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Originally published In Press as doi:10.1074/jbc.M305519200 on July 14, 2003

J. Biol. Chem., Vol. 278, Issue 38, 36505-36512, September 19, 2003
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Oxidized Human Neuroglobin Acts as a Heterotrimeric G{alpha} Protein Guanine Nucleotide Dissociation Inhibitor*,

Keisuke Wakasugi {ddagger} § , Tomomi Nakano § and Isao Morishima {ddagger} 

From the {ddagger}Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, and the §Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation (JST), Kyoto 615-8510, Japan

Received for publication, May 27, 2003 , and in revised form, June 26, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuroglobin (Ngb) is a newly discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. It has been reported that Ngb expression levels increase in response to oxygen deprivation and that it protects neurons from hypoxia in vitro and in vivo. However, the mechanism of this neuroprotection remains unclear. In the present study, we tried to clarify the neuroprotective role of Ngb under oxidative stress in vitro. By surface plasmon resonance, we found that ferric Ngb, which is generated spontaneously as a result of the rapid autoxidation, binds exclusively to the GDP-bound form of the {alpha} subunit of heterotrimeric G protein (G{alpha}i). In GDP dissociation assays or guanosine 5'-O-(3-thio)triphosphate binding assays, ferric Ngb behaved as a guanine nucleotide dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP. The interaction of GDP-bound G{alpha}i with ferric Ngb will liberate G{beta}{gamma}, leading to protection against neuronal death. In contrast, ferrous ligand-bound Ngb under normoxia did not have GDI activities. Taken together, we propose that human Ngb may be a novel oxidative stress-responsive sensor for signal transduction in the brain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuroglobin (Ngb)1 is a recently discovered globin found in the vertebrate brain that has a high affinity for oxygen (1–3). Globins are iron porphyrin complex (heme)-containing proteins that bind reversibly to oxygen and as such play an important role in respiratory function. They have been found in many taxa including bacteria, fungi, plants, and animals (4). The two major globins that have been described in vertebrates are hemoglobin and myoglobin. Hemoglobin (Hb), which consists of four subunits that cooperatively bind oxygen, is present in red blood cells where it is responsible for transporting oxygen from the lungs to the tissues (5). Myoglobin (Mb) is a monomeric intracellular globin that stores oxygen in muscle tissue and facilitates its diffusion from the periphery of the cell to mitochondria, which use it during oxidative phosphorylation (6). Although Ngb shares only 21–25% sequence identity with vertebrate Hb and Mb, it conserves the key amino acid residues that are required for Hb and Mb function (1). Like Hb and Mb, Ngb can reversibly bind oxygen (1, 7, 8). The iron atom in the heme prosthetic group of each globin normally exists in either the ferrous (Fe2+) or ferric (Fe3+) state. In the absence of exogenous ligands, the ferric and ferrous forms of Ngb are hexacoordinated with the endogenous protein ligands, distal histidine and proximal histidine (7) (Fig. 1). Oxygen (O2) or carbon monoxide (CO) can displace the distal histidine of ferrous Ngb to produce ferrous oxygen-bound Ngb (ferrous-O2 Ngb) or ferrous carbon monoxide-bound Ngb (ferrous-CO Ngb) (7). On the other hand, Hb and Mb are normally hexacoordinated in the ferric state, with a water molecule coordinated to iron and pentacoordinated in the ferrous form, leaving the sixth position empty and available for the binding of exogenous ligands such as O2 and CO.



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  		FIG. 1.
Sequence alignment of human Ngb to RGS domains. The multiple sequence alignment was performed by Clustal W and manual adjustments. GRK2, GRK4, GRK5, GRK6, RGS1, RGS4, RGS9, and GAIP share RGS domains. Human Mb was used as a representative protein among globin family members. Consensus amino acids between Ngb and RGS domain are indicated as red letters. Residues highlighted in yellow are conserved residues that form the hydrophobic core of the RGS domain. Numbers on the left and right of the sequences correspond to those at the beginning and end of the sequences, respectively. Gaps in the sequences are indicated by dashes. Boundaries of {alpha}-helices in human Mb or GAIP, based on its crystal structure (52, 53), are depicted as boxes above or below the sequence, respectively. The secondary structure prediction for human Ngb obtained with the program PHD (54) is shown above the sequence (boxes for {alpha}-helices and continuous lines for the rest). Residues in RGS4 and RGS9 that are involved in contacts (<4.0 Å) with G{alpha}i1 and the G{alpha}i/t chimera, respectively, are blue (55, 56). Cysteine residues of Ngb are highlighted in green, and distal and proximal histidine residues of Ngb are highlighted in purple. The primary sequences used in the alignment are human Mb (154-amino acid (aa) protein, GenBankTM accession number NP_005359 [GenBank] ), human Ngb (151 aa, GenBankTM NP_067080 [GenBank] ), human GRK2 (689 aa, GenBankTM accession number P25098 [GenBank] ), human GRK4 (578 aa, GenBankTM accession number P32298 [GenBank] ), human GRK5 (590 aa, GenBankTM accession number P34947 [GenBank] ), human GRK6 (576 aa, GenBankTM accession number P43250 [GenBank] ), human RGS1 (196 aa, GenBankTM accession number Q08116 [GenBank] ), human RGS4 (205 aa, GenBankTM accession number P49798 [GenBank] ), human RGS9 (443 aa, GenBankTM accession number NP_003826 [GenBank] ), and human GAIP (217 aa, GenBankTM accession number CAA62919 [GenBank] ).

