Biomedical subjects

Thomas Hankeln

Publications and source records attributed to Thomas Hankeln.

At least 37 records · Page 2Linked to original sources

PubMed2004

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 cells (HeLa, Vero). Analysis of the neuroglobin genomic sequence from man and mouse identifies sequence motifs with similarity to the neuron-restrictive silencer element, possibly explaining a neuron-specific expression of neuroglobin.

Animals↗

PubMed2004

Mapping protein matrix cavities in human cytoglobin through Xe atom binding.

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 (2.4A 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 to the heme distal site region of CYGB* mapping the protein matrix apolar cavity. Despite the conserved globin fold, the cavity found in CYGB* is structured differently from those recognized to play a functional role in myoglobin, neuroglobin, truncated hemoglobins, and Cerebratulus lacteus mini-hemoglobin.

Binding Sites↗

PubMed2004

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, immune staining, and mRNA in situ hybridization demonstrate neuroglobin expression in the fish central nervous system and the retina but also in the gills. Neurons containing neuroglobin have a widespread distribution in the brain but are also present in the olfactory system. In the fish retina, neuroglobin is mainly present in the inner segments of the photoreceptor cells. In the gills, the chloride cells were identified to express neuroglobin. Neuroglobin appears to be associated with mitochondria-rich cell types and thus oxygen consumption rates, suggesting a myoglobin-like function of this protein in facilitated oxygen diffusion.

Animals↗

PubMed2004

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 (2.1 A resolution, 21.3% R-factor) highlights a three-over-three alpha-helical globin fold, covering residues 18-171; the 1-17 N-terminal, and the 172-190 C-terminal residue segments are disordered in both molecules of the crystal asymmetric unit. Heme hexa-coordination is evident in one of the two cytoglobin chains, whereas alternate conformation for the heme distal region, achieving partial heme penta-coordination, is observed in the other. Human cytoglobin displays a large apolar protein matrix cavity, next to the heme, not related to the myoglobin cavities recognized as temporary ligand docking stations. The cavity, which may provide a heme ligand diffusion pathway, is connected to the external space through a narrow tunnel nestled between the globin G and H helices.

Amino Acid Sequence↗

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

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↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2004

Neuroglobin and other hexacoordinated hemoglobins show a weak temperature dependence of oxygen binding.

Mouse 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 560 nm and the Soret band at 426 nm. 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 association and slow (100 ms-1 s) dissociation. However, the competing protein ligand results in a much lower affinity, on the order of magnitude of 1 torr. In addition to decreasing the affinity for external ligand, the competitive internal ligand leads to a weaker observed temperature dependence of the ligand affinity, since the difference in equilibrium energy for the two ligands is much lower than that of ligand binding to pentacoordinated hemoglobin. This effect could be of biological relevance for certain organisms, since it could provide a globin with an oxygen affinity that is nearly independent of temperature.

Animals↗

PubMed2003

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 present in chondroblasts and osteoblasts and shows a decreased level of expression upon differentiation to chondrocytes and osteocytes. Cytoglobin is located in the cytoplasm of these cell types. Evidence against an exclusively nuclear localization of cytoglobin, as recently proposed, is also provided by transfection assays with green fluorescent protein fusion constructs, which demonstrates the absence of an active nuclear import. The differential expression of cytoglobin argues against a general respiratory function of this molecule, but rather indicates a connective tissue-specific function. We hypothesize that cytoglobin may be involved in collagen synthesis. Cytoglobin expression was also observed in some neuronal subpopulations of the central and the peripheral nervous systems. Surprisingly, cytoglobin is localized in both the cytoplasm and nucleus of neurons, indicating a possible additional role of this protein in neuronal tissues.

Amino Acid Sequence↗

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

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 cerebral cortical regions, subcortical structures such as thalamus and hypothalamus, nuclei of cranial nerves in the brainstem and cerebellum. Thus, the widespread distribution of neuroglobin protein is in good agreement with its mRNA localization. Regionally differing intensities of immunostaining suggest different levels of neuroglobin protein expression, in line with the idea that brain regions show variation in their tolerance towards hypoxic conditions.

Animals↗

PubMed2003

ESTAnnotator: A tool for high throughput EST annotation.

In high throughput sequence analysis, it is often necessary to combine the results of contemporary bioinformatics tools, because no individual tool alone computes all the requested information. ESTAnnotator is a tool for the high throughput annotation of expressed sequence tags (ESTs) by automatically running a collection of bioinformatics applications. In the first step, a quality check is performed and repeats, vector parts and low quality sequences are masked. Then successive steps of database searching and EST clustering are performed. Already known transcripts present within mRNA and genomic DNA reference databases are identified. Subsequently, tools for the clustering of anonymous ESTs, and for further database searches at the protein level, are applied. Finally, the outputs of each individual tool are gathered and the relevant results presented in a descriptive summary. ESTAnnotator was already successfully applied for the systematic identification and characterisation of novel human genes involved in cartilage/bone formation, growth, differentiation and homeostasis. ESTAnnotator is available at http://genome.dkfz-heidelberg.de, contact: genome@dkfz.de.

Cartilage↗

PubMed2003

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 enzymatic instead of respiratory functions.

Amino Acid Sequence↗

PubMed2003

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.

Binding Sites↗

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↗

PubMed2002

How does the eye breathe? Evidence for neuroglobin-mediated oxygen supply in the mammalian retina.

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 neuronal retina, whereas the protein was present mainly in the plexiform layers and in the ellipsoid region of photoreceptor inner segment. The distribution of neuroglobin correlates with the subcellular localization of mitochondria and with the relative oxygen demands, as the plexiform layers and the inner segment consume most of the retinal oxygen. These findings suggest that neuroglobin supplies oxygen to the retina, similar to myoglobin in the myocardium and the skeletal muscle.

Animals↗

PubMed2002

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-cell parameters a = 39.6, b = 94.9, c = 67.5 A, beta = 94.4 degrees, and contain 2-4 protein molecules per asymmetric unit.

Crystallography↗

PubMed2002

Characterization of Drosophila hemoglobin. Evidence for hemoglobin-mediated respiration in insects.

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 fat body. This suggests that oxygen supply in insects may be more complex than thought previously and may depend on hemoglobin-mediated oxygen transport and storage in addition to simple diffusion.

Animals↗