16.7±2.1%). Therefore, we separated GPA+ and GPA- spherical cells from ES sac by magnetic selection before further erythroid differentiation, which resulted in higher ε-globin expression (43.0±16.6% vs 4.4±1.2%, p<0.01) and lower β-globin expression (7.6±5.3x10e-7% vs 19.8±2.7%, p<0.01) from the GPA+ cell fraction. In contrast, after erythroid differentiation from CD34+ or CD34- sorted spherical cells, lower ε-globin expression (3.7±0.3% vs 17.1±0.9%, p<0.01) and higher β-globin expression (17.4±0.7 % vs 0.9±0.4 %, p<0.01) were observed from the CD34+ cell fraction. These data suggest that the ES sac contains both primitive erythroid progenitor cells in the CD34- or GPA+ cell fraction and definitive erythroid progenitor cells in the CD34+ or GPA- cell fraction.In addition, iPS sac-derived erythroid cells were generated from 2 clones of fibroblast-derived iPS cells, which demonstrated 9.0±2.6% (clone #1) and 7.3±3.7% (clone #2) of β-globin expression. These data demonstrate that similar to ES sac-derived erythroid cells, iPS cell-derived erythroid cells can produce β-globin when differentiated from iPS sacs.In conclusion, we demonstrate that human ES and iPS cells can generate both primitive and definitive erythroid progenitor cells when differentiated in ES/iPS sac. CD34 or GPA discriminates between primitive and definitive erythroid progenitor cells in ES sac. The presented differentiation and selection strategy represent an important step to develop in vitro RBC production system from pluripotent stem cells. DisclosuresNo relevant conflicts of interest to declare.

Function and evolution of vertebrate globins.

Globins are haem-proteins that bind O2 and thus play an important role in the animal's respiration and oxidative energy production. However, globins may also have other functions such as the decomposition or production of NO, the detoxification of reactive oxygen species or intracellular signalling. In addition to the well-investigated haemoglobins and myoglobins, genome sequence analyses have led to the identification of six further globin types in vertebrates: androglobin, cytoglobin, globin E, globin X, globin Y and neuroglobin. Here, we review the present state of knowledge on the functions, the taxonomic distribution and evolution of vertebrate globins, drawing conclusions about the functional changes underlying present-day globin diversity.

Generation Of Mature Erythroid Cells From Human Embryonic Stem Cell Derived Sacs and Optimization Of Erythroid Cell Generation

