A GLOBIN IN THE NUCLEUS!

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 analysed 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 globin-folded proteins as transcriptional regulators.


Introduction
The widespread occurrence of hemoglobins (Hbs) 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)(2)(3)(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 6 th 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

Expression cloning and purification of recombinant Ngb and Cygb
Mouse Ngb (mNgb) expression and purification were done as described previously (17). Expression and purification of human cytoglobin (hCYGB) was done as follows. The expression plasmid containing hCYGB in pET3a (13) was transformed into E. coli strain BL21(DE3)pLysS. Except for the omission of 5-aminolevulinic acid hydrochloride, cells were grown and harvested as described for mNgb (17). Cells were suspended in 50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonylfluorid and 5 mM dithiotreitol. After suspension, 1/10 volume 10% triton X-100, 10% deoxycholic acid, 500 mM Tris-HCl pH 7.5, 20 mM EDTA was added and the cells were exposed to three freezethaw cycles and sonication until complete lysis. Inclusion bodies were isolated by centrifugation at 3,300 g for 10 min and the pellet was suspended in and washed three times with 1% triton X-100, 1 mM EDTA and 50 mM Tris-HCl pH 7.5. Inclusion bodies were solubilized in 6 M guanidinium hydrochloride, 50 mM Tris-HCl pH 7.5 and 1% 2mercapto-ethanol during 1 hr at 0°C. After elimination of the insoluble material by centrifugation (10 min 10,000 g), hCYGB was reconstructed by adding a 1.4 M excess of hemin and dialyzed against 5 mM Tris-HCl pH 8.5 at 4°C. The reconstructed hCYGB was further purified as described for mNgb.

Immunohistochemistry
Polyclonal antibodies against purified recombinant mNgb and hCYGB were raised in rabbits and monitored by ELISA using standard protocols. The anti-mNgb and anti-hCYGB antibodies were purified from sera by ammonium sulfate precipitation and affinity chromatography using the corresponding immobilized antigen coupled to Cyanogen Bromide-activated Sepharose TM 4b (Amersham Pharmacia) as matrix (22).
Adult Swiss mice (n=6) were used for immunohistochemistry. All procedures were approved by the local ethics committee of the University of Antwerp and conformed to European Community regulations. After lethal injection with Nembutal (sodium pentobarbital) mice were transcardially perfused with physiological saline, followed by Zamboni's fixative (30 ml for 10 minutes). Tissues (brain, liver, heart, striated muscle, lung, kidney and small bowel) were removed and post-fixed in Zamboni's fixative for 30 min at room temperature, after which they were treated for improving the immunocytochemical conditions according to Llewellyn-Smith et al. (23). After being stored overnight in 20% sucrose in phosphate buffered saline (PBS) at 4°C, tissues were mounted in Tissue Tek (Sakura Finettek, Europe). Twenty-µm-thick cryostat sections were thaw-mounted on poly-L-lysine-coated microscope slides and dried for 2 h at 37°C. All primary and secondary antisera were diluted in PBS containing 10% normal goat serum (DAKO X 0907, Carpinteria, CA) 0.1% bovine serum albumin, 0.05% thimerosal and 0.01% NaN 3 (PBS). All incubations were carried out at room temperature. After preincubation with PBS containing 1% Triton X-100, sections were incubated overnight with rabbit polyclonal antibodies against mNgb Sections were cross-explored using fluorescence (Olympus BX50 or Zeiss Axiophot) microscopy. Fluorescence micrographs of DAPI-stained tissues were made with on the Olympus Microscope with a Sony 9100 VID CAM, and processed with AnalySIS software.
To obtain detailed images of labeled neurons, a confocal laser scanning microscope (Zeiss LSM 410) equipped with image reconstruction facilities (Imaris 2.7 software; Bitplane AG, Zürich, Switzerland; Silicon Graphics Indigo 2 workstation) was used. Excitation of the FITC-fluorophore and the Cy3-fluorophore was achieved with an argon laser (488 nm) and a Helium/Neon laser (543nm), respectively.

