Duplicated cytoglobin genes in teleost fishes

Cytoglobin is a recently discovered myoglobin-related O2-binding protein of vertebrates with uncertain function. It occurs as single-copy gene in mammals. Here, we demonstrate the presence of two paralogous cytoglobin genes (Cygb-1 and Cygb-2) in the teleost fishes Danio rerio, Oryzias latipes, Tetraodon nigroviridis, and Takifugu rubripes. The globin-typical introns at positions B12.2 and G7.0 are conserved in both genes, whereas the C-terminal exon found in mammalian cytoglobin is absent in the fish genes. Phylogenetic analyses show that the two cytoglobin genes diverged early in teleost evolution. This is confirmed by gene synteny analyses, which suggest a large-scale duplication event. …

Molecular and Functional Characterisation of Hemocyanin of the Giant African Millipede Archispirostreptus gigas

SummaryIn contrast to other terrestrial arthropods where gaseous O2 that fuels aerobic metabolism diffuses to the tissues in tracheal tubes, and most other metazoans where O2 is transported to tissues by circulating respiratory proteins, the myriapods (millipedes and centipedes) strikingly have tracheal systems as well as circulating hemocyanin (Hc). In order to elucidate the evolutionary origin and biological significance of millipede Hc we report the molecular structure (subunit composition and amino acid sequence) of multimeric (36-mer) Hc from the forest-floor dwelling giant African millipede Archispirostreptus gigas and its allosteric oxygen binding properties under various physico-che…

A Globin Gene of Ancient Evolutionary Origin in Lower Vertebrates: Evidence for Two Distinct Globin Families in Animals

Hemoglobin, myoglobin, neuroglobin, and cytoglobin are four types of vertebrate globins with distinct tissue distributions and functions. Here, we report the identification of a fifth and novel globin gene from fish and amphibians, which has apparently been lost in the evolution of higher vertebrates (Amniota). Because its function is presently unknown, we tentatively call it globin X (GbX). Globin X sequences were obtained from three fish species, the zebrafish Danio rerio, the goldfish Carassius auratus, and the pufferfish Tetraodon nigroviridis, and the clawed frog Silurana tropicalis. Globin X sequences are distinct from vertebrate hemoglobins, myoglobins, neuroglobins, and cytoglobins.…

A vertebrate globin expressed in the brain.

Haemoglobins and myoglobins constitute related protein families that function in oxygen transport and storage in humans and other vertebrates. Here we report the identification of a third globin type in man and mouse. This protein is predominantly expressed in the brain, and therefore we have called it neuroglobin. Mouse neuroglobin is a monomer with a high oxygen affinity (half saturation pressure, P50 approximately 2 torr). Analogous to myoglobin, neuroglobin may increase the availability of oxygen to brain tissue. The human neuroglobin gene (NGB), located on chromosome 14q24, has a unique exon-intron structure. Neuroglobin represents a distinct protein family that diverged early in metaz…

The respiratory proteins of insects

For a long time, respiratory proteins have been considered unnecessary in most insects because the tracheal system was thought to be sufficient for oxygen supply. Only a few species that survive under hypoxic conditions were known exceptions. However, recently it has become evident that (1) intracellular hemoglobins belong to the standard repertoire of insects and (2) that hemocyanin is present in many "lower" insects. Intracellular hemoglobins have been identified in Drosophila, Anopheles, Apis and many other insects. In all investigated species, hemoglobin is mainly expressed in the fat body and the tracheal system. The major Drosophila hemoglobin binds oxygen with high affinity. This hem…

Complete subunit sequences, structure and evolution of the 6 x 6-mer hemocyanin from the common house centipede, Scutigera coleoptrata.

Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6…

Human Brain Neuroglobin Structure Reveals a Distinct Mode of Controlling Oxygen Affinity

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O(2) supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a hexacoordinated heme. O(2) and CO bind to the heme iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold adapted to host the reversible bis-histidyl heme complex and an elongated protein matrix cavity, held to facilitate O(2) diffusion to the heme. The neuroglobin structure suggests that the classical globin fold is endowed …

Knockdown of Drosophila hemoglobin suggests a role in O2 homeostasis.

Almost all insects are equipped with a tracheal system, which appears to be sufficient for O2 supply even in phases of high metabolic activity. Therefore, with the exception of a few species dwelling in hypoxic habitats, specialized respiratory proteins had been considered unnecessary in insects. The recent discovery and apparently universal presence of intracellular hemoglobins in insects has remained functionally unexplained. The fruitfly Drosophila melanogaster harbors three different globin genes (referred to as glob1-3). Glob1 is the most highly expressed globin and essentially occurs in the tracheal system and the fat body. To better understand the functions of insect globins, the lev…

News from an Ancient World: Two Novel Astacin Metalloproteases from the Horseshoe Crab

In this work, we report the cloning, heterologous expression, and characterization of two novel astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus astacin) and LAST_MAM (Limulus astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphat…

Limulus polyphemus Hemocyanin: 10 Å Cryo-EM Structure, Sequence Analysis, Molecular Modelling and Rigid-body Fitting Reveal the Interfaces Between the Eight Hexamers

Abstract The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its 48 subunits are arranged as eight hexamers (1×6mers) that form the native 8×6mer in a nested hierarchy of 2×6mers and 4×6mers. This quaternary structure is established by eight subunit types (termed I, IIA, II, IIIA, IIIB, IV, V, and VI), of which only type II has been sequenced. Crystal structures of the 1×6mer are available, but for the 8×6mer only a 40 A 3D reconstruction exists. Consequently, the structural parameters of the 8×6mer are not firmly established, and the molecular inte…

A welcome shortage of breath

The respiratory systems of animals must guarantee an efficient oxygen supply. But it seems that, in some insects, they have evolved to restrict the flow of oxygen too.

Exploiting a list of protein sequences

Abstract: We describe a software program to help exploit a database of aligned protein sequences. In addition to the classical lists of sequences, a graphical representation is used to get a better overview of the information. As natural parameters, the type of amino acid and sequence position are used. Various plots or 3D representations are then updated. Examples are shown based on globin sequences from various species and on the abnormal human hemoglobins. The software should be of interest to protein engineers who need to know what variants are already known.

Origin and evolution of arthropod hemocyanins and related proteins.

Arthropod hemocyanins are large, multimeric, (n x 6) copper-containing proteins that deliver oxygen in the haemolymph of many chelicerate, crustacean, myriapod, and also possibly some insect species. The arthropod hemocyanins belong to a large protein superfamily that also includes the arthropod phenoloxidases, certain crustacean and insect storage proteins (pseudo-hemocyanins and hexamerins), and the insect hexamerin receptors. Here I summarise the present knowledge of the origin, functional adaptations, and evolution of these proteins. Arthropod and mollusc hemocyanins are, if at all, only distantly related. As early as in the arthropod stem line, the hemocyanins emerged from a phenoloxid…

Coupling of the heme and an internal disulfide bond in human neuroglobin

Neuroglobin displays a hexacoordination His-Fe-His in the absence of external ligands such as oxygen. The observed oxygen affinity therefore depends on the binding rates of both oxygen and the competing distal histidine. Furthermore, the binding properties depend on the presence of an internal disulfide bond. In the case of human neuroglobin, cysteines at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. For cytoglobin, the cysteine residues at positions A7 and GH4 may also form a disulfide bond. Mass spectrometry, ligand binding, and thiol accessibility studies were used to study the role influence of these disulfide bonds. Mutation of specific cysteines, or r…

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-bi…

Neuroglobin and Other Hexacoordinated Hemoglobins Show a Weak Temperature Dependence of Oxygen Binding