Human embryonic stem cells (hESCs) represent a potential alternative source for red blood cells (RBCs) for transfusion therapy. However, in vitro production of erythroid cells from hESCs has two critical limitations: low-efficiency of erythroid cell generation and absent production of adult type β globin. Previously, hESC-derived erythroid cells were generated by co-culture with feeder cells through embryoid body formation, and these erythroid cells produced embryonic type ε globin or fetal type γ globin. Recently, embryonic stem cell-derived sacs (ES sacs) were generated with hemangioblast markers and generated functional platelets (Takayama, Blood. 2008). We hypothesized that hESC-derived β globin producing erythroid cells could be efficiently produced through the hemangioblast-like ES sac and developed new strategies to generate mature erythroid cells in vitro from hESCs using the ES sac.ES sacs were generated from H1 hESCs using ES sac media containing 15% fetal bovine serum supplemented with 20ng/ml VEGF for 14-16 days. Spherical cells in suspension in these ES sacs were harvested, and erythroid cells were generated using a modified human erythroid massive amplification culture for 2 weeks (Migliaccio, Blood cells Mol Dis. 2002). We evaluated total cell counts, hematopoietic stem/progenitor cell marker (CD34) expression, and erythroid cell marker (CD235a) expression. In addition, we analyzed globin types in erythroid cells by hemoglobin electrophoresis.After erythroid differentiation from suspension cells within ES sacs (containing 12.39% CD34+ cells), we generated CD235a+ erythroid cells (55.47%). Additionally, we confirmed morphology of erythroid cells by cytospin examination and erythroid colony formation by CFU assays using harvested suspension cells within the ES sac. To improve upon erythroid cell differentiation during ES sac production, we added SCF, IL-3, Flt-3L, TPO, and EPO to the ES sac media from day 9-15. The number of CD34+ cells increased 2.7 fold (19x10e3 cells vs. 7x10e3 cells) by supplementation with SCF, IL-3 and EPO while the number of CD235a+ cells increased 6.6 fold (239x10e3 cells vs. 36x10e3 cells) after erythroid differentiation. These data suggest that cytokine stimulation can induce erythroid differentiation during ES sac production.We then hypothesized that efficient erythroid cell generation might be further promoted by shifting the balance between hematopoietic cell differentiation and endothelial cell differentiation in the hemangioblast-like ES sac. Thus, we added BMP4 to ES sac media from day 6-15 to improve hematopoietic cell differentiation, and we compared no VEGF with normal concentration VEGF (20ng/ml) from day 0-15 to reduce endothelial cell differentiation. After supplementation with BMP4, CD34+ cell numbers increased 3.1 fold (125x10e3 cells vs. 40x10e3 cells) among suspension cells in the ES sac, while the number of CD235a+ cells increase 2.6 fold (646x10e3 cells vs. 250x10e3 cells) after erythroid differentiation. In the no VEGF condition, the number of CD34+ cells increased 3.0 fold (118x10e3 cells vs. 40x10e3 cells) among suspension cells in ES sac, while the number of CD235a+ cells increased 2.8 fold (710x10e3 cells vs. 250x10e3 cells). These data suggest that erythroid differentiation during ES sac production was induced by not only stimulating hematopoietic cell differentiation but also suppressing endothelial cell differentiation.Finally, we evaluated hemoglobin types among hESC-derived erythroid cells differentiated from ES sacs without cytokine supplementation. We detected embryonic type hemoglobin (27.9%), fetal type hemoglobin (61.3%), and adult type hemoglobin (10.8%) by hemoglobin electrophoresis. These data suggests that ES sac-derived erythroid cells produce more adult type hemoglobin as compared to previous strategies.In conclusion, we demonstrated for the first time that erythroid cells can be efficiently generated from hESCs derived ES sacs and further improved by cytokine supplementation. Our data suggest that ES sacs have fractions of definitive hematopoiesis which can induce generation of mature red blood cells producing adult type hemoglobin. Disclosures:No relevant conflicts of interest to declare.

Conservation of globin genes in the “living fossil” Latimeria chalumnae and reconstruction of the evolution of the vertebrate globin family

The (hemo-)globins are among the best-investigated proteins in biomedical sciences. These small heme-proteins play an important role in oxygen supply, but may also have other functions. In addition to well known hemoglobin and myoglobin, six other vertebrate globin types have been identified in recent years: neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Analyses of the genome of the “living fossil” Latimeria chalumnae show that the coelacanth is the only known vertebrate that includes all eight globin types. Thus, Latimeria can also be considered as a “globin fossil”. Analyses of gene synteny and phylogenetic reconstructions allow us to trace the evolution and the functional changes of the vertebrate globin family. Neuroglobin and globin X diverged from the other globin types before the separation of Protostomia and Deuterostomia. The cytoglobins, which are unlikely to be involved in O2 supply, form the earliest globin branch within the jawed vertebrates (Gnathostomata), but do not group with the agnathan hemoglobins, as it has been proposed before. There is strong evidence from phylogenetic reconstructions and gene synteny that the eye-specific globin E and muscle-specific myoglobin constitute a common clade, suggesting a similar role in intracellular O2 supply. Latimeria possesses two α- and two β-hemoglobin chains, of which one α-chain emerged prior to the divergence of Actinopterygii and Sarcopterygii, but has been retained only in the coelacanth. Notably, the embryonic hemoglobin α-chains of Gnathostomata derive from a common ancestor, while the embryonic β-chains – with the exception of a more complex pattern in the coelacanth and amphibians – display a clade-specific evolution. Globin Y is associated with the hemoglobin gene cluster, but its phylogenetic position is not resolved. Our data show an early divergence of distinct globin types in the vertebrate evolution before the emergence of tetrapods. The subsequent loss of globins in certain taxa may be associated with changes in the oxygen-dependent metabolism. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Recent advances in the molecular understanding of non-transfusion-dependent thalassemia

