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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↗
PubMed2005

Human neuroglobin protein in cerebrospinal fluid.

BACKGROUND: Neuroglobin is a hexacoordinated member of the globin family of proteins. It is predominantly localized to various brain regions and retina where it may play a role in protection against ischemia and nitric oxide-induced neural injury. Cerebrospinal fluid was collected from 12 chronic regional or systemic pain and 5 control subjects. Proteins were precipitated by addition of 50% 0.2 N acetic acid, 50% ethanol, 0.02% sodium bisulfite. The pellet was extensively digested with trypsin. Peptides were separated by capillary liquid chromatography using a gradient from 95% water to 95% acetonitrile in 0.2% formic acid, and eluted through a nanoelectrospray ionization interface into a quadrapole - time-of-flight dual mass spectrometer (QToF2, Waters, Milford, MA). Peptides were sequenced (PepSeq, MassLynx v3.5) and proteins identified using MASCOT (R). RESULTS: Six different neuroglobin peptides were identified in various combinations in 3 of 9 female pain subjects, but none in male pain, or female or male control subjects. CONCLUSION: This is the first description of neuroglobin in cerebrospinal fluid. The mechanism(s) leading to its release in chronic pain states remain to be defined.

Journal Article↗
PubMed2005

Presence of neuroglobin in cultured astrocytes.

Neuroglobin (Ngb), a recently discovered intracellular respiratory globin in neurons, may play a crucial role in oxygen homeostasis in the brain. We report preliminary findings indicating the presence of functional neuroglobin in primary cultures of cerebral cortical astrocytes. Reverse transcription real-time polymerase chain reaction (RRT-PCR) and immunostaining confirmed such presence in cultured astrocytes isolated from newborn mouse brain. Ngb antisense treatment increased apoptosis in ischemic astrocytes. The discovery of Ngb in astrocytes may provide some insight into how oxygen homeostasis is regulated in the brain.

Animals↗
PubMed2006

Analyzing heme proteins using EPR techniques: the heme-pocket structure of ferric mouse neuroglobin.

In this work, an electron paramagnetic resonance (EPR) strategy to study the heme-pocket structure of low-spin ferric heme proteins is optimized. Frozen solutions of ferric mouse neuroglobin (mNgb) are analyzed by means of electron spin echo envelope modulation and pulsed electron-nuclear double resonance techniques. The hyperfine and nuclear quadrupole couplings of the directly coordinating heme and histidine nitrogens are derived and are discussed in comparison with known data of other ferric porphyrin compounds. In combination with the hyperfine matrices of the imidazole protons, the 14N EPR parameters reveal structural information on the heme pocket of mNgb that is in agreement with previous X-ray diffraction data on neuroglobins.

Animals↗
PubMed2004

Temperature dependence of NO binding modes in human neuroglobin.

Both the ferrous and ferric forms of wild-type neuroglobin are found to be hexacoordinated with axial ligation of the F8-His and E7-His. Rapidly growing Escherichia coli cell cultures with low O2 concentration generate nitric oxide (NO). Combined electron paramagnetic resonance (EPR) and optical measurements show that wild-type human recombinant neuroglobin, overexpressed in such E. coli cells, still favors the F8His-Fe2+ -E7His conformation, whereby only a small fraction of the protein binds NO. Upon mutation of the E7-His to Leu and Gln, the competition with the distal histidine disappears and the nitrosyl ferrous form is readily observed. At low temperature, the EPR spectra of the NO-ligated Ngb proteins consist of contributions from two geometrically different NO-heme conformations. In combination with EPR data of vertebrate hemoglobins and myoglobins, the temperature dependence of the EPR spectra of the NO adducts of ferrous hNgb and its E7-mutants proves a strong stabilization of one isomer by the E7-histidine in wt hNgb. It is shown that this is not related to the polarity of histidine, but to its specific binding characteristics.

Electron Spin Resonance Spectroscopy↗
PubMed2006

The reactions of neuroglobin with CO: evidence for two forms of the ferrous protein.