AbstractMouse and human neuroglobins, as well as the hemoglobins from Drosophila melanogaster and Arabidopsis thaliana, were recombinantly expressed in Escherichia coli, and their ligand-binding properties were studied versus temperature. These globins have a common feature of being hexacoordinated (via the distal histidine) under deoxy conditions, as evidenced by a large amplitude for the alpha absorption band at 560nm and the Soret band at 426nm. The transition from the hexacoordinated form to the CO bound species is slow, as expected for a replacement reaction Fe-His → Fe → FeCO. The intrinsic binding rates would indicate a high oxygen affinity for the pentacoordinated form, due to rapid…

Hyperthermal stability of neuroglobin and cytoglobin

Neuroglobin (Ngb) and cytoglobin (Cygb), recent additions to the globin family, display a hexa-coordinated (bis-histidyl) heme in the absence of external ligands. Although these proteins have the classical globin fold they reveal a very high thermal stability with a melting temperature (Tm) of 100 °C for Ngb and 95 °C for Cygb. Moreover, flash photolysis experiments at high temperatures reveal that Ngb remains functional at 90 °C. Human Ngb may have a disulfide bond in the CD loop region; reduction of the disulfide bond increases the affinity of the iron atom for the distal (E7) histidine, and leads to a 3 °C increase in the Tm for ferrous Ngb. A similar Tm is found for a mutant of human Ng…

Characterization of the hemoglobin of the backswimmer Anisops deanei (Hemiptera).

Abstract While O 2 -binding hemoglobin-like proteins are present in many insects, prominent amounts of hemoglobin have only been found in a few species. Backswimmers of the genera Anisops and Buenoa (Notonectidae) have high concentrations of hemoglobin in the large tracheal cells of the abdomen. Oxygen from the hemoglobin is delivered to a gas bubble and controls the buoyant density, which enables the bugs to maintain their position without swimming and to remain stationary in the mid-water zone where they hunt for prey. We have obtained the cDNA sequences of three Anisops deanei hemoglobin chains by RT-PCR and RACE techniques. The deduced amino acid sequences show an unusual insertion of a…

Globin genes are present in Ciona intestinalis.

The key position of the Ciona intestinalis basal to the vertebrate phylogenetic tree brings up the question of which respiratory proteins are used by the tunicate to facilitate oxygen transport and storage. The publication of the Ciona draft genome sequence suggests that globin genes are completely missing and that-like some molluscs and arthropods-the sea squirt uses hemocyanin instead of hemoglobin for respiration. However, we report here the presence and expression of at least four distinct globin gene/protein sequences in Ciona. This finding is in agreement with the ancestral phylogeny of the vertebrate globins. Moreover, it seems likely that the Ciona hemocyanin-like sequences have enz…

The human brain hexacoordinated neuroglobin three-dimensional structure

Neuroglobin, mainly expressed in vertebrate brain and retina, is a recently identified member of the globin superfamily. Augmenting O2 supply, neuroglobin promotes survival of neurons upon hypoxic injury, potentially limiting brain damage. In the absence of exogenous ligands, neuroglobin displays a six-coordinated heme. O2 and CO bind to the heme-iron, displacing the endogenous HisE7 heme distal ligand. Hexacoordinated human neuroglobin displays a classical globin fold, adapted to host the reversible bis-histidyl heme complex, and an elongated protein matrix cavity, held to facilitate O2 diffusion to the heme. The structure of neuroglobin suggests that the classical globin fold is endowed w…

New insight into the haemoglobin superfamily: preliminary crystallographic characterization of human cytoglobin.

Human cytoglobin, present in almost all tissue types, is a newly identified member of the Hb superfamily. A double mutant, having both cysteines replaced by serines, has been overexpressed in Escherichia coli, purified and crystallized. A highly redundant SAD data set has been collected at the haem Fe-atom absorption edge (lambda = 1.720 A) to 2.60 A resolution. The crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 46.8, b = 73.1, c = 98.9 A and two molecules per asymmetric unit. The anomalous difference Patterson map clearly reveals the position of the haem Fe-atom sites, thus paving the way for SAD structure determination.

The nerve hemoglobin of the bivalve mollusc Spisula solidissima

Abstract Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (μm). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mm), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analo…

Corrigendum to “Living with stress: Regulation of antioxidant defense genes in the subterranean, hypoxia-tolerant mole rat, Spalax” [Gene 500 (2012) 199–206]

Additional file 1 of Diversity, evolution, and function of myriapod hemocyanins

Table S1. List of primer sequences used in this study. (PDF 64 kb)

The hemoglobin genes of Drosophila

We recently reported the unprecedented occurrence of a hemoglobin gene (glob1) in the fruitfly Drosophila melanogaster. Here we investigate the structure and evolution of the glob1 gene in other Drosophila species. We cloned and sequenced glob1 genes and cDNA from D. pseudoobscura and D. virilis, and identified the glob1 gene sequences of D. simulans, D. yakuba, D. erecta, D. ananassae, D. mojavensis and D. grimshawi in the databases. Gene structure (introns in helix positions D7.0 and G7.0), gene synteny and sequence of glob1 are highly conserved, with high ds/dn ratios indicating strong purifying selection. The data suggest an important role of the glob1 protein in Drosophila, which may b…

When the brain goes diving: glial oxidative metabolism may confer hypoxia tolerance to the seal brain.

Deep diving mammals have developed strategies to cope with limited oxygen availability when submerged. These adaptations are associated with an increased neuronal hypoxia tolerance. Brain neurons of the hooded seal Cysto- phora cristata remain much longer active in hypoxic condi- tions than those of mice. To understand the cellular basis of neuronal hypoxia tolerance, we studied neuroglobin and cy- tochrome c in C. cristata brain. Neuroglobin, a respiratory protein typically found in vertebrate neurons, displays three unique amino acid substitutions in hooded seal. However, these substitutions unlikely contribute to a modulation of O2 affinity. Moreover, there is no significant difference i…

Identification, molecular cloning, and phylogenetic analysis of a non-respiratory pseudo-hemocyanin of Homarus americanus.

Copper-containing hemocyanins serve to transport oxygen in many arthropod species. Here I describe the identification and cDNA cloning of a structurally closely related non-respiratory pseudo-hemocyanin (PHc) of the American lobster, Homarus americanus. This protein has lost the ability to bind copper and, therefore, oxygen because a histidine residue in copper-binding site A is replaced by tyrosine. Like many arthropod hemocyanins, PHc forms a hexamer. It consists of two different subunit types of 660 and 661 amino acids, respectively, that share a 94.4% sequence identity. Whereas Homarus hemocyanin is produced in the hepatopancreas, PHc is synthesized by the ovaries and the heart tissue. …

The Globin Gene Repertoire of Lampreys: Convergent Evolution of Hemoglobin and Myoglobin in Jawed and Jawless Vertebrates

Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus )w hich revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin …

Localization of neuroglobin protein in the mouse brain.

Neuroglobin is a recently discovered vertebrate oxygen-binding respiratory protein. In situ hybridization data demonstrated that neuroglobin-mRNA is widely expressed in neuronal cells of the central and peripheral nervous systems as well as in endocrine cells. The present study was conducted to investigate the presence of neuroglobin protein in neurons of the mouse brain. A polyclonal antibody directed against a synthetic peptide of neuroglobin was raised in rabbits and affinity-purified. The specificity of the antibody was demonstrated by ELISA and preabsorption tests. We report here for the first time that neuroglobin is expressed on the protein level in many brain sites including cerebra…

High Pressure Enhances Hexacoordination in Neuroglobin and Other Globins

The techniques of high applied pressure and flash photolysis have been combined to study ligand rebinding to neuroglobin (Ngb) and tomato Hb, globins that may display a His-Fe-His hexacoordination in the absence of external ligands. High pressure induces a moderate decrease in the His association rate and a large decrease in His dissociation rate, thus leading to an enhancement of the overall His affinity. The overall structural difference between penta- and hexacoordinated globins may be rather small and can be overcome by external modifications such as high pressure. Over the pressure range 0.1-700 MPa (7 kbar), the globins may show a loss of over a factor of 100 in the amplitude of the b…

Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia.

