Biomedical subjects

Thorsten Burmester

Publications and source records attributed to Thorsten Burmester.

At least 37 records · Page 2Linked to original sources

PubMed2004

A globin gene of ancient evolutionary origin in lower vertebrates: evidence for two distinct globin families in animals.

Hemoglobin, myoglobin, neuroglobin, and cytoglobin are four types of vertebrate globins with distinct tissue distributions and functions. Here, we report the identification of a fifth and novel globin gene from fish and amphibians, which has apparently been lost in the evolution of higher vertebrates (Amniota). Because its function is presently unknown, we tentatively call it globin X (GbX). Globin X sequences were obtained from three fish species, the zebrafish Danio rerio, the goldfish Carassius auratus, and the pufferfish Tetraodon nigroviridis, and the clawed frog Silurana tropicalis. Globin X sequences are distinct from vertebrate hemoglobins, myoglobins, neuroglobins, and cytoglobins. Globin X displays the highest identity scores with neuroglobin (approximately 26% to 35%), although it is not a neuronal protein, as revealed by RT-PCR experiments on goldfish RNA from various tissues. The distal ligand-binding and the proximal heme-binding histidines (E7 and F8), as well as the conserved phenylalanine CD1 are present in the globin X sequences, but because of extensions at the N-terminal and C-terminal, the globin X proteins are longer than the typical eight alpha-helical globins and comprise about 200 amino acids. In addition to the conserved globin introns at helix positions B12.2 and G7.0, the globin X genes contain two introns in E10.2 and H10.0. The intron in E10.2 is shifted by 1 bp in respect to the vertebrate neuroglobin gene (E11.0), providing possible evidence for an intron sliding event. Phylogenetic analyses confirm an ancient evolutionary relationship of globin X with neuroglobin and suggest the existence of two distinct globin types in the last common ancestor of Protostomia and Deuterostomia.

Amino Acid Sequence↗

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

A respiratory hemocyanin from an insect.

Insects possess an elaborate tracheal system that enables transport of gaseous oxygen from the atmosphere directly to the inner organs. Therefore, the presence of specialized oxygen-transport proteins in the circulatory system of insects has been considered generally unnecessary. Here, we show for the first time, to our knowledge, the presence of an ancestral and functional hemocyanin (Hc) in an insect. In the hemolymph of nymphs and adults of the stonefly Perla marginata, a hexameric Hc was identified, which consists of two distinct subunit types of 659 and 655 amino acids. P. marginata Hc displays cooperative oxygen binding with a moderately high oxygen affinity [(half-saturation pressure, P(50) approximately 8 torr (1 torr = 133 Pa)]. No evidence was found for the presence of Hcs in the more evolutionarily advanced holometabolan insects, suggesting that this type of respiratory protein was lost later in insect evolution. However, our results demonstrate that, in contrast to the accepted paradigm, certain basal insects have retained an ancestral blood-based mechanism of gas exchange.

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

Evolutionary history and diversity of arthropod hemocyanins.

Hemocyanins are copper-containing, multi-subunit proteins that transport oxygen in the hemolymph of many molluscs and arthropods [Markl and Decher, Adv. Comp. Environ. Physiol. 13 (1992) 325; 15563]. Arthropod hemocyanins originated more than 550 million years ago from oxygen-consuming phenoloxidases. Hemocyanins are present in various Onychophora, Chelicerata, Myriapoda, Crustacea, and Hexapoda, but subunit evolution differs striking in these arthropod subphyla. Hemocyanins also gave rise to non-respiratory proteins (crustacean pseudo-hemocyanins, insect hexamerins, and hexamerin receptors), which most likely have storage functions.

Animals↗

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

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

The pro-opiomelanocortin gene of the zebrafish (Danio rerio).

The cDNA and the gene for pro-opiomelanocortin (POMC) in the zebrafish (Danio rerio) were isolated and analyzed. The gene consists of three exons and two short introns and has a similar overall structural organization as in Homo sapiens. Intron 1 (339 bp) divides the 5(') untranslated region from the coding region while intron 2 (1522 bp) is located between the signal peptide and the sequence encoding ACTH. Transcription starts 26 bp downstream of a TATA box and there is one polyadenylation signal in the 3(') untranslated region. The cDNA comprises of 964 bp with an open reading frame encoding a 222 amino acid hormone prepropeptide that is split into six putative hormones. Sequence comparison of zebrafish POMC to sequences of various other vertebrate species reveals four regions that are highly conserved during the evolution of vertebrates-the N-terminal region, ACTH, beta-MSH, and beta-endorphin, whereas the connecting peptides show a much higher degree of variability. Phylogenetic analysis of the POMC sequences of various vertebrate species resulted in the expected pattern of species evolution. In situ hybridization demonstrated POMC expression in a cluster of cells (corticotrophs) in the pituitary of the zebrafish as early as 23 h after fertilization. These findings will facilitate the use of the zebrafish as a model organism in the study of the physiological role of POMC-derived peptides.

Amino Acid Sequence↗

PubMed2003

Quaternary structure of the European spiny lobster (Palinurus elephas) 1x6-mer hemocyanin from cryoEM and amino acid sequence data.

Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8 x 6-mer) in the 75kDa range. A 3D reconstruction of the 1 x 6-mer hemocyanin from the European spiny lobster Palinurus elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8A resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as alpha-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (>80%) of the hemocyanin subunits from the European spiny lobster P.elephas and the American spiny lobster Panulirus interruptus. Comparison of the P.elephas hemocyanin electron microscopy (EM) density map with the known P.interruptus X-ray structure shows a close structural correlation, demonstrating the reliability of both methods for reconstructing proteins. By molecular modelling, we have found the putative locations for the amino acid sequence (597-605) and the C-terminal end (654-657), which are absent in the available P.interruptus X-ray data.

Amino Acid Sequence↗