 

The mammalian brain accounts for up to 20% of the total oxygen consumption, even though it constitutes only 2% of total body weight, and it is the most sensitive organ to the effects of tissue hypoxia (9). Ngb is widely expressed in the cerebral cortex, hippocampus (CA1, CA2, CA3, and CA4, especially in the pyramidal layer), thalamus, hypothalamus, and cerebellum (1, 3, 10) of the rat brain. Recently, it has been suggested that Ngb plays a role in the neuronal response to hypoxia and ischemia (11, 12). Ngb expression was reported to increase in response to neuronal hypoxia in vitro and focal cerebral ischemia in vivo (11, 12). Neuronal survival following hypoxia was reduced by inhibiting Ngb expression with an antisense oligodeoxynucleotide and was enhanced by Ngb overexpression, supporting the notion that Ngb protects neurons from hypoxicischemic insults (11). Moreover, Ngb protected the brain from experimental stroke in vivo (12).

A possible mechanism by which Ngb protects these neurons is by functioning as an O2 carrier, facilitating the diffusion of O2 to the mitochondria within cells that are engaging in active aerobic metabolism, in a manner similar to the way Mb acts in muscle cells. However, Ngb has been estimated to comprise less than 0.01% of the total protein content in the brain (1). The low concentration (in the micromolar range) of Ngb in brain tissue perhaps argues against its role in storing and carrying significant amounts of O2. On the other hand, local concentrations of Ngb may reach sufficiently high levels to allow it to regulate local oxygen consumption (10, 13). Finally, although debatable, the affinity of Ngb for oxygen may be so high as to prevent its release under physiological conditions (7, 8). Thus, the mechanism by which Ngb affords neuroprotection under oxidative stress conditions such as ischemia and reperfusion remains unclear.

The objective of this study was to investigate whether Ngb has novel functions that are related to neuroprotective roles under oxidative stress. On line BLAST searches were performed via the website of the National Center for Biotechnology Information (Conserved Domain Database, www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; www.ncbi.nlm.nih.gov/BLAST). These analyses revealed that human Ngb has 25–35% amino acid sequence homology with regulators of G protein signaling (RGS) and RGS domains of G protein-coupled receptor kinases (GRK) (Fig. 1). The protein that most closely resembles Ngb (24% amino acid identity) is GRK4 (Fig. 1). RGS and GRK proteins are modulators of heterotrimeric G proteins (14–17). Heterotrimeric G proteins (G proteins) consist of an {alpha} subunit (G{alpha}) with GTPase activity and a {beta}{gamma} dimer (G{beta}{gamma}) and belong to a family of proteins, whose signal transduction function depends on the binding of guanine nucleotides (18–23). Ligand- or signal-activated G protein-coupled receptors (GPCRs) induce GDP release from a G{alpha} subunit, which is followed by the binding of GTP. Binding of GTP to G{alpha} "turns on" the system and causes conformational changes that result in dissociation of the GTP-bound G{alpha} from both the receptor and G{beta}{gamma}. The GTP-bound G{alpha} and G{beta}{gamma} can then regulate the activity of different effector molecules, such as adenylyl cyclase, phospholipase C{beta}, and ion channels. Signal transduction is "turned off" by the intrinsic GTPase activity of the G{alpha} protein, which hydrolyzes the bound GTP to GDP, inducing the reassociation of GDP-bound G{alpha} with G{beta}{gamma}. The on/off G protein ratio can be regulated by three groups of protein modulators: guanine nucleotide exchange factors (GEFs), which stimulate GDP dissociation and subsequent GTP binding; guanine nucleotide dissociation inhibitors (GDIs), which inhibit GDP dissociation; and GTPase-activating proteins (GAPs), which enhance GTP hydrolysis (18–23). GPCRs play a role as functional analogues of GEFs (18, 20, 21). GRKs phosphorylate agonist-activated forms of GPCRs to induce homologous desentsitization of signaling pathways (14, 15). RGS proteins act as GAPs for G{alpha}i or G{alpha}o and play a role in desensitization (16, 17).