Cytoglobin is a recently discovered vertebrate globin distantly related to myoglobin, and its function is unknown. Here we present the first detailed analysis of the distribution and expression of cytoglobin. Northern and Western blotting experiments show the presence of cytoglobin mRNA and protein in a broad range of tissues. Quantitative PCR demonstrates an up-regulation of cytoglobin mRNA levels in rat heart and liver under hypoxic conditions (22 and 44 h of 9% oxygen). Immunofluorescence studies with three antibodies directed against different epitopes of the protein consistently show cytoglobin in connective tissue fibroblasts as well as in hepatic stellate cells. Cytoglobin is also pr…

Additional file 6: of Diversity, evolution, and function of myriapod hemocyanins

Table S3. Expression of hemocyanin subunit mRNA in myriapods. (PDF 72 kb)

Subunit sequences of the 4 x 6-mer hemocyanin from the golden orb-web spider, Nephila inaurata. Intramolecular evolution of the chelicerate hemocyanin subunits

The transport of oxygen in the hemolymph of many arthropod and mollusc species is mediated by large copper-proteins that are referred to as hemocyanins. Arthropod hemocyanins are composed of hexamers and oligomers of hexamers. Arachnid hemocyanins usually form 4 x 6-mers consisting of seven distinct subunit types (termed a-g), although in some spider taxa deviations from this standard scheme have been observed. Applying immunological and electrophoretic methods, six distinct hemocyanin subunits were identified in the red-legged golden orb-web spider Nephila inaurata madagascariensis (Araneae: Tetragnathidae). The complete cDNA sequences of six subunits were obtained that corresponded to a-,…

The diversity and evolution of chelicerate hemocyanins

Abstract Background Oxygen transport in the hemolymph of many arthropod species is facilitated by large copper-proteins referred to as hemocyanins. Arthropod hemocyanins are hexamers or oligomers of hexamers, which are characterized by a high O2 transport capacity and a high cooperativity, thereby enhancing O2 supply. Hemocyanin subunit sequences had been available from horseshoe crabs (Xiphosura) and various spiders (Araneae), but not from any other chelicerate taxon. To trace the evolution of hemocyanins and the emergence of the large hemocyanin oligomers, hemocyanin cDNA sequences were obtained from representatives of selected chelicerate classes. Results Hemocyanin subunits from a sea s…

Human neuroglobin: crystals and preliminary X-ray diffraction analysis

Neuroglobin, a recently discovered member of the haemoglobin superfamily, is primarily expressed in the brain of humans and other vertebrates, where it has been proposed to enhance O(2) supply in response to hypoxia or ischaemia, protecting the neuron from hypoxic injury. Neuroglobin is the first example of a vertebrate haemoglobin in which a hexacoordinate haem geometry has been detected. A triple mutant (replacing three Cys residues) of human neuroglobin (151 amino acids) has been expressed in Escherichia coli, purified and crystallized in two crystal forms, the best of which diffracts to 1.95 A resolution using synchrotron radiation. The crystals belong to space group P2(1), with unit-ce…

Neuroglobin, cytoglobin, and myoglobin contribute to hypoxia adaptation of the subterranean mole rat Spalax.

The subterranean mole rat Spalax is an excellent model for studying adaptation of a mammal toward chronic environmental hypoxia. Neuroglobin (Ngb) and cytoglobin (Cygb) are O 2 -binding respiratory proteins and thus candidates for being involved in molecular hypoxia adaptations of Spalax . Ngb is expressed primarily in vertebrate nerves, whereas Cygb is found in extracellular matrix-producing cells and in some neurons. The physiological functions of both proteins are not fully understood but discussed with regard to O 2 supply, the detoxification of reactive oxygen or nitrogen species, and apoptosis protection. Spalax Ngb and Cygb coding sequences are strongly conserved. However, mRNA and …

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 fu…

Hypoxia induces a complex response of globin expression in zebrafish (Danio rerio).

SUMMARY Unlike most mammals, many fish species live and survive in environments with low or changing levels of oxygen. Respiratory proteins like hemoglobin or myoglobin bind or store oxygen, thus enhancing its availability to the respiratory chain in the mitochondria. Here we investigate by means of quantitative real-time PCR the changes of hemoglobin, myoglobin, neuroglobin,cytoglobin and globin X mRNA in zebrafish (Danio rerio) exposed to mild (PO2=∼8.6 kPa) or severe(PO2=∼4.1 kPa) hypoxia. Neuroglobin and myoglobin protein levels were investigated by western blotting. Whereas mild hypoxia caused only minor changes of mRNA levels, strong hypoxia enhanced mRNA levels of the control genes (…

Neuroglobin and cytoglobin: fresh blood to the vertebrate globin family

Neuroglobin and cytoglobin are two recently discovered members of the vertebrate globin family. Both are intracellular proteins endowed with hexacoordinated heme-Fe atoms, in their ferrous and ferric forms, and display O2 affinities comparable with that of myoglobin. Neuroglobin, which is predominantly expressed in nerve cells, is thought to protect neurons from hypoxic–ischemic injury. It is of ancient evolutionary origin, and is homologous to nerve globins of invertebrates. Cytoglobin is expressed in many different tissues, although at varying levels. It shares common ancestry with myoglobin, and can be traced to early vertebrate evolution. The physiological roles of neuroglobin and cytog…

Quaternary structure of the European spiny lobster (Palinurus elephas) 1x6-mer hemocyanin from cryoEM and amino acid sequence data.

Abstract Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8×6-mer) in the 75 kDa range. A 3D reconstruction of the 1×6-mer hemocyanin from the European spiny lobster Palinurus elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8 A resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as α-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (>80%) of the hemocyanin subunits from the European spiny lobster P. elephas and the American spiny lobster Panulirus interruptus . Comparison of…

Characterization of two globin genes from the malaria mosquito Anopheles gambiae: Divergent origin of nematoceran haemoglobins

The chironomid midges are the only insects that harbour true haemoglobin in their haemolymph. Here we report the identification of haemoglobin genes in two other nematoceran species. Two paralogous haemoglobin genes (glob1 and glob2) from the malaria mosquito Anopheles gambiae were cloned and sequenced. Furthermore, we identified two orthologous haemoglobin genes in the yellow fever mosquito Aedes aegypti. All four haemoglobins were predicted to be intracellular proteins, with the amino acids required for heme- and oxygen-binding being conserved. In situ-hybridization studies showed that glob1 and glob2 expression in An. gambiae is mainly associated with the tracheal system. This pattern re…

A Membrane-Bound Vertebrate Globin

The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2)-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and m…

Neuroglobin and Other Nerve Haemoglobins

The nervous system of animals requires huge amounts of metabolic energy and thus oxygen. Intracellular haemoglobins sporadically occur in glial cells and neurons of various invertebrate taxa, including Annelida, Arthropoda, Echiura, Mollusca, Nematoda and Nemertea. At least some of these respiratory proteins sustain the aerobic metabolism and thus the excitability of the nervous system. Recently, we have identified neuroglobin as an oxygen-binding protein of vertebrate neurons. The physiological role of neuroglobin, which is apparently present in much lower amounts than many invertebrate nerve haemoglobins, is less well established. Phylogenetic analyses have shown that neuroglobin is ortho…

Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy, electrophoresis, and sequence similarities with arthropod hemocyanin

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a …

Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily.

