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PubMed2006

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

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% O2 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 increases in the hippocampus as expected. Therefore, it is unlikely that neuroglobin is instrumental in the acute response of neurons to hypoxic or ischemic insults, for which the mammalian brain is not adapted.

Animals↗
PubMed2004

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 with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily.

Globins↗
PubMed2004

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 distribution of neuroglobin and cytoglobin in the mammalian retina suggests distinct functions of these proteins in the vertebrate nervous system. While neuroglobin seems to be associated with oxygen consumption, a respiratory function of cytoglobin is unlikely.

Animals↗
PubMed2003

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 with striking adaptability, indicating that hemoglobin and myoglobin are just two examples within a wide and functionally diversified protein homology superfamily.

Amino Acid Sequence↗
PubMed2004

Reactivity studies of the Fe(III) and Fe(II)NO forms of human neuroglobin reveal a potential role against oxidative stress.

Neuroglobin, recently discovered in the brain and in the retina of vertebrates, belongs to the class of hexacoordinate globins, in which the distal histidine coordinates the iron center in both the Fe(II) and Fe(III) forms. As for most other hexacoordinate globins, the physiological function of neuroglobin is still unclear, but seems to be related to neuronal survival following acute hypoxia. In this study, we have addressed the question whether human neuroglobin could act as a scavenger of toxic species, such as nitrogen monoxide, peroxynitrite, and hydrogen peroxide, which are generated at high levels in the brain during hypoxia; we have also investigated the kinetics of the reactions of its Fe(III) (metNGB) and Fe(II)NO forms with several reagents. Binding of cyanide or NO* to metNGB follows bi-exponential kinetics, showing the existence of two different protein conformations. In the presence of excess NO*, metNGB is converted into NGBFe(II)NO by reductive nitrosylation, in analogy to the reactions of NO* with metmyoglobin and methemoglobin. The Fe(II)NO form of neuroglobin is oxidized to metNGB by peroxynitrite and dioxygen, two reactions that also take place in hemoglobin, albeit at lower rates. In contrast to myoglobin and hemoglobin, metNGB unexpectedly does not generate the cytotoxic ferryl form of the protein upon addition of either peroxynitrite or hydrogen peroxide. Taken together, our data indicate that human neuroglobin may be an efficient scavenger of reactive oxidizing species and thus may play a role in the cellular defense against oxidative stress.

Ferric Compounds↗
PubMed2004

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 chromosomal segments. While neuroglobin is expressed in the chicken's brain and eye, cytoglobin RNA was detected in all investigated tissues. In addition, a novel globin-type has been identified that is only expressed in the chicken's eye. The gene of this eye-globin contains the typical globin introns at B12.2 and G7.0. Phylogenetic analyses suggest that this globin is most closely related to the cytoglobin lineage. Although the function of this eye-globin remains presently uncertain, it adds an additional diversity to the vertebrate globin family.

Amino Acid Sequence↗
PubMed2006

The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c.

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b(5) is relatively slow (k=6 x 10(2)M(-1)s(-1) at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2 x 10(7)M(-1)s(-1)) with an apparent overall equilibrium constant of 1 microM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.

Animals↗
PubMed2006

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death.

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H(2)O(2) resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H(2)O(2) was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 microM H(2)O(2) for 24h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.

Blotting, Western↗
PubMed2001

Human neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxygen.

Neuroglobin is a newly discovered mammalian hemoglobin that is expressed predominately in the brain (Burmester, T., Welch, B., Reinhardt, S., and Hankeln, T. (2000) Nature 407, 520-523). Neuroglobin has less than 25% identity with other vertebrate globins and shares less than 30% identity with the annelid nerve myoglobin it most closely resembles among known hemoglobins. Spectroscopic and kinetic experiments with the recombinant protein indicate that human neuroglobin is the first example of a hexacoordinate hemoglobin in vertebrates and is similar to plant and bacterial hexacoordinate hemoglobins in several respects. The ramifications of hexacoordination and potential physiological roles are explored in light of the determination of an O(2) affinity that precludes neuroglobin from functioning in traditional O(2) storage and transport.