In the present study, we examined the possibility of interaction of Ngb with G{alpha} by surface plasmon resonance (SPR) measurements. Ferric Ngb interacted exclusively with G{alpha}i in their GDP-bound forms. In GDP dissociation assays or GTP{gamma}S binding assays, ferric Ngb exhibited GDI activity, inhibiting the rate of exchange of GDP for GTP by G{alpha}i. Since ferrous ligand-bound Ngb under normoxia did not have GDI activities, human Ngb may function as a novel oxidative stress-responsive sensor for signal transduction in the brain.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Samples—Rat myristoylated G{alpha} subunits (G{alpha}i1, G{alpha}i2, G{alpha}i3, and G{alpha}o; Calbiochem) and bovine G{beta}{gamma} (Calbiochem) were used. [35S]GTP{gamma}S (>1000 Ci/mmol), [8-3H]GDP (10–15 Ci/mmol), and [{alpha}-32P]GTP (~3000 Ci/mmol) were purchased from Amersham Biosciences (Buckinghamshire, England).

Preparation of Proteins—Amplification of human Ngb cDNA was performed by PCR using human universal Quick-clone cDNA (Clontech, Palo Alto, CA). Human Ngb cDNA was cloned into plasmid PET20b (Novagen, Madison, WI) and was sequenced using an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Overexpression of human Ngb was induced in Escherichia coli strain BL 21 (DE 3) (Novagen) by treatment with isopropyl {beta}-D-thiogalactopyranoside for 4 h. Purification of Ngb without His6-tag was carried out as follows (24). Soluble cell extract was loaded onto a DEAE-Sepharose anion-exchange column equilibrated with 20 mM Tris-HCl, pH 8.0. Ngb was eluted from the column with buffer containing 75 mM NaCl and was further purified by passage through a Sephacryl S-200 HR gel filtration column. Human Ngb mutants, including a COOH-terminal tag of six histidine residues (His6-tag), were purified on nickel affinity columns (His·Bind® resin; Novagen) from the supernatant of lysed cells using the protocol provided by Novagen.

Mass spectrometric measurements of purified Ngbs were performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (PerSpective Biosystems VoyagerTM DE PRO-S.D., Applied Biosystems), and Edman degradation was carried out on purified Ngbs using a G1005A protein sequencing system (Hewlett-Packard, Palo Alto, CA) at Takara Biomedicals, Inc. to determine their NH2-terminal sequences.

Ferric Ngbs were incubated with 2 mM dithiothreitol (DTT) for 2 h, and then DTT was removed by chromatography using a PD-10 column (Amersham Biosciences). Ferrous-CO Ngbs were generated after addition of sodium dithionite and CO gas to the DTT-treated ferric Ngb followed by gel filtration.

Site-directed Mutagenesis—A QuikChangeTM site-directed mutagenesis system (Stratagene, La Jolla, CA) was used to alter cysteine residues (amino acid residues 46, 55, and/or 120) in human Ngb. The point mutations were confirmed by DNA sequencing using BigDye terminator cycle sequencing FS (Applied Biosystems) and an ABI 3100 genetic analyzer (Applied Biosystems).

SPR Experiments—SPR measurements were performed on a BIAcore® X Instrument (Biacore, Uppsala, Sweden). Rat myristoylated G{alpha} subunit (G{alpha}i1, G{alpha}i2, G{alpha}i3, or G{alpha}o) was immobilized on the surface of a CM5 sensor chip using an amine coupling kit (Biacore) according to the instructions of the manufacturer. Activation of the carboxymethylated dextran in the CM5 sensor chip was carried out by mixing equal volumes of 400 mM N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide in water and 100 mM hydrochloride/N-hydroxysuccinimide in water, and injecting the mixture into the instrument at 10 µl/min for 7 min. This was followed by the injection of 5 µg/µl G{alpha} protein dissolved in 10 mM acetate buffer, pH 4.5, over the activated surface of the sensor chip for 7 min at a flow rate of 10 µl/min. The unreacted sites of the sensor chip were masked by the injection for 7 min of 1 M ethanolamine, pH 8.5. After immobilization, nonspecifically bound protein was removed by washing with running buffer (10 mM Hepes, 150 mM NaCl, 0.005% Tween 20, pH 7.4) until the value of the resonance units (RU) became nearly constant.