In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157–2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that i…

Divergent Distribution in Vascular and Avascular Mammalian Retinae Links Neuroglobin to Cellular Respiration

The visual function of the vertebrate retina relies on sufficient supply with oxygen. Neuroglobin is a respiratory protein thought to play an essential role in oxygen homeostasis of neuronal cells. For further understanding of its function, we compared the distribution of neuroglobin and mitochondria in both vascular and avascular mammalian retinae. In the vascular retinae of mouse and rat, oxygen is supplied by the outer choroidal, deep retinal, and inner capillaries. We show that in this type of retina, mitochondria are concentrated in the inner segments of photoreceptor cells, the outer and the inner plexiform layers, and the ganglion cell layer. These are the same regions in which oxyge…

Living with stress: regulation of antioxidant defense genes in the subterranean, hypoxia-tolerant mole rat, Spalax.

Lack of oxygen is life threatening for most mammals. It is therefore of biomedical interest to investigate the adaptive mechanisms which enable mammalian species to tolerate extremely hypoxic conditions. The subterranean mole rat Spalax survives substantially longer periods of hypoxia than the laboratory rat. We hypothesized that genes of the antioxidant defense, detoxifying harmful reactive oxygen species generated during hypoxia and hyperoxia, are involved in Spalax underground adaptation. Using quantitative RT-PCR, we analyzed the mRNA expression levels of seven antioxidant defense genes (catalase, glutathione peroxidase 1, glutathione-S-transferase Pi1, heme oxygenase 1, superoxide dism…

Distribution of Cytoglobin in the Mouse Brain

Cytoglobin (Cygb) is a vertebrate globin with so far poorly defined function. It is expressed in the fibroblast cell-lineage but has also been found in neurons. Here we provide, using immunohistochemistry, a detailed study on the distribution of Cygb in the mouse brain. While Cygb is a cytoplasmic protein in active cells of the supportive tissue, in neurons it is located in the cytoplasm and the nucleus. We found the expression of Cygb in all brain regions, although only a fraction of the neurons was Cygb-positive. Signals were of different intensity ranging from faint to very intense. Telencephalic neurons in all laminae of the cerebral cortex (CCo), in the olfactory bulb (in particular pe…

From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

Structure, diversity and evolution of myriapod hemocyanins

Oxygen transport in the hemolymph of many arthropods is mediated by hemocyanins, large copper-containing proteins that are well-studied in Chelicerata and Crustacea, but had long been considered unnecessary in the subphylum of Myriapoda. Only recently has it become evident that hemocyanins are present in Scutigeromorpha (Chilopoda) and Spirostreptida (Diplopoda). Here we present evidence for a more widespread occurrence of hemocyanin in the myriapods. By means of RT-PCR, western blotting and database searches, hemocyanins were identified in the symphylans Hanseniella audax and Symphylella vulgaris, the chilopod Scolopendra subspinipes dehaani and the diplopod Polydesmus angustus. No hemocya…

The Heme Environment of Mouse Neuroglobin

Neuroglobin (Ngb) is a newly discovered oxygen-binding heme protein that is primarily expressed in the brain of humans and other vertebrates. To characterize the structure/function relationships of this new heme protein, we have used resonance Raman spectroscopy to determine the structure of the heme environment in Ngb from mice. In the Fe2+CO complex, two conformations of the Fe–CO unit are present, one of which arises from an open conformation of the heme pocket in which the CO is not interacting with any nearby residue, and the other arises from a closed conformation where a positively charged residue near the CO group stabilizes the complex. For the Fe2+O2 complex, we detect a single νF…

Androglobin: a chimeric globin in metazoans that is preferentially expressed in mammalian testes

Abstract: 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 O2 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 glo…

Neuroglobin and Cytoglobin

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we summarize the current state of knowledge of the structures, ligand-binding kinetics, evolution, and expression patterns of these two proteins. These data provide working hypotheses with regard to the possible physiological roles of these globins in the animal's metabolism. Both neuroglobin and cytoglobin are structurally similar to myoglobin, but they contain distinct features like extraordinarily high temperature resistances and unusual cavities inside the molecules. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the…

How Does the Eye Breathe?

Visual performance of the vertebrate eye requires large amounts of oxygen, and thus the retina is one of the highest oxygen-consuming tissues of the body. Here we show that neuroglobin, a neuron-specific respiratory protein distantly related to hemoglobin and myoglobin, is present at high amounts in the mouse retina (approximately 100 microm). The estimated concentration of neuroglobin in the retina is thus about 100-fold higher than in the brain and is in the same range as that of myoglobin in the muscle. Neuroglobin is expressed in all neurons of the retina but not in the retinal pigment epithelium. Neuroglobin mRNA was detected in the perikarya of the nuclear and ganglion layers of the n…

Regulation and Role of Neuroglobin and Cytoglobin Under Hypoxia

Neuroglobin (Ngb) and cytoglobin (Cygb) are two novel members of the globin superfamily that are ubiquitously present in vertebrates. Their exact physiological roles are still uncertain. Here we review the expression of Ngb and Cygb, with particular emphasis on their regulation and potential role under hypoxia. Ngb expression is confined to neurons and some endocrine tissues. At the subcellular level, Ngb is associated with the presence of mitochondria and thus linked to the oxidative metabolism. Hypoxia or ischemic insults most likely do not strongly increase Ngb levels in the rodent brain. This might be explained by the fact that most mammals are not adapted to low oxygen levels. In zebra…

The amphibian globin gene repertoire as revealed by the <i>Xenopus </i>genome

The draft genome sequence of the Western clawed frog <i>Xenopus (Silurana) tropicalis</i> facilitates the identification, expression analysis and phylogenetic classification of the amphibian globin gene repertoire. Frog and mammalian neuroglobin display about 67% protein sequence identity, with the expected predominant expression in frog brain and eye. Frog and mammalian cytoglobins share about 69% of their amino acids, but the frog protein lacks the mammalian-type extension at the C-terminus. Like in mammals, <i>X. tropicalis</i> cytoglobin is expressed in many organs including neural tissue. Neuroglobin and cytoglobin genomic regions are syntenically conserved in a…

Sequence and evolution of a hexamerin from the ant Camponotus festinatus.

In the ant Camponotus festinatus, two different hexamerins accumulate stage-specifically during the late larval period and at various times in adults. These hexamerins serve as storage proteins and play important roles in brood nourishment and colony founding. We report an analysis of the cDNA sequence of C. festinatus hexamerin 2 (CfeHex2). The native protein contains 732 amino acids, which are moderately enriched in aromatic amino acids, aspartate and asparagine. Phylogenetic analyses show a close relationship of CfeHex2 to a putative toxin of the braconid wasp, Bracon hebetor. The divergence of Formicidae and Braconidae hexamerins was calculated to have begun 187 MYA, an estimate consist…

Neuroglobins from the Zebrafish Danio rerio and the Pufferfish Tetraodon nigroviridis

Neuroglobin is a recently discovered respiratory, porphyrin-containing protein that is expressed in the brain of mouse and man. Here we show that neuroglobin is also present in the teleost fish. Complete cDNA sequences are reported from the pufferfish Tetraodon nigroviridis and the zebrafish Danio rerio. In addition, the neuroglobin gene of T. nigroviridis was sequenced, demonstrating the conservation of the B12.2, E11.0 and G7.0 introns plus the presence of an additional intron in the 5' noncoding region. The fish neuroglobins each comprise 159 amino acids and are 84.3% identical. Phylogenetic analyses show a basal position of the neuroglobins within the metazoan globin tree. An enhanced a…

Zebrafish Reveals Different and Conserved Features of Vertebrate Neuroglobin Gene Structure, Expression Pattern, and Ligand Binding

Neuroglobin has been identified as a respiratory protein that is primarily expressed in the mammalian nervous system. Here we present the first detailed analysis of neuroglobin from a non-mammalian vertebrate, the zebrafish Danio rerio. The zebrafish neuroglobin gene reveals a mammalian-type exon-intron pattern in the coding region (B12.2, E11.0, and G7.0), plus an additional 5'-non-coding exon. Similar to the mammalian neuroglobin, the zebrafish protein displays a hexacoordinate deoxy-binding scheme. Flash photolysis kinetics show the competitive binding on the millisecond timescale of external ligands and the distal histidine, resulting in an oxygen affinity of 1 torr. Western blotting, i…

EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A conc…

Neuroglobin: A Respiratory Protein of the Nervous System

Nerve tissues exhibit some of the highest oxygen consumption rates found in the body. Neuroglobin, a heme protein distantly related to hemoglobin, is thought to enhance the supply of oxygen to the neurons, the eye, and some endocrine tissues. Neuroglobin may promote neuronal survival under hypoxic conditions as they occur, for example, in stroke.

