Advertisement

Neurotrophic Activity of Neudesin, a Novel Extracellular Heme-binding Protein, Is Dependent on the Binding of Heme to Its Cytochrome b5-like Heme/Steroid-binding Domain*

Open AccessPublished:December 04, 2007DOI:https://doi.org/10.1074/jbc.M706679200
      Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme-binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b5-like heme/steroid-binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402 nm. The addition of exogenous hemin extensively increased the amount of hemin-bound neudesin. In contrast, neudesinΔHBD, a mutant lacking the heme-binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin-hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The neurotrophic activity of neudesin was enhanced by the binding of Fe(III)-protoporphyrin IX, but neither Fe(II)-protoporphyrin IX nor protoporphyrin IX alone. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti-neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme-affinity column at all. Neudesin is the first extracellular heme-binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme-binding proteins.
      Heme is a key component of many biochemical reactions. Heme serves as a prosthetic group of heme-binding proteins such as hemoglobins, cytochromes, and guanylate cyclases. It is involved in important transportation, catalytic, electron transfer, and signaling activities. On the other hand, as heme is able to intercalate into lipid membranes and participate in the Feton reaction in the production of hydroxyl radicals, it is a potent catalyst for injury due to hydrogen peroxide, oxidized low-density lipoprotein, and activated neutrophils (
      • Balla G.
      • Jacob H.S.
      • Balla J.
      • Rosenberg M.
      • Nath K.
      • Apple F.
      • Eaton J.W.
      • Vercellotti G.M.
      ,
      • Balla G.
      • Jacob H.S.
      • Eaton J.W.
      • Belcher J.D.
      • Vercellotti G.M.
      ). Heme-mediated oxidative stress is suggested to contribute to various pathologic inflammatory conditions including neuro-degenerative disorders (
      • Smith M.A.
      • Hirai K.
      • Hsiao K.
      • Pappolla M.A.
      • Harris P.L.
      • Siedlak S.L.
      • Tabaton M.
      • Perry G.
      ). However, heme is almost always trapped by proteins in living cells to avert its toxicity (
      • Hentze M.W.
      • Muckenthaler M.U.
      • Andrews N.C.
      ). Under physiological conditions, free heme in plasma binds to the sole extracellular heme-binding protein hemopexin. The binding of extracellular heme to hemopexin prevents strong oxidative features and proinflammatory effects of free heme (
      • Hrkal Z.
      • Vodrazka Z.
      • Kalousek I.
      ,
      • Camejo G.
      • Halberg C.
      • Manschik-Lundin A.
      • Hurt-Camejo E.
      • Rosengren B.
      • Olsson H.
      • Hansson G.I.
      • Forsberg G.B.
      • Ylhen B.
      ,
      • Jeney V.
      • Balla J.
      • Yachie A.
      • Varga Z.
      • Vercellotti G.M.
      • Eaton J.W.
      • Balla G.
      ).
      The intracellular heme-binding proteins are involved in many important cellular functions. For example, hemoglobin and myoglobin are among the most abundant heme-binding proteins, associated with oxygen carriers (
      • Burmester T.
      • Weich B.
      • Reinhardt S.
      • Hankeln T.
      ). Cytochromes are involved in electron transport and steroidogenesis (
      • Black S.M.
      • Harikrishna J.A.
      • Szklarz G.D.
      • Miller W.L.
      ). Guanylate cyclases catalyze the conversion of GTP to cGMP (
      • Denninger J.W.
      • Marletta M.A.
      ). Recently, it has been reported that the binding of heme to DGCR8, another heme-binding protein, is involved in microRNA processing (
      • Faller M.
      • Matsunaga M.
      • Yin S.
      • Loo J.A.
      • Guo F.
      ). Thus, heme-binding proteins may have unknown roles in biological functions.
      Neudesin is the novel secreted protein that we previously identified as a neurotrophic factor (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ,
      • Kimura I.
      • Konishi M.
      • Miyake A.
      • Fujimoto M.
      • Itoh N.