All binding experiments were performed at 25 °C at a flow rate of 5 µl/min. Ferric or ferrous-CO Ngb in the running buffer was injected for 5 min, during which association occurred. Dissociation then took place in the running buffer over the next 10 min. The BIAcore response was expressed in relative RU, i.e. the difference in response between flow cell with immobilized protein and the control flow channel. 1000 RU corresponded to 1 ng/mm2 of bound ligand. For binding analyses in the presence of guanine nucleotides, the running buffer containing 5 mM MgSO4 and either 500 µM GDP, or 500 µM GDP plus 500 µM AlCl3 and 10 mM NaF was loaded for 60 min to allow binding of guanine nucleotide to immobilized G{alpha}, after which the samples were injected. After each binding cycle, the sensor chip was regenerated with 5 µl of 0.05% SDS in the running buffer and was washed with running buffer for 5–10 min prior to the next injection. Experimental curves (sensorgrams) were analyzed by means of the BIAevaluation 3.1 software package using the model A + B {Leftrightarrow} AB to estimate the association and dissociation rate constants ka and kd.

GTP{gamma}S Binding Assays—100 nM G{alpha}i1 or G{alpha}o was incubated for 3 min at 25 °C in buffer A (20 mM Tris-HCl, 100 mM NaCl, and 10 mM MgSO4 at pH 8.0) with 10 µM GDP in the absence or presence of Ngb (5 µM). Binding assays were initiated with additions of 50 nM [35S]GTP{gamma}S (>1000 Ci/mmol). Aliquots (10 µl) were withdrawn from the binding mixtures and were passed through nitrocellulose filters (0.45 µm) (Millipore, Bedford, MA). The filters were then washed three times with 1 ml of ice-cold buffer A and were counted in a liquid scintillation counter (LSC-6100; Aloka, Tokyo, Japan). The apparent rate constant (kapp) values for the binding reactions were calculated by fitting the data to the following equation: GTP{gamma}S binding (%) = 100% x (1–e–kt).

GDP Dissociation Assays—G{alpha}i1 complexed with [3H]GDP (0.3 µM) was prepared by incubating 0.3 µM G{alpha}i1 with 2 µM [3H]GDP in buffer A for 1.5 h at 25 °C. Excess unlabeled GTP or GDP (200 µM) was added to monitor dissociation of [3H]GDP from G{alpha}i1 in the absence or presence of Ngb (5 µM). Aliquots were withdrawn at the indicated times and were passed through nitrocellulose filters (0.45 µm) (Millipore, Bedford, MA). The filters were then washed three times with 1 ml of ice-cold buffer A and were counted in a liquid scintillation counter (LSC-6100; Aloka). As for preparation of G{alpha}i1 complexed with [{alpha}-32P]GDP (1 µM), 1 µM G{alpha}i1 and 2 µM [{alpha}-32P]GTP were incubated in buffer A for 1.5 h at 25 °C. Experiments using G{alpha}i1 complexed with [{alpha}-32P]GDP were also performed, as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SPR Detection of Human Ferric Ngb Binding to G{alpha}—Proteins that interacted with human Ngb were sought by SPR experimentation. SPR is a powerful tool for real time measurement of direct protein-protein interactions that do not require labeling of the proteins. We covalently coupled rat G{alpha}i1, which is highly expressed in the brain (18), to a sensor chip. As positive and negative controls we used G{beta}{gamma} and Mb, respectively, and confirmed that G{beta}{gamma} interacts with GDP-bound G{alpha}i1, but Mb does not bind to G{alpha}i1 by SPR (see Supplemental Material). Then we characterized the interaction between Ngb and G{alpha}i1 by SPR. A representative sensorgram in Fig. 2A shows that the resonance response reflecting G{alpha}i1-ferric Ngb interaction occurred in an analyte concentration-dependent manner. In the association phase (0~300 s), the intensity of SPR increased, indicating that ferric Ngb bound to G{alpha}i1 specifically, while in the dissociation phase (300~900 s), the intensity of SPR decreased, indicating that ferric Ngb dissociated from the immobilized G{alpha}i1. Binding parameters for the interaction of ferric Ngb with G{alpha}i1 were determined to be as follows: association rate constant, ka = 5.0 x 102 M–1 s–1; dissociation rate constant, kd = 3.0 x 10–4 s–1; and equilibrium dissociation constant, Kd = kd/ka = 6.0 x 102 nM. No significant resonance signals were obtained from sensor chip surfaces that did not have attached ligands (data not shown), indicating an absence of nonspecific interactions between the sensor chip surfaces and analytes.