Mapping protein matrix cavities in human cytoglobin through Xe atom binding

Abstract Cytoglobin is the fourth recognized globin type, almost ubiquitously distributed in human tissues; its function is still poorly understood. Cytoglobin displays a core region of about 150 residues, structurally related to hemoglobin and myoglobin, and two extra segments, about 20 residues each, at the N- and C-termini. The core region hosts a large apolar cavity, held to provide a ligand diffusion pathway to/from the heme, and/or ligand temporary docking sites. Here we report the crystal structure (2.4 A resolution, R -factor 19.1%) of a human cytoglobin mutant bearing the CysB2(38) → Ser and CysE9(83) → Ser substitutions (CYGB*), treated under pressurized xenon. Three Xe atoms bind…

Cyanide binding and heme cavity conformational transitions in **Drosophila melanogaster** hexacoordinate hemoglobin

The reason for the presence of hemoglobin-like molecules in insects, such as Drosophila melanogaster, that live in fully aerobic environments has yet to be determined. Heme endogenous hexacoordination (where HisE7 and HisF8 axial ligands to the heme Fe atom are both provided by the protein) is a recently discovered mechanism proposed to modulate O-2 affinity in hemoglobins from different species. Previous results have shown that D. melanogaster hemoglobin 1 (product of the glob1 gene) displays heme endogenous hexacoordination in both the ferrous and ferric states. Here we present kinetic data characterizing the exogenous cyanide ligand binding process, and the three-dimensional structure (a…

Expression analysis of neuroglobin mRNA in rodent tissues

Neuroglobin is a respiratory protein which was reported to be preferentially expressed in the vertebrate brain. Here we present the first detailed analysis of the expression of neuroglobin in mouse and rat tissues. Neuroglobin mRNA was detected in all brain areas studied. Most, but not all, nerve cells were labeled, suggesting differential expression of Ngb. Neuroglobin mRNA was detected in the peripheral nervous system, explaining previous northern hybridization signals in organs other than the brain. Substantial neuroglobin expression was also found in metabolically active endocrine tissues such as the adrenal and pituitary glands. The granule localization of neuroglobin transcripts in va…

Cerebral expression of neuroglobin and cytoglobin after deep hypothermic circulatory arrest in neonatal piglets

Deep hypothermic circulatory arrest (DHCA) is used in corrective cardiac surgery for complex congenital heart disease. Endogenous protective mechanisms may be responsible for the prevention of brain damage after hypothermic ischemia. Neuroglobin and cytoglobin are expressed in brain cells and appear to modulate hypoxic-ischemic brain injury. However, their neuroprotective potency is still not understood. Thus the aim of this study was to detect the influence exerted by DHCA on their expression.The effects of DHCA were analyzed in a neonatal piglet model with cardiopulmonary bypass, DHCA of 60 and 120 min and subsequent reperfusion of 6h. Complete histological analysis and changes in the mRN…

Oxygen-induced changes in hemoglobin expression in Drosophila

The hemoglobin gene 1 (dmeglob1) of the fruit fly Drosophila melanogaster is expressed in the tracheal system and fat body, and has been implicated in hypoxia resistance. Here we investigate the expression levels of dmeglob1 and lactate dehydrogenase (a positive control) in embryos, third instar larvae and adult flies under various regimes of hypoxia and hyperoxia. As expected, mRNA levels of lactate dehydrogenase increased under hypoxia. We show that expression levels of dmeglob1 are decreased under both short- and long-term hypoxia, compared with the normoxic (21% O2) control. By contrast, a hypoxia/reoxygenation regime applied to third instar larvae elevated the level of dmeglob1 mRNA. A…

A hemocyanin from the Onychophora and the emergence of respiratory proteins

The velvet worms (Onychophora) are considered living fossils and are closely related to the Euarthropoda. Onychophora possess a tracheal system for respiratory function, but oxygen-transport proteins have been considered unnecessary. Here, we show that the hemolymph of the Epiperipatus sp. (Onychophora: Peripatidae) contains an arthropod-type hemocyanin, demonstrating that such protein exists outside the Euarthropoda. Thus, the evolution of oxygen carriers preceded the divergence of the Onychophora and Euarthropoda and was most likely linked to the evolution of an efficient circulatory system in a low-oxygen environment. The cDNA of the Epiperipatus hemocyanin subunit comprises 2,287 bp an…

Complete Hemocyanin Subunit Sequences of the Hunting SpiderCupiennius salei

Hemocyanins are large copper-containing respiratory proteins found in many arthropod species. Scorpions and orthognath spiders possess a highly conserved 4 x 6-mer hemocyanin that consists of at least seven distinct subunit types (termed a to g). However, many "modern" entelegyne spiders such as Cupiennius salei differ from the standard arachnid scheme and have 2 x 6-mer hemocyanins. Here we report the complete primary structure of the 2 x 6-mer hemocyanin of C. salei as deduced from cDNA sequencing, gel electrophoresis, and matrix-assisted laser desorption spectroscopy. Six distinct subunit types (1 through 6) and three additional allelic sequences were identified. Each 1 x 6-mer half-mole…

Genomic Organization and Molecular Evolution of the Genes for Neuroglobin and Cytoglobin in the Hypoxiatolerant Israeli Mole Rat, Spalax Carmeli

The genes for the two respiratory proteins neuroglobin (Ngb) and cytoglobin (Cygb) in the subterranean Israeli mole rat Spalax carmeli have been sequenced and compared to other mammals including human. Coding regions of both Spalax genes are highly conserved on the nucleotide and amino acid level. The ratios of non-synonymous to synonymous nucleotide substitutions suggest strong purifying selection acting on Ngb and Cygb in all mammals. Thus, there appears to be no special sequence level adaptation in the two respiratory proteins within the hypoxia-tolerant mole rat. On the genomic level, Spalax Ngb and Cygb gene regions revealed the conserved 4-exon-3-intron structure and conserved CpG-ric…

Complete sequence, expression and evolution of two members of the hexamerin protein family during the larval development of the rice moth, Corcyra cephalonica

Three distinct types of storage hexamerins are expressed in the "last-instar" larvae of the rice moth, Corcyra cephalonica. A cDNA expression library was constructed from fat body-RNA and screened with a polyclonal antibody raised against purified hexamerin (SP2) of Corcyra cephalonica. Two slightly different "full-length" hexamerin cDNA clones (Hex2a and Hex2b) were isolated and sequenced. Both include open reading frames of 2109 bp which are translated into polypeptides of 703 amino acids with 92.5% identity. Signal peptides of 19 amino acids are present at the N-termini. The 684 amino acids native proteins have a high content of aryl groups (17.6%). According to both the criteria for ami…

Interspecies comparison of neuroglobin, cytoglobin and myoglobin: Sequence evolution and candidate regulatory elements

Neuroglobin and cytoglobin are two novel members of the vertebrate globin family. Their physiological role is poorly understood, although both proteins bind oxygen reversibly and may be involved in cellular oxygen homeostasis. Here we investigate the selective constraints on coding and non-coding sequences of the neuroglobin and cytoglobin genes in human, mouse, rat and fish. Neuroglobin and cytoglobin are highly conserved, displaying very low levels of non-synonymous nucleotide substitutions. An oxygen supply function predicts distinct modes of gene regulation, involving hypoxia-responsive transcription factors. To detect conserved candidate regulatory elements, we compared the neuroglobin…

Identification, structure, and properties of hemocyanins from Diplopod myriapoda.