      ). Mouse neudesin exhibited high similarity (∼90% identity) to human and rat neudesins. Neudesin was expressed in heart, kidney, brain, and lung, but not in liver, in adult mice. Neudesin exhibited significant neurotrophic activity in primary cultured cortical neurons and neural precursor cells. The neurotrophic activity was mediated through the activation of mitogen-activated protein (MAP) and phosphatidylinositol 3-kinase (PI-3K) pathways. However, the molecular property of neudesin still remained to be elucidated.
      Neudesin possesses a predicted cytochrome b5-like heme/steroid-binding domain in its primary structure. Therefore, we examined whether neudesin binds hemin and, if so, whether the binding exerts a significant effect on the activity of neudesin in neuronal cells. We found that hemin bound to the cytochrome b5-like heme/steroid-binding domain of the neudesin molecule, and that Fe(III)- protoporphyrin IX is essential for the neurotrophic activity of neudesin but not Fe(II)- protoporphyrin IX or protoporphyrin IX. The present results suggested that neudesin is the first extracellular heme-binding protein found to be involved in intercellular signal transduction. These findings have revealed a new potential role of extracellular heme-binding proteins.

      EXPERIMENTAL PROCEDURES

      Production of Recombinant Mouse Neudesin Protein–Recombinant baculoviruses containing mouse neudesin cDNA or neudesinΔHBD cDNA with an identical tag sequence were obtained by cotransfection of Sf9 cells with a recombinant pBacPAK9 and a Bsu36I-digested expression vector, BacPAK6 (Clontech), as described elsewhere (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ). High Five cells infected with the recombinant baculovirus were cultured at 27 °C for 96 h in Grace's insect medium (Invitrogen) with 10% fetal bovine serum (FBS,
      The abbreviations used are: FBS
      fetal bovine serum
      DMEM
      Dulbecco's modified Eagle's medium
      BSA
      bovine serum albumin
      PBS
      phosphate-buffered saline
      TUNEL
      terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling
      MAPR
      membrane-associated progesterone receptor
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      ERK
      extracellular signal-regulated kinase
      MAP2
      microtubule-associated protein 2
      GFAP
      glial fibrillary acidic protein.
      Invitrogen). Recombinant mouse neudesin and neudesinΔHBD proteins were purified from the culture medium by affinity chromatography using Ni-NTA agarose (Qiagen) as described elsewhere (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ).
      Culture of Neuro2a Cells–Mouse brain neuroblastoma cells (Neuro2a) were seeded in DF (1:1 Dulbecco's modified Eagle's medium (DMEM), Ham's F12; Invitrogen) containing 1% penicillin-streptomycin solution (Invitrogen) on 35-mm dishes or 96-well plates. The cells were incubated at 37 °C in the conditioned medium containing 10% FBS in an atmosphere of 5% CO2. The cells were further cultured under various conditions.
      Culture of Mouse Cerebral Cortical Neurons–Mouse cerebral cortical neurons were cultured as described previously (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ,
      • Sawada H.
      • Kawamura T.
      • Shimohama S.
      • Akaike A.
      • Kimura J.
      ). In brief, mouse embryonic cerebral cortexes at E15.5 were mechanically dissociated into single cells in Hank's balanced salt solution. The dissociated cells were seeded in DF (1:1 DMEM, Ham's F12) containing 0.1 mg/ml kanamycin monosulfate (MP Bio Inc.) on 35-mm dishes or 96-well plates coated with 0.1% polyethyleneimine (MP Bio Inc.). The cells were incubated at 37 °C in conditioned medium containing 10% FBS in an atmosphere of 5% CO2. From the fourth day of culture, the cells were incubated in DMEM containing 10% FBS. The cells on the fifth day of culture were further incubated under various conditions indicated elsewhere.