Thalassemias are a group of inherited autosomal recessive hematologic disorders that occur because of defects in the alpha (α)- and beta (β)-globin genes of adult hemoglobin (Hb). An imbalance in the synthesis of one or more of the globin chains can result in a wide spectrum of phenotypes depending on the type and amount of globin synthesized and additional genetic modifiers. In patients with thalassemia intermedia, a condition known as non-transfusion-dependent thalassemia (NTDT), transfusion requirements are absent or episodic. Non-transfusion-dependent thalassemia includes β-thalassemia intermedia, HbE β-thalassemia, and α-thalassemia intermedia, also known as Hb H disease. This article focuses on the molecular features and genetic mutations specific to NTDT.

Androglobin: A Chimeric Globin in Metazoans That Is Preferentially Expressed in Mammalian Testes

Comparative genomic studies have led to the recent identification of several novel globin types in the Metazoa. They have revealed a surprising evolutionary diversity of functions beyond the familiar O(2) supply roles of hemoglobin and myoglobin. Here we report the discovery of a hitherto unrecognized family of proteins with a unique modular architecture, possessing an N-terminal calpain-like domain, an internal, circular permuted globin domain, and an IQ calmodulin-binding motif. Putative orthologs are present in the genomes of many metazoan taxa, including vertebrates. The calpain-like region is homologous to the catalytic domain II of the large subunit of human calpain-7. The globin domain satisfies the criteria of a myoglobin-like fold but is rearranged and split into two parts. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom. Molecular evolutionary analyses indicate that this chimeric globin family is phylogenetically ancient and originated in the common ancestor to animals and choanoflagellates. In humans and mice, the gene is predominantly expressed in testis tissue, and we propose the name "androglobin" (Adgb). Expression is associated with postmeiotic stages of spermatogenesis and is insensitive to experimental hypoxia. Evidence exists for increased gene expression in fertile compared with infertile males.

Oxygen Supply from the Bird's Eye Perspective: GLOBIN E IS A RESPIRATORY PROTEIN IN THE CHICKEN RETINA

The visual process in the vertebrate eye requires high amounts of metabolic energy and thus oxygen. Oxygen supply of the avian retina is a challenging task because birds have large eyes, thick retinae, and high metabolic rates but neither deep retinal nor superficial capillaries. Respiratory proteins such as myoglobin may enhance oxygen supply to certain tissues, and thus the mammalian retina harbors high amounts of neuroglobin. Globin E (GbE) was recently identified as an eye-specific globin of chicken (Gallus gallus). Orthologous GbE genes were found in zebra finch and turkey genomes but appear to be absent in non-avian vertebrate classes. Analyses of globin phylogeny and gene synteny showed an ancient origin of GbE but did not help to assign it to any specific globin type. We show that the photoreceptor cells of the chicken retina have a high level of GbE protein, which accumulates to ∼10 μM in the total eye. Quantitative real-time RT-PCR revealed an ∼50,000-fold higher level of GbE mRNA in the eye than in the brain. Spectroscopic analysis and ligand binding kinetics of recombinant chicken GbE reveal a penta-coordinated globin with an oxygen affinity of P(50) = 5.8 torrs at 25 °C and 15 torrs at 41 °C. Together these data suggest that GbE helps to sustain oxygen supply to the avian retina.

Haemoglobin in normal and neoplastic canine mammary glands

Four types of globins for oxygen transport are known in vertebrates, and the haemoglobin is responsible for carrying oxygen in blood. In this study, we found that haemoglobin was also expressed in canine mammary glands. Samples were taken from 26 malignant mammary tumors, 16 normal mammary glands and 10 other normal tissues. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and mass spectrometry were used to investigate haemoglobin in mammary tissues. The results indicated that normal canine mammary glands expressed high levels of haemoglobin protein as shown by Coomassie blue staining. The identity of haemoglobin was confirmed by immunoblotting and mass spectrometry, and the mass spectrometry data revealed that both alpha-haemoglobin and beta-haemoglobin were expressed. Relative to normal mammary glands, the levels of haemoglobin expression in mammary tumors were lower. Our results also indicated that the haemoglobin was endogenously produced in mammary gland tissues and was not derived from the erythroid cells.