Hemocyanins are copper-containing, respiratory proteins that occur in the hemolymph of many arthropod species. Here we report for the first time the presence of hemocyanins in the diplopod Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two immunologically distinct subunits in the 75-kDa range that are most likely arranged in a 36-mer (6 x 6) native molecule. It has a high oxygen affinity (P(50) = 4.7 torr) but low cooperativity (h = 1.3 +/- 0.2). Spirostreptus hemocyanin is structurally similar to the single known hemocyanin from the myriapod taxon,…

Molecular characterization of hemoglobin from the honeybee Apis mellifera

Due to the prevailing importance of the tracheal system for insect respiration, hemoglobins had been considered rare exceptions in this arthropod subphylum. Here we report the identification, cloning and expression analysis of a true hemoglobin gene in the honeybee Apis mellifera (Hymenoptera). The deduced amino acid sequence covers 171 residues (19.5 kDa) and harbors all globin-typical features, including the proximal and the distal histidines. The protein has no signal peptide for transmembrane transport and was predicted to localize in the cytoplasm. The honeybee hemoglobin gene shows an ancient structure, with introns in positions B12.2 and G7.0, while most other insect globins have div…

Oxygen Supply from the Bird's Eye Perspective

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 sho…

Critical re-evaluation of neuroglobin expression reveals conserved patterns among mammals.

Neuroglobin (Ngb) is a respiratory protein that is almost exclusively expressed in the vertebrate nervous system. Despite many years of research, the exact function and even the expression sites of Ngb are still a matter of debate. However, to investigate hypotheses surrounding the potential roles of Ngb, a detailed knowledge of its major and minor expression sites is indispensable. We have therefore evaluated Ngb expression by extensive bioinformatic analysis using publicly available transcriptome data (RNA-Seq). During mammalian brain development, we observed low embryonic expression of Ngb mRNA and an increase after birth, arguing against a role of Ngb in fetal hypoxia tolerance. In adul…

Divergent roles of the Drosophila melanogaster globins.

In contrast to long-held assumptions, the gene repertoire of most insects includes hemoglobins. Analyses of the genome of the fruitfly Drosophila melanogaster identified three distinct hemoglobin genes (glob1, glob2, and glob3). While glob1 is predominantly associated with the tracheal system and fat body, glob2 and glob3 are almost exclusively expressed in the testis. The physiological role of globins in Drosophila is uncertain. Here, we studied the functions of the three globins in a cell culture system. Drosophila Schneider 2 (S2) cells were stably transfected with each of the three globins and the empty vector as control. Under hypoxia (1% atmospheric O2), only glob1 overexpression enha…

Protection of islets in culture by delivery of oxygen binding neuroglobin via protein transduction.

Islet transplantation has become an accepted method to treat type 1 diabetes. To succeed and achieve normal levels of glucose in transplant recipients, the quality of the transplanted islets is of the utmost importance. Lack of oxygen during organ procurement, islet isolation, and subsequent culture triggers apoptosis or necrosis and loss of islet function, causing the yield and quality to diminish. A promising candidate for cytoprotection against oxygen deprivation is neuroglobin (Ngb). Ngb is a recently described member of globin family and is expressed in neurons, retina, and pancreatic islets. To overexpress this protein in the islets and study its ability to protect them, we utilized p…

Neuroglobin, cytoglobin, and a novel, eye-specific globin from chicken

Neuroglobin and cytoglobin are two recently discovered respiratory proteins of vertebrates. Here we report the first identification and expression analyses of these proteins in bird species. Neuroglobin from the domestic chicken Gallus gallus differs in approximately 30% from the mammalian proteins, but its genome structure shows the conservation of the B12.2, E11.0, and G7.0 intron positions. The chicken cytoglobin protein is shorter than the mammalian orthologs, from which it differs overall by approximately 25%, due to the absence of the C-terminal exon in the gene. Comparison of chicken and mammalian gene order shows that neuroglobin and cytoglobin are located on conserved syntenic chro…

What is the function of neuroglobin?

SUMMARY 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 correla…

Neuron-specific expression of neuroglobin in mammals.

Neuroglobin, a vertebrate oxygen-binding protein, is expressed in many regions of the adult brain. We examined the cell type-specific expression of neuroglobin in neurons and astroglial cells in primary cultures of fetal hippocampal cells and sections of the adult mouse brain using neuroglobin-specific polyclonal antibodies and cell type-specific markers NeuN and GFAP to differentiate between neurons and glial cells. Neuroglobin is exclusively expressed in neurons, but not in astroglial cells. Accordingly, neuroglobin was detected in two neuroblastoma cell lines (N2a, SH-SY5Y) and the pheochromocytoma cell line PC-12, but not in glioblastoma cell lines (DKMG, GAMG) or other, non-neural cell…

Diversity of stonefly hexamerins and implication for the evolution of insect storage proteins

Hexamerins are large storage proteins of insects in the 500 kDa range that evolved from the copper-containing hemocyanins. Hexamerins have been found at high concentration in the hemolymph of many insect taxa, but have remained unstudied in relatively basal taxa. To obtain more detailed insight about early hexamerin evolution, we have studied hexamerins in stoneflies (Plecoptera). Stoneflies are also the only insects for which a functional hemocyanin is known to co-occur with hexamerins in the hemolymph. Here, we identified hexamerins in five plecopteran species and obtained partial cDNA sequences from Perla marginata (Perlidae), Nemoura sp. (Nemouridae), Taeniopteryx burksi (Taeniopterygid…

Additional file 2 of Diversity, evolution, and function of myriapod hemocyanins

Table S2. List of sequences used in this study. (DOCX 24 kb)

Complete Sequence of the 24-mer Hemocyanin of the TarantulaEurypelma californicum

Hemocyanins are large oligomeric respiratory proteins found in many arthropods and molluscs. The hemocyanin of the tarantula Eurypelma californicum is a 24-mer protein complex with molecular mass of 1,726,459 Da that consists of seven different polypeptides (a–g), each occupying a distinct position within the native molecule. Here we report the complete molecular structure of the E. californicumhemocyanin as deduced from the corresponding cDNAs. This represents the first complex arthropod hemocyanin to be completely sequenced. The different subunits display 52–66% amino acid sequence identity. Within the subunits, the central domain, which bears the active center with the copper-binding sit…

Old proteins - new locations: myoglobin, haemoglobin, neuroglobin and cytoglobin in solid tumours and cancer cells

Aim:  The unexpected identification of myoglobin (MB) in breast cancer prompted us to evaluate the clinico-pathological value of MB, haemoglobin (HB) and cytoglobin (CYGB) in human breast carcinoma cases. We further screened for the presence of neuroglobin (NGB) and CYGB in tumours of diverse origin, and assessed the O2-response of HB, MB and CYGB mRNAs in cancer cell lines, to better elicit the links between this ectopic globin expression and tumour hypoxia. Methods:  Breast tumours were analysed by immunohistochemistry for HB, MB and CYGB and correlated with clinico-pathological parameters. Screening for CYGB and NGB mRNA expression in tumour entities was performed by hybridization, quant…

Neuroglobin mRNA expression after transient global brain ischemia and prolonged hypoxia in cell culture.

Abstract Neuroglobin is a nerve-specific respiratory protein that has been proposed to play an important role in the protection of brain neurons from ischemic and hypoxic injuries. Here, we investigated the regulation of neuroglobin expression after transient global ischemia in the rat brain using mRNA in situ hybridization and under hypoxic stress in cultured neuronal cell lines (PC12, HN33) by quantitative RT-PCR. While neuroglobin mRNA expression was significantly enhanced in cell culture after severe prolonged hypoxia (0–1% O 2 for 24 h), we did not find any significant increases in neuroglobin mRNA levels in the rat brain after transient global ischemia. Vegf and Glut1 mRNAs showed inc…

Molecular characterization of hemocyanin and hexamerin from the firebrat Thermobia domestica (Zygentoma).