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  		FIG. 2.
SPR analyses of human Ngb binding to G protein {alpha}-subunit G{alpha}i1. A, concentration dependence of ferric Ngb on binding affinities with G{alpha}i1.G{alpha}i1 was immobilized to a CM5 sensor chip. The immobilization level of G{alpha}i1 was 10,000 RU. The on and off processes for ligand binding were recorded on a BIAcoreX. The bars at 0 and 300 s indicate the start of injection of ligand (association phase) and the start of injection of buffer alone (dissociation phase), respectively. Concentrations of Ngb used were 1, 2, 4, 6, 8, and 10 µM. B, SPR analyses of ferric or ferrous-CO Ngb binding to G{alpha}i1. The concentrations of Ngbs were 4 µM. C, effects of a guanine nucleotide (GDP or GDP plus ) on the interaction between Ngb and G{alpha}i1. The concentration of Ngb was 3 µM.

 

Next we investigated the possibility of interaction of ferrous-O2 Ngb with G{alpha}i. Since ferrous-O2 Ngb is unstable and is converted into ferric Ngb very rapidly due to its autoxidation (7), stable ferrous-CO Ngb was used for SPR experiments. As shown in Fig. 2B, the binding affinity of ferrous-CO Ngb to G{alpha}i1 was significantly low (Kd > 1 mM) as compared with that of ferric Ngb.

Moreover, further SPR measurements clarified that human ferric Ngb binds to G{alpha}i2, G{alpha}i3, and G{alpha}o (Kd = 5.8 x 102, 5.5 x 102, and 6.1 x 102 nM, respectively), whereas ferrous-CO Ngb does not bind them (Kd > 1 mM).

Ferric Ngb Interacts Exclusively with the GDP-bound Form of G{alpha}—Next we investigated guanine nucleotide dependence of the binding of G{alpha}i1 to Ngb by SPR measurements. As shown in Fig. 2C, ferric Ngb bound to G{alpha}i1, even in the presence of Mg2+ and GDP. The binding parameters (ka = 1.1 x 103 M–1 s–1, kd = 6.8 x 10–4 s–1, Kd = 6.0 x 102 nM) were almost the same as those seen in the absence of Mg2+ and GDP. Aluminum tetrafluoride , together with Mg +, can interact with G{alpha}i1-bound GDP and mimic GTP and thereby activate G{alpha}i1 (21, 22). In the presence of Mg2+, GDP, and , ferric Ngb did not bind to the activated G{alpha}i1 (Fig. 2C). Therefore, human ferric Ngb clearly interacts exclusively with the inactive (GDP-bound) form of G{alpha}i1.

Effects of Ngb on GTP{gamma}S Binding to G{alpha}—Since our SPR data suggested that human ferric Ngb interacts with GDP-bound G{alpha}i1 but does not interact with activated GTP-bound G{alpha}i1, we hypothesized that Ngb may function as a GEF or GDI for G{alpha}i1. To determine whether ferric Ngb functions as a GEF or a GDI, we performed GTP{gamma}S (a nonhydrolyzable analog of GTP) binding experiments. Increased GTP{gamma}S binding to G{alpha}i1 would imply ferric Ngb is a GEF, whereas decreased binding would imply that ferric Ngb is a GDI.

As shown in Fig. 3A, G{alpha}i1 bound GTP{gamma}S due to spontaneous guanine nucleotide exchange (kapp = 0.081 min–1). In the presence of ferric Ngb, the rate of GTP{gamma}S binding to G{alpha}i1 was reduced 6.2-fold (kapp = 0.013 min–1) (Fig. 3A), implying that ferric Ngb functions as a GDI for G{alpha}i. On the other hand, ferrous-CO Ngb had no effect on the GTP{gamma}S binding (kapp = 0.078 min–1) (Fig. 3A).



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  		FIG. 3.
Effects of human Ngb on GTP{gamma}S binding to G{alpha}i/o. The binding of GTP{gamma}S to 100 nM G{alpha}i1 (A) and G{alpha}o (B) in the absence ({circ}) or presence of 5 µM ferric (•) or ferrous-CO Ngb ({blacktriangleup}) was initiated by the addition of 50 nM [35S]GTP{gamma}S (>1000 Ci/mmol). G{alpha}i1- or G{alpha}o-bound GTP{gamma}S was counted by withdrawing aliquots at the indicated times and passing through nitrocellulose filters (0.45 µm).

 

Moreover, ferric Ngb inhibited the rate of GTP{gamma}S binding to G{alpha}o by 10-fold; in contrast ferrous-CO Ngb had no effect (kapp = 0.040, 0.004, and 0.038 min–1 in the absence and presence of ferric and ferrous-CO Ngb, respectively) (Fig. 3B). These results imply that ferric Ngb is a GDI for G{alpha}o as well as G{alpha}i.