      RNA Extraction and Reverse Transcription Polymerase Chain Reaction–Total RNA was extracted from Neuro2a cells by using an RNeasy Mini Kit (Qiagen). cDNA was transcribed from the RNA as a template with Molony murine leukemia virus reverse transcriptase (Invitrogen). The cDNA was amplified by polymerase chain reaction (PCR) with TaqDNA polymerase (Japan Gene) and primers specific for the genes examined. The primers used were 5′-GCCTGCTCCTCGCTGTCTAT-3′ and 5′-CCTAGAACCGGCTGCTTCTC-3′ for neudesin, and 5′-ACGCTGAGCCAGTCAGTGTA-3′ and 5′-CTTAGAGGGACAAGTGGCG-3′ for 18S ribosomal RNA as a control (
      • Zhu L.J.
      • Altmann S.W.
      ). The amplified DNA was analyzed by 1.5% agarose gel electrophoresis, and the gel was stained with ethidium bromide.
      Knockdown of Neudesin Expression by siRNA–Neuro2a cells were transfected with 10 nm siRNA (stealth, Invitrogen) against the first target neudesin (5′-AUCCAGUGACAUCUUGGCCACACCU-3′), the first target control neudesin (5′-AUCACCGUGAUACAGUUCACCGCCU-3′), the second target neudesin (5′-UGCUGAAGACGUCAUCGAGGGCCUC-3′), the second target control neudesin (5′-UGCCGUAGGACAUGACCUAGGGCUC-3′), the third target neudesin (5′-UCUUCAGGCUUGAAGUCCAGGUUGG-3′) and the third target control neudesin (5′-UCUUGUACGGUCGUAAUGCCGAUGG-3′) by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen). The knockdown of neudesin expression was examined by RT-PCR. The transfected cells were cultured in DF containing 10% FBS for 24 h and then in DF containing 1% N2 Supplement.
      Western Blotting–Neuro2a cells were seeded at a density of 0.4 × 105 cells/cm2 in 35-mm dishes. After being cultured in serum-free DF containing 0.1% BSA for 24 h, the cells were further cultured for 30 min in the presence of recombinant neudesin (10 ng/ml), neudesin-hemin (10 ng/ml), or hemin (100 nm). siRNA-transfected Neuro2a cells were cultured in DF containing 10% FBS for 24 h and then in DF containing 1% N2 supplement for 1 or 2 days. Mouse cerebral cortical neurons were seeded at a density of 1.3 × 105 cells/cm2 in 35-mm dishes coated with polyethyleneimine. The cells on the fifth day of culture were incubated in the conditioned medium with 0.1% bovine serum albumin (BSA) for 2 h and then treated with recombinant neudesin (10 ng/ml), neudesin-hemin (10 ng/ml), hemin (100 nm) or FGF2 (10 ng/ml) (Amgen) for 30 min. The cells were lysed in TNE buffer containing 10 mm Tris-HCl (pH7.4), 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 50 mm NaF, 2 mm Na3VO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 μg/ml pepstatin. Proteins in the cell lysate were resolved by SDS gel electrophoresis and blotted onto a nitrocellulose membrane. ERK1/2, Akt, and their phosphorylated forms were detected by Western blotting using antibodies. Primary antibodies used were as follows: rabbit antibodies against ERK1/2 (1:1000) (Cell Signaling), phosphorylated ERK1/2 (1:1000) (Cell Signaling), Akt (1:1000) (Cell Signaling), and phosphorylated Akt (1:1000) (Cell Signaling). The secondary antibody used was a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000) (VECTOR). Immunoreactive bands were visualized using an enhanced chemiluminescence detection system as described (
      • Sawada H.
      • Kawamura T.
      • Shimohama S.
      • Akaike A.
      • Kimura J.
      ,
      • Yamashita T.
      • Konishi M.
      • Miyake A.
      • Inui K.
      • Itoh N.
      ). ImageJ (National Institutes of Health) was used to quantify the integrated density of each band. Results are means ± S.E. from four independent experiments as described (
      • Shinohara H.
      • Udagawa J.
      • Morishita R.
      • Ueda H.
      • Otani H.
      • Semba R.
      • Kato K.
      • Asano T.
      ).