Hexapods possess a tracheal system that enables the transport of oxygen to the inner organs. Although respiratory proteins have been considered unnecessary in most Hexapoda for this reason, we recently showed the presence of a functional hemocyanin in the stonefly Perla marginata. Here we report the identification and molecular characterization of a hemocyanin from Zygentoma (Thysanura). We obtained the full length cDNA of two distinct subunit types from the firebrat Thermobia domestica, and partial sequences of the orthologs from the silverfish Lepisma saccharina. The native T. domestica hemocyanin subunits both consist of 658 amino acids, but a signal peptide for transmembrane transport i…

Molecular evolution of the arthropod hemocyanin superfamily.

Arthropod hemocyanins are members of a protein superfamily that also comprises the arthropod phenoloxidases (tyrosinases), crustacean pseudohemocyanins (cryptocyanins), and insect storage hexamerins. The evolution of these proteins was inferred by neighbor-joining, maximum-parsimony, and maximum-likelihood methods. Monte Carlo shuffling approaches provided evidence against a discernible relationship of the arthropod hemocyanin superfamily and molluscan hemocyanins or nonarthropodan tyrosinases. Within the arthropod hemocyanin superfamily, the phenoloxidase probably emerged early in the (eu-)arthropod stemline and thus form the most likely outgroup. The respiratory hemocyanins evolved from t…

Molecular characterisation and evolution of the hemocyanin from the European spiny lobster, Palinurus elephas.

The hemocyanin of the European spiny lobster Palinurus elephas (synonym: Palinurus vulgaris) is a hexamer composed by four closely related but distinct subunits. We have obtained the full cDNA sequences of all four subunits, which cover 2275-2298 bp and encode for native polypeptides of 656 and 657 amino acids. The P. elephas hemocyanin subunits belong to the alpha-type of crustacean hemocyanins, whereas beta- and gamma-subunits are absent in this species. An unusual high ratio of non-synonymous versus synonymous nucleotide substitutions was observed, suggesting positive selection among subunits. Assuming a constant evolution rate, the P. elephas hemocyanin subunits emerged from a single he…

Globins and hypoxia adaptation in the goldfish, Carassius auratus

Goldfish (Carassius auratus) may survive in aquatic environments with low oxygen partial pressures. We investigated the contribution of respiratory proteins to hypoxia tolerance in C. auratus. We determined the complete coding sequence of hemoglobin α and β and myoglobin, as well as partial cDNAs from neuroglobin and cytoglobin. Like the common carp (Cyprinus carpio), C. auratus possesses two paralogous myoglobin genes that duplicated within the cyprinid lineage. Myoglobin is also expressed in nonmuscle tissues. By means of quantitative real-time RT-PCR, we determined the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and cytoglobin in goldfish exposed to prolonged hypoxia (48…

Diplopod hemocyanin sequence and the phylogenetic position of the Myriapoda

Hemocyanins are copper-containing respiratory proteins of the Arthropoda that have so far been thoroughly investigated only in the Chelicerata and the Crustacea but have remained unstudied until now in the Myriapoda. Here we report the first sequence of a myriapod hemocyanin. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two distinct subunits that are arranged in a 6 x 6 native molecule. The cloned hemocyanin subunit cDNA codes of for a polypeptide of 653 amino acids (75.5 kDa) that includes a signal peptide of 18 amino acids. The sequence closely resembles that of the chelicerate hemocyanins. Molecular phylogenetic analyses reject with high statistical con…

Diversity, evolution, and function of myriapod hemocyanins.

Background Hemocyanin transports O2 in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the presence of hemocyanin in Myriapoda has long been overlooked. We analyzed transcriptome and genome sequences from all major myriapod taxa – Chilopoda, Diplopoda, Symphyla, and Pauropoda – with the aim of identifying hemocyanin-like proteins. Results We investigated the genomes and transcriptomes of 56 myriapod species and identified 46 novel full-length hemocyanin subunit sequences in 20 species of Chilopoda, Diplopoda, and Symphyla, but not Pauropoda. We found in Cleidogona sp. (Diplopoda, Chordeumatida) a hemocyanin-…

Divergent distribution of cytoglobin and neuroglobin in the murine eye

Neuroglobin (Ngb) and cytoglobin (Cygb) are two vertebrate globins with yet poorly defined functions. Previous studies had demonstrated a high expression level of neuroglobin in the mammalian retina, being in line with a respiratory function. Here we show that in the mouse eye, cytoglobin is localised in fibroblasts of the ciliary processes and the choroidea. In the neuronal retina, cytoglobin is expressed in a subset of neurons of the ganglion cell and inner nuclear layers. Cytoglobin is also present in the inner plexiform layer, but absent from the pigment cells. Neuroglobin is localised in photoreceptor inner segments, the plexiform layers and the ganglion cell layer. The divergent distr…

Cytoglobin: A Novel Globin Type Ubiquitously Expressed inVertebrate Tissues

Vertebrates possess multiple respiratory globins that differ in terms of structure, function, and tissue distribution. Three types of globins have been described so far: hemoglobin facilitates the transport of oxygen in the blood, myoglobin serves oxygen transport and storage in the muscle, and neuroglobin has a yet unidentified function in nerve cells. Here we report the identification of a fourth and novel type of globin in mouse, man, and zebrafish. It is expressed in apparently all types of human tissue and therefore has been called cytoglobin (CYGB). Mouse and human CYGBs comprise 190 amino acids; the zebrafish CYGB, 174 amino acids. The human CYGB gene is located on chromosome 17q25. …

Evaluating the Hypoxia Response of Ruffe and Flounder Gills by a Combined Proteome and Transcriptome Approach

Hypoxia has gained ecological importance during the last decades, and it is the most dramatically increasing environmental factor in coastal areas and estuaries. The gills of fish are the prime target of hypoxia and other stresses. Here we have studied the impact of the exposure to hypoxia (1.5 mg O2/l for 48 h) on the protein expression of the gills of two estuarine fish species, the ruffe (Gymnocephalus cernua) and the European flounder (Platichthys flesus). First, we obtained the transcriptomes of mixed tissues (gills, heart and brain) from both species by Illumina next-generation sequencing. Then, the gill proteomes were investigated using two-dimensional gel electrophoresis and mass sp…

Evolutionary history and diversity of arthropod hemocyanins

Hemocyanins are copper-containing, multi-subunit proteins that transport oxygen in the hemolymph of many molluscs and arthropods [Markl and Decher, Adv. Comp. Environ. Physiol. 13 (1992) 325; van Holde et al., J. Biol. Chem. 276 (2001) 15563]. Arthropod hemocyanins originated more than 550 million years ago from oxygen-consuming phenoloxidases. Hemocyanins are present in various Onychophora, Chelicerata, Myriapoda, Crustacea, and Hexapoda, but subunit evolution differs striking in these arthropod subphyla. Hemocyanins also gave rise to non-respiratory proteins (crustacean pseudo-hemocyanins, insect hexamerins, and hexamerin receptors), which most likely have storage functions.

Neuroglobin and cytoglobin:two new entries in the hemoglobin superfamily.