Erythropoietin-responsive sites in normal and malignant human lung tissues

Preliminary findings of various types of globin expressed in the respiratory bronchiolar and alveolar epithelium prompted us to compare the expression of erythropoietin (Epo) and its receptor (EpoR) in normal (healthy) human lung tissues with that in malignant lung tissues. The expression of Epo and EpoR was examined at the transcriptional and protein levels in normal and malignant lung tissues by reverse transcription-PCR, western blot, and immunohistochemical analyses. EpoR mRNA, but not Epo mRNA, was detected in all samples. In normal tissues, EpoR was detected in the mesothelium, chondrocytes, alveolar cells, vascular endothelial cells, smooth muscle fibers, macrophages, and neutrophils, while in malignant foci, the cancer cells of five malignant types showed various intensities of EpoR immunoreactivity. The pattern of staining of EpoR protein was generally stronger in the malignant tissues than in the normal samples. Phosphorylation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK-ERK1/2) was frequently seen in malignant cells, but not in the normal tissues, with the exception of macrophages. Based on the expression of Epo and EpoR mRNA with the EpoR in almost all cell components in normal tissues, we suggest that the normal lung may produce various types of globin through the autocrine and/or paracrine role of Epo. When the Epo signal is upregulated by hypoxic stress, the normal cells appear to transform into malignant cells and proliferate through activated MAPK signaling.

Identification of high oxygen affinity hemoglobin variants in the investigation of patients with erythrocytosis

Erythrocytosis is a disorder of red cell production that can arise from several different causes. Familial cases have been classified into four groups (ECYT1-4) by Online Mendelian Inheritance in Man (OMIM) as listed at the website: http://www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM. Erythrocytosis associated with mutations in the erythropoietin receptor belong to group ECYT1 and arise from a primary cause that is intrinsic to the red cell.1 The other three categories of erythrocytosis, ECYT2-4, are secondary and are characterized by dysregulated erythropoietin (Epo) production resulting from defects in the oxygen sensing pathway.1 OMIM classification does not consider other secondary causes of erythrocytosis that are related to abnormal oxygen delivery such as hemoglobin (Hb) variants or 2,3-biphosphoglycerate mutase (BPG) deficiencies. Approximately 100 hemoglobin variants with a high affinity for oxygen have been described that cause a decrease in the supply of oxygen to tissues.2 These Hb variants are inherited in an autosomal dominant manner with an associated family history.2 Both α and β globin genes can be affected2 and the serum Epo would be inappropriately normal for the associated raised hematocrit. During the differential diagnosis of erythrocytosis, a high oxygen affinity Hb can be excluded by studying the oxygen binding properties of freshly drawn blood.2–4 Blood gas analyzers available in all hospitals will calculate P50 values. High affinity Hbs cause a left shift in the oxygen dissociation curve and thus lower the P50 value. Not all high affinity Hb variants are detected by routine Hb electrophoresis and isoelectric focusing in polyacry-lamide and hence a number are misdiagnosed. Over the last decade we have developed a data base of erythrocytosis patients referred from centers throughout the UK and Ireland.5 Before patients can be included in the registry all secondary causes of erythrocytosis should be excluded. During an audit of the data base we decided it may be useful to confirm the absence of high oxygen affinity Hb variants. Patients with a strong family history and serum Epo within the reference range or above were selected for β globin gene sequencing. Those patients negative for a β chain variant were further screened for an alpha globin gene variant. Four different high oxygen affinity Hb variants, Olympia (β20Va→Met),1,6,7 Pierre-Benite (β90Glu→Asp),6,8 Santa Clara (β97His→Asn)6,9 and Heathrow (β103Phe→Leu),6,10 were detected in 6 families from a data base of 205 individuals giving a prevalence of 3%. All are electrophoretically silent and hence difficult to detect by routine laboratory tests.6 Variants can be confirmed by high performance liquid chromatography or by mass spectrometry and identified by PCR-direct sequencing. 6 The hematological indices of the patients are listed in Table 1. In all cases the hematocrit and the hemoglobin level was raised, except for UPN224 which was found to be iron deficient. White blood cell and platelet counts were in the normal range. The serum Epo level was either elevated or inappropriately normal for the elevated Hb. Thrombotic events have been reported for only Patients 214 and 225. Table 1. Hematologic indices of erythrocytosis patients with hemoglobin variants. The hemoglobin tetramer is composed of 2 chains of α and 2 chains of β globin arranged in two identical halves, containing one chain of each globin type. As Hb transfers from the low oxygen affinity state (known as T or tense) to the high affinity state (R or relaxed) there is an accompanying movement within the α1β2 subunit.1 Many of the amino acid changes associated with a high affinity phenotype map to the α1β2 interface or the C-terminal of the β chain.1 These mutations prevent the transition of the Hb molecule to the T state and hence are unable to release the bound oxygen molecules. The Santa Clara variant cannot achieve the T state9 while Pierre-Benite is thought to stabilize Hb in the T quaternary structure8,11 and Hb Olympia12 affects the surface of the protein causing it to self-associate. Other mutations cause structural alteration of the hemepocket. The presence of several aromatic amino acids around the hememoiety is crucial for the correct orientation of the hemegroup. Replacement of Phe103 with Leu (Hb Heathrow) causes the affinity of the hemegroup for oxygen to be increased.1 During the differential diagnosis of erythrocytosis the first step would be the calculation of P50 using the routine blood gas analyzer. In those patients with a low value for P50 sequencing of the globin gene to establish the actual variant would be performed. In the absence of P50 values, as with patients in this study, it is important to eliminate the possibility of Hb variants by sequencing t he globin genes, which confirmed 6 positive cases on the registry.