      Peroxidase Reaction Staining–Recombinant neudesin (1 mg/ml) was incubated with hemin (50 μm) at 24 °C for 30 min. Then, recombinant neudesin, recombinant neudesin with hemin and hemin were subjected to SDS-PAGE without reducing by dithiothreitol. Hemin-containing protein bands were visualized with the peroxidase substrates Chemiluminescence Reagent Plus (PerkinElmer) as described (
      • Thomas P.E.
      • Ryan D.
      • Levin W.
      ).
      Preparation of Neudesin-hemin and Neudesin-protoporphyrin IX–Recombinant neudesin (10 μg) was incubated with hemin (500 μm, if not indicated) or protoporphyrin IX (PP IX, 100 μm) at 24 °C for 30 min. The mixture was applied to a Sephadex™ PD-10 column (GE Healthcare) pre-equilibrated with phosphate-buffered saline (PBS) containing 10% dimethyl sulfoxide (Me2SO). Bound forms (neudesinhemin and neudesin-PP IX) were eluted from the column with PBS containing 10% Me2SO to be separated from hemin and PP IX, respectively. Reduced hemin [Fe(II)-protoporphyrin IX] was produced by treating hemin with sodium dithionite.
      MTT Assay–The survival of Neuro2a cells was evaluated by measuring the activity to reduce MTT. Neuro2a cells were seeded at a density of 0.75 × 105 cells/cm2 in 35-mm dishes. After being cultured in DF containing 10% FBS for 24 h, the cells were further cultured for 3 days in serum-free DF with recombinant neudesin, neudesin-hemin, or hemin. siRNA-transfected Neuro2a cells were seeded at a density of 0.2 × 105 cells/cm2 in 96-well plates. The cells were cultured in DF containing 10% FBS for 24 h and then in DF containing 1% N2 Supplement for 5 days. For MTT-reducing activity, the cells were treated with 0.5 mg/ml MTT (MTT Cell Count Kit; Nakalai) at 37 °C for 4 h. The reduction product, MTT-formazan, was solubilized with isopropyl alcohol. The absorption at 570 nm of each sample solution was measured as the MTT reducing activity of the cells.
      Cell Survival Activity Assay–Cultured mouse cerebral cortical cells were seeded at a density of 1.3 × 105 cells/cm2 in 35-mm dishes coated with polyethyleneimine. The cells on the fifth day of culture were treated with recombinant neudesin (10 ng/ml), neudesin-hemin (10 ng/ml), hemin (100 nm), or 10% horse serum (HS) for 4 days. For immunostaining, the cells were fixed with 4% paraformaldehyde (PFA), washed in PBS, and treated with 5% BSA in PBS. The cells were permeabilized with 0.1% Triton X-100 (Sigma) and immunostained with primary antibodies and secondary antibodies. The cells were treated with primary antibodies for 2 h at room temperature or overnight at 4 °C. After three washes for 5 min each in PBS, the cells were further treated with the secondary antibodies for 1 h at room temperature. The primary antibodies used were an anti-microtubule-associated protein 2 (MAP2) mouse monoclonal antibody (1:400; Sigma) and an anti-glial fibrillary acidic protein (GFAP) rabbit polyclonal antibody (1:80; Sigma). Anti-MAP2 antibody and anti-GFAP antibody were used as neuronal and astrocytic markers, respectively. The secondary antibodies used were rhodamine red-conjugated anti-rabbit IgG antibodies, rhodamine red-conjugated anti-mouse IgG antibodies, and fluorescein isothiocyanate-conjugated anti-mouse IgG antibodies (1:200 each; Sigma). Cell nuclei were counterstained with TOPRO3 (Molecular Probes), and the stained cells were observed under a confocal laser scanning microscope as described previously (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ,
      • Kimura I.
      • Konishi M.
      • Miyake A.
      • Fujimoto M.
      • Itoh N.
      ). Results are means ± S.E. for five different fields from four independent slides.
      TUNEL Assay–The presence of DNA breaks was evaluated by a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) procedure as described previously (
      • Druzhyna N.M.
      • Musiyenko S.I.
      • Wilson G.L.
      • LeDoux S.P.