Western blotting
mCygb was isolated from a nuclear extract of mouse liver. The nuclei were isolated according to the method of Blobbel and Potter as cited by Tata (26) and mCygb was isolated by affinity chromatography on a matrix-immobilized anti-hCYGB antibody column.
Extracts of the nuclear and the cytosolic fraction were analyzed by SDS-PAGE. For immunodetection the anti-hCYGB antibody (1:1000) was used.

Results and Discussion
ELISA and Western blotting confirmed the specificity of the antibodies against mNgb and hCYGB, which did not cross-react or produce any reaction against recombinant Mb and Hb, and no cross-reaction was seen between mNGB and hCYGB (additional Fig * ).
Immunoreactivity of mCygb was detected in all studied tissues (Fig. 1). Within the brain, mCygb immunoreactivity was uniformly distributed over the different brain regions. No colocalization of mCygb-and GFAP-immunoreactivity was observed but some mCygbimmunoreactivity positive cells also displayed immunoreactivity for calbindin, a calciumbinding protein known to be present in neurons within the central nervous system (Fig. 1), and calretinin, corroborating that, mCygb immunoreactivity is limited to neurons within the brain. In all mCygb-positive cells the immunoreactivity was specifically confined to the cell nucleus, as evidenced by combined detection of mCygb-immunoreactivity and 4'-6-Diamidino-2-phenylindole (DAPI), a specific nuclear staining (Fig. 2).
The nuclear localization of mCygb was independently confirmed by isolating mCygb from a nuclear liver extract by affinity chromatography on a matrix-immobilized anti-hCYGB antibody column, followed by Western blot analysis (Fig. 3).
In contrast, mNgb was expressed in distinct tissues: no immunoreactivity was detected in liver, heart, striated muscle, lung, small bowel or kidney. In pancreas, mNgb immunoreactivity was restricted to the islets of Langerhans. It was visible in the brain, as such confirming the results of Mammen et al. (27) and Reuss et al. (28). Within the brain, mNgb immunoreactivity was found focally having different staining intensities. The most intense mNgb immunoreactivity was found in the medial vestibular nucleus (Fig. 4) and the ________________________ * supplemental data paraolivary nucleus, less intense reactivity was seen in the thalamic and subthalamic regions and in the cortex, whereas none was detected in the hippocampus and corpus callosum. These results mainly confirm previous in situ hybridization data (27;28). However, in contrast to With the exception of FixL, which is a high-spin and pentacoordinated protein, the heme-iron atom occurring in these proteins is always in the hexacoordinated low-spin state, displaying cytochrome-like spectra.
The overall structure of these molecules is variable. They may exist as a single molecule containing a heme-based sensor domain and an effector domain with enzymatic activity. This is the case for FixL and EcDos, where a PAS-folded heme-based sensor domain is covalently linked to a kinase and a phosphodiesterase domain, respectively (38).
CooA, a homodimer, consists of a heme-containing cAMP receptor protein (CRP) linked to a small DNA-binding domain with a typical helix-turn-helix motif (42). In contrast, NPAS2 is part of a non-covalent tetrameric complex, containing two NPAS2 subunits and two BMAL1 subunits, the latter allowing specific DNA binding (39;43).
All these molecules are, upon activation, involved in different signal transduction pathways: FixL will control the expression of N 2 fixation genes in Rhizobia; EcDos will regulate the aerobic respiration in E. coli; CooA, a bacterial CO sensor, will regulate the transcription of two operons encoding a CO oxidizing-system and NPAS2 is a gasresponsive transcription factor involved in the regulation of the circadian rhythm (38;39).
In bacterial flavohemoglobins, globin-folded domains occur linked to a FAD/NADP binding dehydrogenase domain and are used in the detoxification of NO as a dioxygenase (44). In contrast, the hemoglobin of Vitreoscilla binds non-covalently to a dehydrogenase containing a flavin domain to form its functional complex (45)(46)(47).   Detection was done with anti-hCYGB antibodies. In lanes 2 and 3 similar protein concentrations (10 µg) of an affinity-purified nuclear extract (2) and a cytosolic extract (3) were analyzed, whereas in lane 1 recombinant mCygb is used as a positive control.