News Physiol Sci 19: 110-113, 2004;
doi:10.1152/nips.01513.2003
1548-9213/04 $5.00
News in Physiological Sciences, Vol. 19, No. 3, 110-113,
June 2004
© 2004 Int. Union Physiol. Sci./Am. Physiol. Soc.
Neuroglobin: A Respiratory Protein of the Nervous System
Thorsten Burmester1 and
Thomas Hankeln2
1 Institute of Zoology and
2 Institute of Molecular Genetics, Johannes Gutenberg University of Mainz, D-55099 Mainz, Germany.
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Abstract
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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.
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Introduction
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Animal life usually requires large amounts of oxygen, mainly to sustain aerobic ATP production in the respiratory chain of the mitochondria. Globins are small globular proteins that have the ability to bind oxygen and that supply the cells with oxygen (4). Vertebrates possess four types of globins that differ in terms of structure, tissue distribution, and function (Fig. 1
). Hemoglobin consists of four polypeptide chains and is used for the transport of oxygen in the red blood cells of the vertebrate animal’s circulatory system (4). Myoglobin is a monomeric protein in striated and cardiac muscles that acts as a temporary oxygen store and that may facilitate oxygen diffusion (for review, see Ref. 17). Myoglobin is also involved in the detoxification of nitric oxide (NO). Due to their high concentrations in the blood and muscles, respectively, hemoglobin and myoglobin are among the best-studied proteins in terms of structure, function, and evolution. Recently, two novel globin types have been added to the vertebrate globin family: cytoglobin, which is present in many different organs (11), and neuroglobin (Ngb), which is mainly expressed in nerve cells (2). The presence of Ngb as a respiratory protein in the central nervous system and other nerve tissues may have important physiological and medical implications. Here we will review the available data, which allow a first solid assessment of the possible Ngb function(s).
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The functions of (neuro)globins
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Globins are usually considered either oxygen transport or storage proteins (4). Thus Ngb may function as a "neuronal myoglobin," providing oxygen to the respiratory chain (Fig. 2A). However, in recent years several other globin functions have been discovered that are conceivable for Ngb too (see, e.g., Ref. 8). Similar to some nonsymbiotic plant hemoglobins, Ngb may be a terminal oxidase that regenerates NAD+ under anaerobic conditions, thereby sustaining ATP production (Fig. 2B). Ngb may also be involved in the detoxification of harmful reactive oxygen species (ROS), which are known to be generated, for example, under hypoxic conditions (Fig. 2C). Another noxious reactive molecule is NO, which is detoxified in the heart muscle by myoglobin, and Ngb may have a similar role in neurons (Fig. 2D). Finally, Ngb may act as a sensor to detect cellular oxygen concentration (Fig. 2E).
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FIGURE 2. Possible function(s) of neuroglobin (Ngb). Ngb may facilitate oxygen diffusion within the cells (A), regenerate NAD+ to sustain glycolysis under anaerobic conditions (B), decompose reactive oxygen species (C), act as nitric oxide (NO) dioxygenase (D), or be an oxygen sensor (E). Refer to the text for further discussion.
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Ngb is a conserved protein of ancient evolutionary origin
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Structural analyses show that human Ngb displays the typical globin fold (10). Ngb is a particularly highly conserved protein, with mouse and human Ngb differing in only 6% of the amino acid positions. On average, the amino acid substitution rates are three- to fourfold lower than those of the vertebrate hemo- and myoglobins. The coding regions of mammalian and fish Ngb genes carry three introns positioned at B12.2 (i.e., between codon positions 2 and 3 of the 12th amino acid of the 
-helix B), E11.0, and G7.0 (Fig. 3). Although the introns B12.2 and G7.0 are also present in hemoglobins and myoglobins, as well as in many other globin genes, the intron E11.0 is unique to Ngb and may have been acquired in the early evolution of the gene (1, 2). Ngb shares as little as 25% of the amino acids with vertebrate hemoglobins and myoglobins but resembles some nerve-specific invertebrate globins (2). This observation is in line with phylogenetic analyses, which suggest that Ngb is a derivative of an ancient branch of the metazoan globins that emerged before the separation of Deuterostomia and Protostomia. These facts hint at an important and conserved function of Ngb, at least in vertebrates and possibly in some invertebrate taxa as well.