Ferric Ngb Acts as a GDI—We then addressed the mechanism by which human ferric Ngb inhibited GTP{gamma}S binding to G{alpha}i and G{alpha}o. The inhibition of GTP{gamma}S binding to G{alpha}i/o by ferric Ngb may reflect a reduction in the rate of nucleotide exchange. To examine the effects of ferric Ngb on the release of GDP from G{alpha}i1, we measured the rates of GDP dissociation in the absence or presence of ferric Ngb. In the presence of an excess amount of unlabeled GTP, [3H]GDP release from [3H]GDP-bound G{alpha}i1 was inhibited by ferric Ngb (Fig. 4A). The inhibition of GDP dissociation by ferric Ngb suggests that ferric Ngb diminished the rates of spontaneous GTP{gamma}S binding to G{alpha}i and G{alpha}o by blocking the GDP release. In other words, ferric Ngb functions as a GDI for G{alpha}i.



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  		FIG. 4.
Effects of Ngb on dissociation of GDP from GDP-bound G{alpha}i1. A, experiments performed in the presence of an excess amount of unlabeled GTP. G{alpha}i1 complexed with [3H]GDP was obtained as described under "Experimental Procedures." An excess of unlabeled GTP (200 µM) was added to the G{alpha}i1:[3H]GDP complex in the absence or presence of 5 µM Ngb. Aliquots were withdrawn at 0, 5, and 10 min and passed through nitrocellulose filters (0.45 µm). Each error bar represents the S.D. of three to four independent experiments. B, experiments performed in the presence of an excess amount of unlabeled GDP. Experimental conditions were as in A, except for the addition of an excess amount of unlabeled GDP (200 µM).

 

The most representative GDIs for heterotrimeric G proteins share conserved sequence repeats named the G protein regulatory (GPR) (25) or GoLoco motifs (26). One of the family, Purkinje cell protein-2 (Pcp2), can modulate GDP binding to G{alpha}o and G{alpha}i (27, 28). In the presence of excess unlabeled GTP, Pcp2 preferentially interacts with the GDP-bound conformation of G{alpha} and serves exclusively as a GDI as does human ferric Ngb (28, 29). On the other hand, in the presence of excess unlabeled GDP, Pcp2 was reported to stimulate GDP release from G{alpha}o (27). To further characterize properties of Ngb as a GDI, we performed GDP dissociation assays in the presence of excess GDP. As shown in Fig. 4B, ferric Ngb stimulated [3H]GDP release from [3H]GDP-bound G{alpha}i1 in the presence of an excess amount of unlabeled GDP, suggesting that the mechanism of ferric Ngb as a GDI is similar to that of Pcp2. Experiments using [{alpha}-32P]GDP instead of [3H]GDP also supported these results (data not shown).

Functional Analyses of Ngb with an Intra- or Intermolecular Disulfide Bond—Cysteine (Cys) residues are particularly sensitive to oxidation by almost all forms of reactive oxygen species during ischemia and reperfusion (30). Under even mild oxidative conditions, Cys residues are converted to disulfides (31). Human Ngb has three Cys residues at positions 46, 55, and 120 (Fig. 1). Cys55 and Cys120 are conserved among mammalian Ngbs, whereas Cys46 is specific for human Ngb. We investigated whether human wild-type Ngb before DTT treatment forms a disulfide bond. Fig. 5A shows the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) that was run under nonreducing conditions. The protein treated with DTT migrated slower than the untreated protein. Since it has been reported that a protein containing an intramolecular disulfide linkage has a smaller radius of gyration and migrates further down a gel (32), these data suggest that ferric Ngb forms a disulfide bond between Cys46, Cys55, or Cys120 spontaneously upon exposure to air.



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  		FIG. 5.
SDS-PAGE analyses of human Ngbs under nonreducing conditions. A, analysis of human wild-type Ngb. Human wild-type Ngb was incubated in the absence or presence of 2 mM DTT for 2 h at 25 °C. The samples were analyzed under nonreducing conditions on 12.5% SDS-polyacrylamide gels and stained by Coomassie Blue. Molecular size markers are given to the left (in kilodaltons). B, analysis of human Ngb mutants (triple mutant (C46S,C55S,C120S) and double mutant (C55S,C120S)) with COOH-terminal His6-tag under nonreducing conditions. Experimental conditions were the same as in A.