Biological Transport↗
PubMed2003

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 involved, or the use of reducing agents to break the S-S bond, led to a decrease in the observed oxygen affinity of human neuroglobin by an order of magnitude. The critical parameter is the histidine dissociation rate, which changes by about a factor of 10. The same effect is observed with human cytoglobin, although to a much lesser extent (less than a factor of 2). These results suggest a novel mechanism for the regulation of oxygen binding; contact with an appropriate electron donor would provoke the release of oxygen. Hence the oxygen affinity would be directly linked to the redox state of the cell.

Cysteine↗
PubMed2003

Residues in the distal heme pocket of neuroglobin. Implications for the multiple ligand binding steps.

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the low-spin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His64 is the endogenous ligand and that Lys67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the nu(Fe-His) stretching frequency appears at 221 cm(-1), which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the nu(Fe-CO) stretching frequency was detected at 521 and 494 cm(-1) in WT, M69L, and K67T, while only the 494 cm(-1) component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm(-1) component is attributed to the presence of polar His64. The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His64 and Lys67 comprise a unique distal heme pocket in neuroglobin.

Amino Acid Sequence↗
PubMed2004

Allosteric regulation and temperature dependence of oxygen binding in human neuroglobin and cytoglobin. Molecular mechanisms and physiological significance.

Two new globin proteins have recently been discovered in vertebrates, neuroglobin in neurons and cytoglobin in all tissues, both showing heme hexacoordination by the distal His(E7) in the absence of gaseous ligands. In analogy to hemoglobin and myoglobin, neuroglobin and cytoglobin are supposedly involved in O2 storage and delivery, although their physiological role remains to be solved. Here we report O2 equilibria of recombinant human neuroglobin (NGB) and cytoglobin (CYGB) measured under close to physiological conditions and at varying temperature and pH ranges. NGB shows both alkaline and acid Bohr effects (pH-dependent O2 affinity) and temperature-dependent enthalpy of oxygenation. O2 and CO binding equilibrium studies on neuroglobin mutants strongly suggest that the bound O2 is stabilized by interactions with His(E7) and that this residue functions as a major Bohr group in the presence of Lys(E10). As shown by the titration of free thiols with 4,4'-dithiodipyridine and by mass spectrometry, this mechanism of modulating O2 affinity is independent of formation of an internal disulfide bond under the experimental conditions used, which stabilize thiols in the reduced form. In CYGB, O2 binding is cooperative, consistent with its proposed dimeric structure. Similar to myoglobin but in contrast to NGB, O2 binding to CYGB is pH-independent and exothermic throughout the temperature range investigated. Our data support the hypothesis that CYGB may be involved in O2-requiring metabolic processes. In contrast, the lower O2 affinity in NGB does not appear compatible with a physiological role involving mitochondrial O2 supply at the low O2 tensions found within neurons.

Allosteric Site↗
PubMedDate not supplied

The cellular and subcellular localization of neuroglobin and cytoglobin -- a clue to their function?

Neuroglobin and cytoglobin are recently discovered respiratory proteins of vertebrates with yet ill-defined physiological functions. Neuroglobin is widely expressed in neurons, but not glia, in the vertebrate central and peripheral nervous systems. Other major expression sites are the retina and endocrine tissues. This distribution is indicative of a function of neuroglobin in metabolically most active, oxygen-consuming cell types, but does not yet allow to safely distinguish between different cellular roles, such as oxygen homeostasis, scavenging of reactive oxygen species or sustaining energy metabolism. Cytoglobin is predominantly expressed in connective tissue fibroblasts and related cell types in the body organs. Its main function may therefore be related to the specific amounts of extracellular matrix. Cytoglobin may hypothetically be involved in the oxygen-consuming maturation of collagen proteins. Cytoglobin is also expressed in distinct cell types of brain and retina. Its distribution strikingly differs from neuroglobin, suggesting an independent, yet unknown function.