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FIGURE 3. Comparison of human globins. NGB, neuroglobin; CYGB, cytoglobin; MB, myoglobin; HBA, HBB, HBD, HBE, HBG, and HBZ, hemoglobin chains HB , ß,  ,  ,  , and  , respectively. The secondary structure myoglobin and the putative disulfide bridge (7) are given at the top, and the globin consensus numbering and the intron positions are shown below the sequences. Conserved residues are shaded in gray.
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Ngb is preferentially but not exclusively expressed in nervous tissues
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Another clue for Ngb function derives from its occurrence in the organisms. Ngb was initially identified in the databases of human and murine brain cDNAs (2). Its identification in fishes (1), amphibians, and birds (unpublished data) suggests that Ngb is present in all vertebrates. In mouse and rat, Ngb is expressed in most brain neurons but is apparently absent from glia cells (5, 10, 19). Ngb mRNA and protein are not equally distributed throughout the brain, although in our studies we did not observe a distinct focal expression (10, 19) as proposed by others (5, 7). Ngb expression also occurs in endocrine tissues, as represented by the adenohypophysis of the pituitary gland, the adrenal gland, the testis (spermatogonia and primary spermatocytes) (10), and the islets of Langerhans in the pancreas (5). A particularly high expression level was observed in the mammalian retina (12). The estimated concentration of Ngb in this tissue is ~100 µM, thus ~100-fold higher than that in total brain and in the same range as the concentration of myoglobin in the skeletal muscle. Within the retina, Ngb protein is accumulated in the plexiform layers and the inner segment of the photoreceptor cells, thus correlating with the high oxygen demands of these regions. On the subcellular level, Ngb is concentrated in regions containing many mitochondria, i.e., the apical region of the inner segments and the synapses in the plexiform layers. Thus both cellular and subcellular distributions support the notion that in the eye Ngb expression levels are positively correlated with cellular oxygen consumption rates.
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Ngb is a hexacoordinated globin that binds O2 with a similar affinity to myoglobin
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The iron ion (Fe2+) in the heme of myoglobin and hemoglobin is pentacoordinated in the absence of oxygen. Four ligands are provided by the heme itself; the fifth ligand is the proximal histidine of the protein chain. On oxygenation, O2 provides the sixth ligand. Ngb binds O2 with a half-saturation pressure (P50) of ~1–2 Torr, which is comparable with that of a typical myoglobin (2, 3). However, like some other intracellular and plant hemoglobins, Ngb is hexacoordinated in the absence of oxygen, with the distal histidine bound to the Fe2+ as the sixth ligand (3, 9). Thus O2 must compete on binding to the iron with the proximal histidine. This competition results in biphasic ligand-binding kinetics. Reduction of an internal disulfide bridge, which may form in human Ngb, lowers oxygen affinity by a factor of 10 (6). Oxygen binding of Ngb may therefore be linked to the redox state of the cell: low oxygen levels lead to an increase of reduction equivalents (e.g., NADH), which may then reduce the cysteines and break the disulfide bridge, thereby enhancing the release of O2 from Ngb.
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Ngb may help neurons to survive hypoxia
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The central nervous system is particularly susceptible to low-oxygen conditions (hypoxia) that occur under cerebral ischemia or stroke, and a reduced supply of oxygen to the brain will inevitably lead to death of the neurons. Thus an enhanced expression of a protein that helps to sustain cellular respiration would increase the likelihood of neuronal survival under hypoxia and on ischemic injury. However, to date it is unclear whether Ngb is regulated under varying oxygen concentrations. Although Sun et al. (13) reported an ~2.5-fold upregulation of Ngb mRNA and protein levels after 24 h anoxia-reperfusion of a cerebral tissue culture, Mammen et al. (7) did not observe any increased expression of Ngb mRNA under long-term hypoxia (10% O2) in living mice. If confirmed, these conflicting data might mean that Ngb is mainly involved in an acute hypoxia response. A comparison of the mouse and human Ngb genes did not reveal conserved hypoxia-responsive elements that would allow for a hypoxic induction of the Ngb gene by the hypoxia-inducible factor (HIF) pathway (18). However, hypoxia activation of Ngb transcription was suggested to depend on the MAPK signal transduction pathway (20), which may activate HIF-1 by recruitment of the coactivating p300/CREB-binding protein.