 

To investigate the role of a disulfide bond in the functioning of Ngb, Ngb Cys -> Ser mutants (three single mutants (C46S; C55S; C120S), three double mutants (C46S,C55S; C46S,C-120S; C55S,C120S), and a triple mutant (C46S,C55S,C120S)) with COOH-terminal His6-tag were prepared. The C120S Ngb mutant formed a disulfide bond as did wild-type Ngb, while the C46S and C55S mutants did not (data not shown), suggesting that the intramolecular disulfide bond between Cys46 and Cys55 is present in human wild-type Ngb. As shown in Fig. 5B, we found that a double mutant (C55S,C120S) forms a dimer. This dimer was converted into a monomer by incubation with DTT (Fig. 5B), indicating that the intermolecular disulfide bond Cys46-Cys46 was present in this mutant.

Next we investigated GDI activities of these Ngbs with the intra- or intermolecular disulfide bond. As shown in Fig. 6A, human wild-type ferric Ngb with the intramolecular disulfide bond inhibited GDP release as did the DTT-reduced ferric Ngb. Double mutant (C55S,C120S) homodimer linked by the intermolecular disulfide bond did not inhibit GDP/GTP exchange of G{alpha}i1, whereas its DTT-reduced monomeric form inhibited GDP/GTP exchange of G{alpha}i1 (Fig. 6A), implying that formation of the intermolecular Cys46-Cys46 disulfide bond in the double mutant blocks binding sites with G{alpha}i1. As a control, triple mutant (C46S,C55S,C120S), which cannot form a disulfide bond, acted as a GDI with or without DTT (Fig. 6A). As shown in Fig. 6B, in the presence of excess unlabeled GDP, only double mutant (C55S,C120S) homodimer could not stimulate [3H]GDP release from [3H]GDP-bound G{alpha}i1. These data also imply that residues around Cys46 are important for GDI activities of human ferric Ngb.



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  		FIG. 6.
Effects of disulfide bond formation on GDI activities of ferric Ngbs. A, experiments were performed in the presence of an excess amount of unlabeled GTP. Human wild-type Ngb and Ngb mutants (triple mutant (C46S,C55S,C120S) and double mutant (C55S,C120S) with COOH-terminal His6-tag) were used. Percentages of [3H]GDP-bound G{alpha}i1 at 5 min are shown. The concentrations of Ngbs were 5 µM. Experimental conditions were the same as in Fig. 4A. B, experiments performed in the presence of an excess amount of unlabeled GDP. Experimental conditions were the same as in Fig. 4B.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
During ischemia and reperfusion, overproduction of nitric oxide (NO) occurs due to induction of expression of NO synthetases (33). Reactive oxygen species, which have been identified as central mediators in certain signaling events, are also generated (34). Reaction of ferrous-O2 Mb or Hb with NO and/or reactive oxygen species converts these proteins to their ferric forms (35–38). Thus, ferrous-O2 Ngb may become ferric Ngb during ischemia and reperfusion. In fact, Ngb has been reported to have a surprisingly high rate of auto-oxidation (7). Moreover, a recent histochemical study suggested that Ngb transcript exists in the brain areas important for adaptive responses to stressful events and that Ngb and NO synthetase are co-expressed in a number of nuclei (39). Our results showed that ferric Ngb functions as a novel GDI for G{alpha}i/o in vitro. The interaction of G{alpha}i with ferric Ngb will liberate G{beta}{gamma}, leading to protection against neuronal death. Since ferrous ligand-bound Ngb, which is a normal form under normoxia, does not have GDI activities, Ngb may act as an oxidative stress-responsive sensor for signal transduction in the brain (Fig. 7).



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  		FIG. 7.
Human Ngb as an oxidative-stress responsive sensor for signal transduction in the brain.

 

Structure and Function of Ngb, the Hypoxia-regulated Sensor—In this study, we showed that human Ngb, which contains weak homology to RGS domains, functions as a novel GDI in vitro. Human ferric Ngb binds exclusively to the GDP-bound form of G{alpha}i and inhibits GDP/GTP exchange of G{alpha}i. On the other hand, ferrous-CO Ngb did not interact with G{alpha}i and did not have GDI activities. Therefore, it is theorized that the regulation of interaction of Ngb with G{alpha}i is dependent on oxidation/reduction state and ligand binding of Ngb.

Nonsymbiotic plant hemoglobins (nsHbs) have some characteristics that are similar to Ngb including the fact they are both members of a newly discovered class of "hexacoordinated" globins and are expressed at low levels (1, 40–43). Hypoxia induces the expression of nsHbs as well as Ngb (44, 45). While the three-dimensional structure of Ngb has not as yet been determined, that of a nsHb (rice Hb1) has been determined (40). In the hexacoordinated structure (Fe3+) of rice Hb1, electron density of the Phe B10 in the distal pocket is very low because of substantial disorder, and it has been suggested that the entire CD corner is disordered because of distal His binding to the heme iron (40). Crystallographic modeling suggests that ligand binding occurs by an upward and outward movement of the E helix, a concomitant dissociation of the distal histidine, a possible repacking of the CD corner, and folding of the D helix (40).