The normally hexa coordinate ferrous form of neuroglobin binds CO by replacement of the heme-linked distal histidine residue. We have studied this reaction in detail using stopped flow techniques. The reaction time courses are complex at all the wavelengths studied. Specifically the reaction with CO occurs in two temporally separable phases, each of which shows a hyperbolic dependence of rate on CO concentration, indicating they each arise from histidine replacement by CO. Analysis of the observed rates as a function of the CO concentration, measured in the pH range 6.0-8.0, allows us to determine both the rate of histidine-heme ligand binding and dissociation for each of the two forms of the protein present in solution at each pH value. The pH dependence of the histidine association and dissociation rates is complex, as are the derived equilibrium constants for distal histidine binding. The spectral change associated with each reaction phase is very similar and independent of the CO concentration, showing that the two protein forms responsible for the two observed kinetic processes are not in equilibrium on the time scale of our investigations. Our data suggests that, unlike many other heme proteins, neuroglobin displays complex reactivity with ligands in the ferrous form due to heme rotational disorder, as has previously been reported for the ferric form of the protein.

Animals↗
PubMed2006

p53-mediated apoptosis, neuroglobin overexpression, and globin deposits in a patient with hereditary ferritinopathy.

The apoptotic death of putaminal neurons and glia in a patient with hereditary ferritinopathy is studied immunohistochemically with antibodies to p53, activated caspase-3, PUMA, BAX, cytochrome c, and inducible nitric oxide synthase. In addition to the overexpression of ferritin and the iron accumulations assumed to result from the genetically incompetent ferritin molecule, additional contributions to the iron, heme, and hyaline deposits in this disease are sought with antibodies to 2 recently discovered globins in humans, neuroglobin and cytoglobin. The "pathognomonic" swollen to vacuolated nuclei are immunoreactive for both p53 and activated caspase-3, indicating the intervention of the p53-mediated apoptotic pathway. The immunohistochemical demonstration of neuroglobin in the swollen nuclei and both globins in the hyaline deposits highlights the potential pathogenic importance of 2 other iron-containing proteins in this disease that is largely restricted to brain. Hereditary ferritinopathy is the first human disease in which abnormalities in these heme-containing proteins are demonstrated.

Apoptosis↗
PubMed2005

Hyperthermal stability of neuroglobin and cytoglobin.

Neuroglobin (Ngb) and cytoglobin (Cygb), recent additions to the globin family, display a hexa-coordinated (bis-histidyl) heme in the absence of external ligands. Although these proteins have the classical globin fold they reveal a very high thermal stability with a melting temperature (Tm) of 100 degrees C for Ngb and 95 degrees C for Cygb. Moreover, flash photolysis experiments at high temperatures reveal that Ngb remains functional at 90 degrees C. Human Ngb may have a disulfide bond in the CD loop region; reduction of the disulfide bond increases the affinity of the iron atom for the distal (E7) histidine, and leads to a 3 degrees C increase in the T(m) for ferrous Ngb. A similar Tm is found for a mutant of human Ngb without cysteines. Apparently, the disulfide bond is not involved directly in protein stability, but may influence the stability indirectly because it modifies the affinity of the distal histidine. Mutation of the distal histidine leads to lower thermal stability, similar to that for other globins. Only globins with a high affinity of the distal histidine show the very high thermal stability, indicating that stable hexa-coordination is necessary for the enhanced thermal stability; the CD loop which contains the cysteines appears as a critical region in the neuroglobin thermal stability, because it may influence the affinity of the distal histidine.

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

Human neuroglobin interacts with flotillin-1, a lipid raft microdomain-associated protein.

Neuroglobin (Ngb) is a newly discovered vertebrate globin that is expressed in the brain and that can reversibly bind oxygen. It has been reported that Ngb levels increase in neurons in response to oxygen deprivation, and that it protects neurons from hypoxia. However, the mechanism of this neuroprotection remains unclear. Recently, we found that oxidized human Ngb bound to the alpha-subunits of heterotrimeric G proteins (Galpha) and acted as a guanine nucleotide dissociation inhibitor for Galpha. To identify other Ngb-binding proteins, we herein screened a human brain cDNA library by using a yeast two-hybrid system. Among the plasmids isolated from positive clones, one contained an insert with 100% sequence identity to human flotillin-1. The interaction of Ngb with flotillin-1 was confirmed by glutathione S-transferase pull-down experiments. Since Galpha exists within lipid rafts critical for signal transduction and flotillin-1 recruits signaling proteins to lipid rafts, flotillin-1 might recruit Ngb to lipid rafts as a means of preventing neuronal death.