      ). For detection and quantitation of apoptotic cells, the DeadEnd fluorometric TUNEL system (Promega) was used. It measures the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP at 3′-OH DNA ends using the enzyme terminal deoxynucleotidyltransferase. The assay was performed according to the manufacturer's direction. Briefly, Neuro2a cells were seeded at a density of 0.75 × 105 cells/cm2 in 35-mm dishes. The cells were cultured for 3 days in the presence of recombinant neudesin, neudesin-hemin, or hemin. siRNA-transfected Neuro2a cells were seeded at a density of 0.2 × 105 cells/cm2 in 35-mm dishes. The transfected Neuro2a cells were cultured in DF containing 10% FBS for 24 h and then in DF containing 1% N2 Supplement for 3 days. Cultured mouse cerebral cortical cells were seeded at a density of 1.3 × 105 cells/cm2 in 35-mm dishes coated with polyethyleneimine. The cells on the fifth day of culture were treated with neudesin (10 ng/ml), neudesin-hemin (10 ng/ml), hemin (100 nm), or 10% HS for 4 days. The cells were fixed in 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Thereafter, DNA strand breaks were labeled with fluorescein-12-dUTP by the terminal deoxynucleotidyltransferase enzyme, neurons were stained with anti-MAP2 mouse monoclonal primary antibody (1:400; Sigma) and rhodamine red-conjugated anti-mouse IgG secondary antibody (1:200; Sigma), and the nuclei were stained with TOPRO3 (Molecular Probes). The cells were observed under a confocal laser scanning microscope. Results are means ± S.E. for five different fields from four independent slides.
      BrdU Assay–Neuro2a cells were plated onto 96-well plates (1 × 103 cells/well) and cultured in DF supplemented with 10% FBS. After 24 h in culture, the cells were cultured in DF supplemented with 0.1% BSA for 24 h. The cells were treated with recombinant neudesin, neudesin-hemin, or hemin. After 16 h, the cells were further cultured for 8 h in the presence of 5-bromo-2′-deoxyuridine (BrdU; 10 μm). The mitogenic activity was determined from five independent wells using 5-bromo-2′-deoxyuridine Labeling and Detection Kit III (Roche Applied Science) as described elsewhere (
      • Hoshikawa M.
      • Yonamine A.
      • Konishi M.
      • Itoh N.
      ).
      Detection of Endogenous Mouse Neudesin–A rabbit anti-neudesin polyclonal antibody was raised against a peptide corresponding to amino acids 153-171 (PNLDFKPEDQPHFDIKDEF) of mouse neudesin. Endogenous neudesin was detected by using this anti-neudesin antibody by Western blotting. Neuro2a cells were plated onto 10-cm dishes (0.2 × 105 cells/cm2) and cultured in DF supplemented with 10% FBS. After 24 h, the cells were cultured in DF containing 1% N2 supplement for 4 days. The collected culture medium was concentrated by Amicon Centricon YM-10 (Millipore) (1:10). Heme affinity chromatography was performed as described previously (
      • Hvidberg V.
      • Maniecki M.B.
      • Jacobsen C.
      • Hojrup P.
      • Moller H.J.
      • Moestrup S.K.
      ). The concentrated culture medium was loaded on a matrix of cross-linked 4% beaded hemin-agarose (Sigma). After washing with HEPES buffer containing 2 mm CaCl2, 1 mm MgCl2, 10mm HEPES, and 0.14 m NaCl, pH7.8, bound proteins were eluted in 0.2 m CH3CO2Na, containing 250 mm imidazole (pH 4.0).

      RESULTS

      Primary Structure of Neudesin Includes a Heme/Steroid-binding Domain and Recombinant Mouse Neudesin Contains Hemin–The domain structure of neudesin was explored by inputting its amino acid sequence into a protein domain structure prediction program (Pfam) at the website. The results suggested that neudesin contains a heme/steroid-binding domain similar to the heme-binding domain of cytochrome b5 neudesin belongs to the membrane-associated progesterone receptor (MAPR) family, a subfamily of the cytochrome b5 family, along with PGC1 (membrane-associated progesterone receptor component 1; 25-Dx) (
      • Mifsud W.