It is tempting to speculate that Ngb undergoes large tertiary structural changes around the CD corner and D helix when its ferrous-O2 form is converted into bis-His conformation (hemichromogen or hemochromogen), as described for rice Hb1 (40). Furthermore, it can be speculated that the structural change around the CD corner and D helix triggers changes in affinity for the binding of G{alpha}i if that binding occurs in the CD region and D helix. In fact, in this study we have demonstrated that Ngb double mutant (C55S,C120S) homodimer linked by the intermolecular disulfide bond at Cys46-Cys46 cannot function as a GDI, whereas its DTT-reduced monomeric form acts as a GDI, suggesting that the CD corner in which Cys46 exists is important for protein-protein interaction between ferric Ngb and G{alpha}i1 and is responsible for GDI activities for G{alpha}.

It should also be noted that a disulfide bond between Cys46 and Cys55 of Ngb is located at the CD corner and D helix. Therefore, the S–S bond formed during hypoxia may contribute to the stabilization of structures near the CD corner and D helix of oxidized Ngb, which is the active form for signal transduction in the brain, since it has been reported that introduction of a disulfide bond enhances the thermal and conformational stability of proteins (46, 47). Because Cys46 is present in human Ngb but is not present in other mammalian Ngbs, human Ngb might have evolved to stabilize the active form during hypoxia.

Novel Brain-specific Signaling Pathway under Oxidative Stress—It has recently been reported that G{alpha}i and G{alpha}o are direct target proteins of reactive oxygen species generated during ischemia and reperfusion and that they are activated in the absence of GPCR-mediated signaling (48, 49). Reactive oxygen species modify two cysteine residues of G{alpha}i and G{alpha}o (49). Modification of G{alpha}i and G{alpha}o accelerates GDP release from G{alpha} and increases the formation of the GTP-bound form of G{alpha} without receptor activation (48, 49).

We have shown here that human ferric Ngb, which may be produced under oxidative stress conditions such as ischemia and reperfusion, functions as a GDI that keeps G{alpha}i or G{alpha}o in its inactive state as does Pcp-2 (GPR, GoLoco). The interaction of G{alpha}i/o with ferric Ngb under oxidative stress is a novel brain-specific signaling pathway (Fig. 7). Since GPR/G{alpha}i/o interaction prevents G{beta}{gamma} from returning to G{alpha} and thus leads to enhanced G{beta}{gamma}-dependent signaling (28, 50), human ferric Ngb would selectively shut off signaling pathways linked to G{alpha} effectors and favor G{beta}{gamma} effector pathways. The intracellular signal transduction induced by G{beta}{gamma} protects the cells against oxidative stress (51): G{beta}{gamma} stimulates proliferation via mitogen-activated protein kinase (MAPK) pathways and promotes cell survival by the activation of phosphotidylinositol 3-kinase. Taken together, the characteristic of human ferric Ngb as a GDI may play important roles in neuroprotective function of human Ngb in the brain. Further study will be necessary to understand the physiological significance of the interaction of Ngb with G{alpha} in the brain.


   FOOTNOTES
 
* This work was supported in part by Grants-in-aid 13780532 and 15770085 for Young Scientists (B) (to K. W.), a Grant-in-aid 12215077 for Scientific Research on Priority Areas (to K. W.), and a Grant-in-aid 12002008 for Specially Promoted Research (to I. M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure. Back

To whom correspondence should be addressed: Dept. of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan. Tel.: 81-75-383-2537; Fax: 81-75-383-2541; E-mail: kei{at}wakasugi.mbox.media.kyoto-u.ac.jp.

1 The abbreviations used are: Ngb, neuroglobin; ferrous-O2 Ngb, ferrous oxygen-bound Ngb; ferrous-CO Ngb, ferrous carbon monoxide-bound Ngb; G protein, guanine nucleotide-binding protein; Hb, hemoglobin; nsHb, nonsymbiotic plant Hb; Mb, myoglobin; GRK, G protein-coupled receptor kinase; RGS, regulator of G protein signaling; GPCR, G protein-coupled receptor; GEF, guanine nucleotide exchange factor; GDI, guanine nucleotide dissociation inhibitor; GAP, GTPase-activating protein; DTT, dithiothreitol; SPR, surface plasmon resonance; RU, resonance units; GTP{gamma}S, guanosine 5'-O-(3-thio)triphosphate; Pcp2, Purkinje cell protein-2; GPR, G protein regulatory; GoLoco, G{alpha}i/o-Loco interaction; GAIP, G{alpha}-interacting protein; aa, amino acids. Back



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