Cytoglobin and neuroglobin are recently discovered members of the globin family. In situ hybridization localized neuroglobin mainly in brain and retina, while cytoglobin was expressed ubiquitously in all analyzed tissues. In the present study, polyclonal antibodies were raised against both proteins and the distribution of them was studied by immunocytochemistry at tissue and subcellular level. Cytoglobin immunoreactivity was uniformly distributed and found in all tissues studied. At the subcellular level, cytoglobin immunoreactivity was exclusively detected in the cell nucleus. In contrast, neuroglobin immunoreactivity was detected in specific brain regions with varying intensities and in the islet of Langerhans in the pancreas. The immunoreactivity was restricted to the cytoplasm of neurons and endocrine beta cells. The nuclear localization of cytoglobin opens new perspectives for possible function(s) of globinfolded proteins as transcriptional regulators.The widespread occurrence of hemoglobins (Hbs) 1 in virtually all kingdoms indicates that the gene for Hb is very ancient and that Hbs may serve functions other than simple O 2 carriers (1-4). The actual function of the proteins of this superfamily is mainly associated with O 2 transport/storage. However, the reported involvement of bacterial (5), invertebrate (6) and vertebrate Hbs/myoglobins (Mbs) (7,8) in the detoxification of NO, might illustrate a more primitive function of these molecules (3). Involvement in other functions as O 2 scavenger (4), O 2 sensor (9), O 2 consuming enzymes (10), or shadow pigments (11) have been suggested and may illustrate the coincidental use of a stable protein fold during evolution.Recently, two new members of the vertebrate globin family, namely neuroglobin (Ngb) and cytoglobin (Cygb), have been discovered (12)(13)(14)(15)(16). Both are monomeric (151 and 190 amino acid residues, respectively), intracellular proteins, displaying all determinants of the globin fold. Sequence analyses reveal low sequence identity with vertebrate Hb and Mb (20 -25%), as well as a very ancient origin, i.e. much older than Mb (16). The heme-iron atom of Ngb and Cygb is hexacoordinated, showing a His-Fe-His binding scheme, both histidines being the proximal and the distal histidine, respectively (13,15,17). Ngb has a high recombination rate (k on ) and a slow dissociation rate (k off ), indicating a high intrinsic affinity for the ligands (O 2 / CO). Before binding, the external ligands must compete with the internal sixth ligand, resulting in an observed O 2 affinity of the recombinant proteins similar to that of Mb (1 torr at 37°C).The function of Ngb and Cygb is a matter of debate. In response to hypoxia, Ngb is up-regulated in vivo and in vitro and protects the neurons against hypoxic damage (18). High concentrations (ϳ100 M) of Ngb are observed in the retina and its subcellular distribution correlates with the localization of the mitochondria (19). Both observations suggest a role in intracellular O 2 supply.A protein, identical to Cygb a...

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A very short hemoglobin (CerHb; 109 amino acids) binds O(2) cooperatively in the nerve tissue of the nemertean worm Cerebratulus lacteus to sustain neural activity during anoxia. Sequence analysis suggests that CerHb tertiary structure may be unique among the known globin fold evolutionary variants. The X-ray structure of oxygenated CerHb (R factor 15.3%, at 1.5 A resolution) displays deletion of the globin N-terminal A helix, an extended GH region, a very short H helix, and heme solvent shielding based on specific aromatic residues. The heme-bound O(2) is stabilized by hydrogen bonds to the distal TyrB10-GlnE7 pair. Ligand access to heme may take place through a wide protein matrix tunnel connecting the distal site to a surface cleft located between the E and H helices.

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O 2 dissociation and ultra-high O 2 affinity, which suggest O 2 sensing or NO scavenging functions. CerHb, however, has high rates of O 2 dissociation (k O 2 ‫؍‬ 200 -600 s ؊1 ) and moderate O 2 affinity (K O 2 Ϸ1 M ؊1 ) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, k O 2 decreases 1000-fold and K O 2 increases 130-fold at pH 7.0, 20°C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 3 Val CerHbO 2 at 1.70 Å resolution is almost identical to that of the wildtype protein (root mean square deviation of 0.12 Å). The dramatic functional and spectral effects of the Thr-E11 3 Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O 2 affinity, a decrease of the rate coefficient for O 2 dissociation, a 40 cm ؊1 decrease in CO of hemebound CO, and an increase in ligand migration toward more remote intermediate sites.Globins are found in all kingdoms of living organisms. Their functions have been the subject of active debate. In addition to O 2 transport and storage (1-3), several novel functions have been proposed recently, including control of NO levels in microorganisms and nerve tissue, O 2 sensing, and dehaloperoxidase activity (4 -8). Nerve tissue Hbs are found in both vertebrates and invertebrates. Neuroglobin is a recently discovered member of the globin family, whose in vivo function is still unknown (9 -14). It is expressed in specific regions of vertebrate brains, displays low sequence identity to conventional Hbs or Mbs, 1 and is characterized by a bis-His-Fe hexacoordinate heme structure (11,12,15,16). In contrast, the nerve tissue Hbs found in mollusc, annelid, arthropod, nemertean, and nematode species (17, 18) appear to store and/or transport O 2 to support brain and axon function during temporary hypoxia (18 -21).The nerve tissue and body wall Hbs of the nemertean worm Cerebratulus lacteus (CerHb) 2 are the smallest naturally occurring Hbs, composed of only 109 amino acids. Analysis of the three-dimensional structure of nerve tissue CerHb has shown that the typical 3-over-3 globin fold is edited markedly (22). The N-terminal A-helix is deleted; the GH region is extended; and the C-terminal H-helix is shortened. Both sequence and fold comparisons suggest that CerHb is equally distant from all known globin tertiary structures, supporting its identification with a new superfamily, the mini-Hbs (22). CerHb contains a large elongated tunnel in its interior. Ligands may enter and exit CerHb through this apolar tunnel between th...

SummaryIn analogy to hemoglobin (Hb) and myoglobin (Mb), neuroglobin (Ngb) and cytoglobin (Cygb) are supposed to be involved in oxygen (O 2 ) storage and delivery. The Cygb gene harbours both conserved HREs and mRNA stabilization sites, strongly suggestive of an oxygen-dependent regulation. We examined the relative transcriptional changes of Ngb and Cygb in a situation of chronic hypoxia using real-time quantitative PCR. We could conclude that Cygb is a hypoxia-induced gene, which is transcriptionally upregulated during chronic hypoxia in a hippocampal neuronal cell line and in multiple murine metabolically active tissues. The mechanism of induction of Cygb is HIF-1a dependent. HIF-1 is unique among mammalian transcription factors with respect to the specificity and sensitivity of its induction by hypoxia. Ngb expression seems to be regulated using other response elements and is less influenced by hypoxia.