Amino Acid Sequence↗
PubMed2005

Preparation and characterization of a chimeric zebrafish-human neuroglobin engineered by module substitution.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that can reversibly bind oxygen that is expressed in the brain. Zebrafish and human Ngb share about 50% amino acid sequence identity. These Ngb proteins consist of four compact protein structural unit "modules" referred to as M1-M4. In the present study, we investigated the effects of module substitution on the properties of Ngb. Specifically, we prepared and characterized a chimeric ZHZZ Ngb in which the heme-binding module M2 of zebrafish Ngb was replaced by the comparable human Ngb module. Our results showed that the chimeric ZHZZ was stable and formed almost the identical heme-environmental and alpha-helical structure as the human and zebrafish Ngb proteins, suggesting that the structure of Ngb has been evolutionarily conserved.

Amino Acid Sequence↗
PubMed2006

Increased neuroglobin levels in the cerebral cortex and serum after ischemia-reperfusion insults.

Neuroglobin (NGB) is a newly discovered protein localized in neurons of the central and peripheral nervous systems in vertebrates. It functions to bind, store, and facilitate the utilization of oxygen in neuronal cells. Recent studies suggest that it may modulate hypoxic and ischemic injury. The major goal of the present study is to characterize the dynamic changes of NGB protein in the brain and serum in a global forebrain ischemia-reperfusion model using gerbils. The sensitivity and validity of serum NGB as a potential biomarker for brain injury were further evaluated. Global cerebral ischemia-reperfusion models were induced by bilateral carotid occlusion for 20 min followed with 2-, 8-, 16-, 24-, 48-, or 72-h reperfusion in forty-six Mongolian gerbils. Sham-operated and operated animals were sacrificed at the designated time after reperfusion. Brains were fixed for immunocytochemical study to evaluate the time-dependent expression of NGB, and the concentration of NGB in serum was measured by enzyme-linked immunosorbent assay. Our results showed that the expression of NGB was upregulated in the cerebral cortex but significantly downregulated in the hippocampus from 2 to 72 h of reperfusion after 20 min of bilateral common carotid arteries occlusion. The concentration of NGB in serum was significantly increased at 8 h and reached a peak at 48 h of reperfusion. There is a significant correlation between NGB levels in the serum and severity of neuronal damage in the gerbil brain. In summary, the upregulation of NGB in cerebral cortex and downregulation in hippocampus after reperfusion insults in the gerbil brain are consistent with the fact that cerebral cortex is more tolerant to hypoxic or ischemic injury than the hippocampus. Moreover, the changes of NGB levels in serum may be used to monitor the extent of brain damage in ischemic brain diseases.

Analysis of Variance↗
PubMed2006

Hypoxia differentially regulates the expression of neuroglobin and cytoglobin in rat brain.

Neuroglobin (Ngb) and Cytoglobin (Cygb) are new members of the globin family and display heterotopic expression patterns. To examine the effect of different hypoxia profiles on expression of Ngb and Cygb in rodent brain, rats were exposed to either sustained hypoxia (SH; 10% O(2)) or intermittent hypoxia (IH; 10% and 21% O(2) alternating every 90 s) for 1, 3, 7 and 14 days, and mRNA and protein expression of Ngb and Cygb were assessed in brain cortex. SH increased Ngb mRNA and protein expression throughout the exposure, while IH only elicited slight increases in Ngb expression at day 1. Neither SH nor IH elicited increases in Cygb expression. Thus, hypoxic stimulus presentation is a major determinant of the regulation of hypoxic sensitive genes such as Ngb. Furthermore, disparities between Ngb and Cygb responses to hypoxia further suggest that these two globins may play divergent roles in brain.

Animals↗
PubMed2005

Effect of aging on neuroglobin expression in rodent brain.