Most interestingly, Sun et al. (13) also reported that the survival of cultured neuronal cells under anoxic conditions in vitro was significantly reduced on inhibition of Ngb expression with an antisense oligodeoxynucleotide, whereas enhanced expression of Ngb promoted cell survival. Most recently, the same authors (14) have shown that, under conditions of experimental stroke, infusion of Ngb antisense oligodeoxynucleotide increases infarct size and worsens neurological deficit of the rat brain. On the other hand, injection of Ngb-expressing adeno-associated virus constructs reduces infarct size and improves the neurological outcome.
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Is Ngb an NO-dioxygenase?
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Besides binding oxygen, myoglobin and other globins may act as NO-dioxygenases involved in the detoxification of NO (17). Such an NO scavenger function is theoretically conceivable also for Ngb, which could, for example, protect neuronal cells from the potentially harmful NO molecule. However, no correlation of the expression patterns of Ngb and NO synthases has been observed in the mouse brain (10). In addition, Ngb antisense oligonucleotides have no effect on the toxicity of NO donors in cultured cells (13). NO binding kinetics of Ngb do not support the idea of Ngb being an NO dioxygenase, mainly because in the hexacoordinate structure the distal histidine prevents the efficient binding of NO to Ngb (15).
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Ngb as oxygen sensors?
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It is well established that in some Archaea and Bacteria transcription factors with globin domains are involved in aerotaxis. By analogy, Ngb may be integrated in an oxygen-sensing pathway. However, there is at the moment no evidence in animals that globins are involved in a similar mechanism. Moreover, the high oxygen affinity of Ngb rather precludes an oxygen-sensing function. Recently, Wakasugi et al. (16) suggested that Ngb is involved in a brain-specific signaling pathway that protects neurons under oxidative stress as it occurs, for example, under hypoxia. They proposed that oxidized Ngb (Fe3+ form) inhibits the release of GDP from G protein . This interaction should trigger the release of the Gß complex, which is known to enhance cell survival. The interaction of Ngb and G was proposed on the basis of an alleged sequence similarity of Ngb with regulators of G protein signaling (RGS) and RGS domains of G protein-coupled receptor kinases (GRK) and evaluated in vitro by surface plasmon resonance. However, in contrast to the statements of Wakasugi et al. (16), there is no discernible similarity of Ngb with RGS or GRK sequences. It remains uncertain whether the interactions of Ngb with G and the suggested protection mechanism actually exist in vivo.
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Summary and conclusions: what do we know about Ngb function?
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Theoretical considerations support various functions of Ngb (Fig. 2
) (8). The actual data discussed here should help to rule out some of these hypotheses. Given the concerns mentioned above, the evidence of Ngb being a sensing or signaling molecule (Fig. 2E) is weak. The present data rather exclude a function of Ngb in NO detoxification (Fig. 2D). It is still conceivable that one role of Ngb is the degradation of ROS (Fig. 2C). Nerve tissues consume large amounts of oxygen, and high oxygen consumption rates are usually linked with the production of ROS. So far, no study has addressed the question of a possible involvement of Ngb in ROS metabolism. However, it is possible that the protective role of Ngb observed by Sun et al. (13, 14) is not related to an enhanced oxygen supply under hypoxia but is due to the Ngb-promoted degradation of ROS that had been generated on the hypoxia/reperfusion regime used by these authors. Other enzymatic activities of Ngb, such as its function as terminal oxidase (Fig. 2B), remain to be investigated.
Although the total concentration of Ngb in the mammalian brain is low (~1 µM; Ref. 2), local concentrations are probably much higher and may be sufficient to contribute to cellular oxygen supply (Fig. 2A) (10, 12). A myoglobin-like role of Ngb is also corroborated by the correlation of Ngb expression with oxygen consumption levels (12) and its capability to enhance neuronal survival (13, 14). Although we consider oxygen supply to be the most likely physiological role of Ngb, other functions are still conceivable and further studies are required for full characterization of this neuronal vertebrate globin.
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Acknowledgments
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We thank our colleagues M. Bolognesi, S. Dewilde, M. Marden, L. Moens, S. Reuss, S. Saaler-Reinhardt, and R. Weber for discussions and cooperation.
Our work is supported by the Deutsche Forschungsgemeinschaft (Ha 2103/3 and Bu 956/5) and by the European Union (QLRT-2001-01548).
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References
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Introduction
The functions of (neuro)globins