Animals↗
PubMedDate not supplied

Tracing the structure-function relationship of neuroglobin and cytoglobin using resonance Raman and electron paramagnetic resonance spectroscopy.

The physiological role of neuroglobin and cytoglobin, two vertebrate globins discovered in the last 5 years, is not yet clearly understood. In this work, we review the structural information on these globins and its implication on the possible protein function, obtained by electron paramagnetic resonance and resonance Raman spectroscopy. All studies reveal a high flexibility in the heme-pocket region of neuroglobin. Together with the observation that the distal ligand of the heme iron is the endogenous E7-histidine in both the ferric and ferrous form of neuroglobin and cytoglobin, the flexibility of the heme environment in neuroglobin will play a crucial role in the globins' ability to bind and stabilize exogenous ligands.

Animals↗
PubMed2002

Neuroglobin and cytoglobin. Fresh blood for 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 cytoglobin are not completely understood. Although supplying cells with O2 is the likely function, it is also possible that both globins act as O2-consuming enzymes or as O2 sensors. Here, we review what is currently known about neuroglobin and cytoglobin in terms of their function, tissue distribution and relatedness to the well-known hemoglobin and myoglobin. Strikingly, the data reveal that O2 metabolism in cells is more complicated than was thought before, requiring unexpected O2-binding proteins with potentially novel functional features.

Amino Acid Sequence↗
PubMed2003

[Identification of interaction and interaction domains between neuroglobin and Na(+), K(+)-ATPase beta2 subunit].

The pre-transformed human fetal brain cDNA library was used to screen the protein interacting with neuroglobin by using yeast two hybrid system III from ClonTech Inc. The protein encoded by one of the clones interacting with neuroglobin (NGB) was confirmed to be the C terminus of the Na(+), K(+)-ATPase beta2 subunit (NKA1b2) based on amino acid sequences. Then the full-length coding region cDNA sequence of NKA1b2 was obtained from human fetal brain cDNA library by PCR. A set of experiments were designed to test the interaction between NGB and NKA1b2. Interaction between NGB and NKA1b2 was confirmed by binding assay in vitro. Furthermore, the interaction was also proved by co-immunoprecipitation test in vivo. Moreover, the structure integrity of neuroglobin was found to be essential for the interaction between NGB and NKA1b2 by yeast two hybrid method with a series of neuroglobin truncated mutants.

Amino Acid Sequence↗
PubMed2004

The structure of murine neuroglobin: Novel pathways for ligand migration and binding.

Neuroglobin, a recently discovered globin predominantly expressed in neuronal tissue of vertebrates, binds small, gaseous ligands at the sixth coordination position of the heme iron. In the absence of an exogenous ligand, the distal histidine (His64) binds to the heme iron in the ferrous and ferric states. The crystal structure of murine ferric (met) neuroglobin at 1.5 A reveals interesting features relevant to the ligand binding mechanism. Only weak selectivity is observed for the two possible heme orientations, the occupancy ratio being 70:30. Two small internal cavities are present on the heme distal side, which enable the His64(E7) side chain to move out of the way upon exogenous ligand binding. Moreover, a third, huge cavity (volume approximately 290 A3) connecting both sides of the heme, is open towards the exterior and provides a potential passageway for ligands. The CD and EF corners exhibit substantial flexibility, which may assist ligands in entering the protein and accessing the active site. Based on this high-resolution structure, further structure-function studies can be planned to elucidate the role of neuroglobin in physiological responses to hypoxia.

Animals↗
PubMed2004

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 reduction to break the S-S bond, led to a large decrease in the observed oxygen affinity of human neuroglobin, mainly due to a decrease in the histidine dissociation rate. This suggests a novel mechanism for the oxygen binding; reduction of the disulfide bond would provoke the release of oxygen.

Cysteine↗