Abstract: Neuroglobin (Ngb) and cytoglobin (Cygb) are two newly discovered intracellular members of the vertebrate hemoglobin (Hb) family. Ngb, predominantly expressed in nerve cells, is of ancient evolutionary origin and is homologous to nerve-globins of invertebrates. Cygb, present in many different tissues, shares common ancestry with myoglobin (Mb) and can be traced to early vertebrate evolution. Ngb and Cygb display the classical three-on-three -helical globin fold and are endowed with a hexa-coordinate heme Fe atom, in both their ferrous and ferric forms, having the heme distal HisE7 residue as the endogenous sixth ligand. Reversible intramolecular hexa- to penta-coordination of the h…

Bishistidyl heme hexacoordination, a key structural property in Drosophila melanogaster hemoglobin

Hemoglobins at high concentration have been isolated long ago from some insect larvae living in hypoxic environments. Conversely, a monomeric hemoglobin has been discovered recently in the fruit fly Drosophila melanogaster as intracellular protein expressed both in larvae and in the adult fly. Such a finding indicates that the oxygen supply in insects may be more complex than previously thought, relying not only on O2 diffusion through the tubular tracheal system, but also on carrier-mediated transport and storage. We present here the crystal structure of recombinant D. melanogaster hemoglobin at 1.20 A resolution. Spectroscopic data show that the protein displays a hexacoordinated heme, wh…

Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family.

Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O(2) supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe(2+)) form, indicating a His-Fe(2+)-His binding scheme. O(2) or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe(3+)) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k(on)) and a slow dissociation rate (k(off)) for both O(2) and CO, indicating…

A respiratory hemocyanin from an insect.

Insects possess an elaborate tracheal system that enables transport of gaseous oxygen from the atmosphere directly to the inner organs. Therefore, the presence of specialized oxygen-transport proteins in the circulatory system of insects has been considered generally unnecessary. Here, we show for the first time, to our knowledge, the presence of an ancestral and functional hemocyanin (Hc) in an insect. In the hemolymph of nymphs and adults of the stonefly Perla marginata , a hexameric Hc was identified, which consists of two distinct subunit types of 659 and 655 amino acids. P. marginata Hc displays cooperative oxygen binding with a moderately high oxygen affinity [(half-saturation pressu…

Ligands and receptors: common theme in insect storage protein transport.

The passage of macromolecules through biological membranes is an essential process for all multicellular organisms. Insects have developed a mechanism different from that known for all other eukaryotes investigated so far. This review discusses the function and evolution of this mechanism. Insect pupae do not feed during metamorphosis. Therefore they depend on material that has been accumulated during the larval life. At the end of this period, shortly before pupariation, a rise in titer of ecdysteroid hormones induces the incorporation of a large fraction of storage proteins (hexamerins) from the body fluid into the fat body cells. The transport of hexamerins across the cell-membrane is me…

Crystal Structure of Cytoglobin: The Fourth Globin Type Discovered in Man Displays Heme Hexa-coordination

Cytoglobin is a recently discovered hemeprotein belonging to the globin superfamily together with hemoglobin, myoglobin and neuroglobin. Although distributed in almost all human tissues, cytoglobin has not been ascribed a specific function. Human cytoglobin is composed of 190 amino acid residues. Sequence alignments show that a protein core region (about 150 residues) is structurally related to hemoglobin and myoglobin, being complemented by about 20 extra residues both on the N and C termini. In the absence of exogenous ligands (e.g. O2), the cytoglobin distal HisE7 residue is coordinated to the heme Fe atom, thus decreasing the ligand affinity. The crystal structure of human cytoglobin (2…

Characterization of DrosophilaHemoglobin

In contrast to previous assumptions, the fruit fly Drosophila melanogaster possesses hemoglobin. This respiratory protein forms a monomer of about 17 kDa that is not exported into the hemolymph. Recombinant Drosophila hemoglobin displays a typical hexacoordinated deoxy spectrum and binds oxygen with an affinity of 0.12 torr. Four different hemoglobin transcripts have been identified, which are generated by two distinct promoters of the hemoglobin (glob1) gene but are identical in their coding regions. Putative binding sites for hypoxia-regulated transcription factors have been identified in the gene. Hemoglobin synthesis in Drosophila is mainly associated with the tracheal system and the fa…

The pro-opiomelanocortin gene of the zebrafish (Danio rerio)

The cDNA and the gene for pro-opiomelanocortin (POMC) in the zebrafish (Danio rerio) were isolated and analyzed. The gene consists of three exons and two short introns and has a similar overall structural organization as in Homo sapiens. Intron 1 (339 bp) divides the 5(') untranslated region from the coding region while intron 2 (1522 bp) is located between the signal peptide and the sequence encoding ACTH. Transcription starts 26 bp downstream of a TATA box and there is one polyadenylation signal in the 3(') untranslated region. The cDNA comprises of 964 bp with an open reading frame encoding a 222 amino acid hormone prepropeptide that is split into six putative hormones. Sequence comparis…

Genomic response of the rat brain to global ischemia and reperfusion

To identify genes that are involved in ischemia response of the brain, we have evaluated changes of gene expression in rat cerebrum after 15 min complete global ischemia, followed by reperfusion for 1 h, 6 h or 24 h. The expression profiles of approximately 30,000 transcripts from three subjects in each group (including sham-operated controls) were monitored employing oligonucleotide microarrays. About 20,000 transcripts were detectable in rat brains. The levels of 576 transcripts (approximately 2.9%) were significantly altered in response to experimental ischemia. 419 transcripts were up- and 157 downregulated; 39 transcripts changed after 1 h reperfusion, 174 after 6 h and 462 after 24 h.…

Molecular cloning and evolution of lobster hemocyanin.

In the American lobster, Homarus americanus, oxygen is transported by a hemocyanin that is composed 2 x 6 subunits. N-terminal sequencing show the presence of three distinct subunit types (alpha, beta and gamma). We cloned the cDNA of one of these subunits that belong to the alpha-type. It encodes a hemocyanin subunit of 654 amino acids with a molecular mass of 84.8 kDa, which is synthesized in the hepatopancreas. Phylogenetic analyses of the crustacean hemocyanin sequences show two well-separated clades, which correspond to the alpha and gamma-type subunits. Sequences of beta-type subunits are still unknown. The gamma-sequences have evolved about 15% faster than the alpha-subunits, consist…

Oxygen binding properties of non-mammalian nerve globins

Oxygen-binding globins occur in the nervous systems of both invertebrates and vertebrates. While the function of invertebrate nerve haemoglobins as oxygen stores that extend neural excitability under hypoxia has been convincingly demonstrated, the physiological role of vertebrate neuroglobins is less well understood. Here we provide a detailed analysis of the oxygenation characteristics of nerve haemoglobins from an annelid (Aphrodite aculeata), a nemertean (Cerebratulus lacteus) and a bivalve (Spisula solidissima) and of neuroglobin from zebrafish (Danio rerio). The functional differences have been related to haem coordination: the haem is pentacoordinate (as in human haemoglobin and myogl…

The redox state of the cell regulates the ligand binding affinity of human neuroglobin and cytoglobin.

Neuroglobin and cytoglobin reversibly bind oxygen in competition with the distal histidine, and the observed oxygen affinity therefore depends on the properties of both ligands. In the absence of an external ligand, the iron atom of these globins is hexacoordinated. There are three cysteine residues in human neuroglobin; those at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. Both cysteine residues in cytoglobin, although localized in other positions than in human neuroglobin, may form a disulfide bond as well. The existence and position of these disulfide bonds was demonstrated by mass spectrometry and thiol accessibility studies. Mutation of the cysteines …

Additional file 3: of Diversity, evolution, and function of myriapod hemocyanins

Figure S1. Multiple sequence alignment of myriapod hemocyanins and phenoloxidases in FASTA format. (PDF 101 kb)

Additional file 4: of Diversity, evolution, and function of myriapod hemocyanins

Data S1. Nucleotide sequences of the Hc and PPO cDNAs and gene identified in this study. (ZIP 81 kb)

Additional file 5: of Diversity, evolution, and function of myriapod hemocyanins

Figure S2. The mRNA levels of Hc subunits were determined by RNA-Seq based on the transcriptomes (Table 1) and displayed as log RPKM values. (TIF 3 mb)