Neuroglobin (Ngb), a recently discovered O2-binding heme protein related to hemoglobin and myoglobin, protects neurons from hypoxic-ischemic injury in vitro and in vivo. In immunostained mouse brain sections, we found widespread expression of Ngb protein in neurons, but not astrocytes, of several brain regions that are prominently involved in age-related neurodegenerative disorders. Western blots from young adult (3 month), middle-aged (12 month), and aged (24 month) rats showed an age-related decline in Ngb expression in cerebral neocortex, hippocampus, caudate-putamen, and cerebellum. Loss of this neuroprotective protein may have a role in increasing susceptibility to age-related neurological disorders.

Age Factors↗
PubMed2004

Functional adaptation and its molecular basis in vertebrate hemoglobins, neuroglobins and cytoglobins.

Hemoglobin (Hb), the paradigm for allosteric proteins through decades, has gained renaissance in recent years following discovery of globins or their genes in all living organisms and in all tissues of higher animals, and of new members of the globin family, such as neuroglobins, Ngb, found predominantly in neural and nerve tissues and cytoglobins, Cygb, that has unprecedented nuclear location. The recent progresses in this field have been prompted by the development of sophisticated techniques to probe molecular structure and functions, which have revealed novel functions, such as the scavenging and release of vasoactive nitric oxide and the regulation of cellular metabolism. This review deals with the functional adaptations and the underlying molecular mechanisms in globins and presents case examples of molecular adaptations encountered in vertebrates and agnathans.

Acclimatization↗
PubMedDate not supplied

Protection of islets in culture by delivery of oxygen binding neuroglobin via protein transduction.

Islet transplantation has become an accepted method to treat type 1 diabetes. To succeed and achieve normal levels of glucose in transplant recipients, the quality of the transplanted islets is of the utmost importance. Lack of oxygen during organ procurement, islet isolation, and subsequent culture triggers apoptosis or necrosis and loss of islet function, causing the yield and quality to diminish. A promising candidate for cytoprotection against oxygen deprivation is neuroglobin (Ngb). Ngb is a recently described member of globin family and is expressed in neurons, retina, and pancreatic islets. To overexpress this protein in the islets and study its ability to protect them, we utilized protein transduction. Protein transduction is achieved by fusing Ngb to the TAT/PTD transduction domain, a peptide originated from the HIV transcriptional transactivator protein. Our study proved that TAT-Ngb is an efficient fusion protein capable of protecting the human islets in culture from loss of cell mass and function, thus increasing the quality of transplantable islets. If the islets could be cultured for a longer period of time without suffering harmful effects, it would be possible to precondition the recipient and there would be more time to assess their quality and function before transplantation.

Automation↗
PubMed2005

Identification of residues in human neuroglobin crucial for Guanine nucleotide dissociation inhibitor activity.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. We previously demonstrated that ferric human Ngb binds to the alpha-subunits of heterotrimeric G proteins (Galpha) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Galpha. Here we have investigated the interaction between Ngb and Galpha in more detail. We report that zebrafish Ngb, which shares about 50% amino acid sequence identity with human Ngb, does not have a GDI activity for Galpha. By carrying out exon swapping between zebrafish and human Ngb and site-directed mutagenesis, we have identified several residues that are crucial for the GDI activity of human Ngb.

Amino Acid Sequence↗
PubMed2004

Association of human neuroglobin with cystatin C, a cysteine proteinase inhibitor.

Neuroglobin (Ngb) is a newly discovered globin that is expressed in vertebrate brain. It has been reported that Ngb levels increase in neurons in response to oxygen deprivation, and that Ngb protects neurons from hypoxia. However, the mechanism of this neuroprotection remains unclear. In the present study, we identified human cystatin C, a cysteine proteinase inhibitor, as an Ngb-binding protein by using a yeast two-hybrid system. Surface plasmon resonance experiments verified that Ngb binds to cystatin C dimers, not to the monomers. Because both intracellular cystatin C and the amyloidogenic variant of cystatin C form dimers, Ngb may modulate the intracellular transport (or secretion) of cystatin C to protect against neuronal death under conditions of oxidative stress and/or it may have a role in the development of neurodegenerative diseases.

Amino Acid Sequence↗