A case of hereditary persistence of fetal haemoglobin

Thalassaemia syndromes comprise a large clinical spectrum and a well recognized observation is the inverse link between clinical severity and quantity of fetal haemoglobin (HbF) present 1 . It is now known that three types of globin that produce haemoglobin (Hb) change from e-globin (epsilone) to g-globin (gamma) in the fetus and to b-globin (beta) around birth. This process of change is termed “globin switching”. Rarely, due to a genetic defect, the gglobin gene continues to function, producing gamma chains. Therefore HbF is present throughout life giving rise to the condition hereditary persistence of fetal haemoglobin (HPFH) 2 . Such persons are often asymptomatic and lead normal lives. Persons with thalassaemia major who have concomitant HPFH have low transfusion requirements due to the persistence of HbF, making up for the deficiency of b-globin 3 . HPFH provides a basis for a genetic approach to cure haemoglobinapathies in the future.

What is the function of neuroglobin?

For a long time, haemoglobin and myoglobin had been assumed to represent the only globin types of vertebrates. In 2000, however, we discovered a third globin type by mining the genome sequence data. Based on a preferential expression in the nervous system, this globin is referred to as neuroglobin. Despite nine years of research, its function is still uncertain and a number of hypotheses have been put forward. Neuroglobin enhances cell viability under hypoxia and under various types of oxidative stress in transgenic systems, but does not appear to be strongly upregulated in response to stress. A close phylogenetic relationship with invertebrate nerve globins and its positive correlation with the oxidative metabolism and mitochondria suggest a role in O(2) supply. In vitro studies and cell culture experiments imply that neuroglobin may detoxify reactive oxygen or nitric oxide. Still other studies propose neuroglobin as being part of a signalling chain that transmits the redox state of the cell or that inhibits apoptosis. Although some functions are more probable than others, we conclude that it is still too early to definitively decide what may be the physiological role(s) of neuroglobin in vertebrates. Nevertheless, there is no doubt that neuroglobin has an essential, conserved function and is beneficial to neurons.