      • Bateman A.
      ,
      • Krebs C.J.
      • Jarvis E.D.
      • Chan J.
      • Lydon J.P.
      • Ogawa S.
      • Pfaff D.W.
      ). The 134-amino acid human cytochrome b5 has a heme-binding domain (in green in Fig. 1A) in which His-44 and His-68 act as the ligand for the heme iron (
      • Nakanishi N.
      • Takeuchi F.
      • Okamoto H.
      • Tamura A.
      • Hori H.
      • Tsubaki M.
      ). The 195-amino acid human PGC1 has a heme-binding domain exhibited (in blue in Fig. 1A) in which Tyr-81, Tyr-87, and Tyr-111 act as the predicted ligands for the heme iron (
      • Min L.
      • Strushkevich N.V.
      • Harnastai I.N.
      • Iwamoto H.
      • Gilep A.A.
      • Takemori H.
      • Usanov S.A.
      • Nonaka Y.
      • Hori H.
      • Vinson G.P.
      • Okamoto M.
      ). The 172-amino acid human and 171-amino acid mouse neudesins have a signal sequence (31 and 30 amino acids, respectively) at the N terminus. Mouse neudesin has a predicted heme-binding domain (45∼143 amino acids). Moreover, we predicted that Tyr-81, Tyr-87, and His-111 in mouse neudesin act as the ligand for the heme iron (Fig. 1A).
      Figure thumbnail gr1
      FIGURE 1Primary structure of neudesin includes a heme/steroid-binding domain, and some recombinant mouse neudesin proteins contain heme. A, alignment of the entire sequences of mouse neudesin and human neudesin. Sequences of the heme-binding region of PGRC1 and cytochrome b5 were also aligned (blue and green, respectively). Homology was indicated by putting common residues in boxes. Asterisks indicate putative ligands for heme iron in neudesin. B, outline of the primary structure of mouse neudesin. The predicted signal sequence and heme/steroid-binding domain. neudesinΔHBD indicates a mutant neudesin lacking an essential part of the heme-binding domain. C, absorbance scan of recombinant neudesin protein before and after reduction with sodium dithionite and neudesinΔHBD protein. Neudesin exhibited a peak at 402 nm (blue) that shifted to 420 nm after reduction (red). D, solutions of recombinant neudesin and/or hemin were subjected to SDS/PAGE and stained by the peroxidase reaction.
      Therefore, we produced recombinant His6-fused mouse neudesin and neudesinΔHBD proteins. The recombinant His6-fused mouse neudesinΔHBD protein lacks the heme-binding domain (73-125 amino acids) that includes Tyr-81, Tyr-87, and His-111 (Fig. 1B). Recombinant mouse neudesin, but not neudesinΔHBD, was tinged with green and had a sharp absorbance peak at 402 nm. When recombinant neudesin was treated with sodium dithionite, the peak shifted to 420 nm, as is characteristic of the heme-binding protein (Fig. 1C).
      Several heme-binding proteins are associated with hemin so tightly that they can be detected as peroxidase reaction-stained bands even when subjected to electrophoresis in SDS-containing gels. Hence, recombinant neudesin was subjected to SDS-PAGE and subsequent staining by the peroxidase reaction after incubation with or without hemin. The 20-kDa band was only slightly detected in the case of recombinant neudesin, whereas, recombinant neudesin incubated with exogenous hemin was stained more without a change in its molecular mass (Fig. 1D).
      Association with Hemin Enhances Neurotrophic Activity of Neudesin in Neuro2a Cell–Next, to compare the neurotrophic activity of neudesin-hemin with that of recombinant neudesin, we produced neudesin-hemin by incubating recombinant neudesin with hemin in vitro (
      • Taketani S.
      • Adachi Y.
      • Kohno H.
      • Ikehara S.
      • Tokunaga R.
      • Ishii T.
      ). Recombinant neudesin exhibited significant neurotrophic activity in primary cultured mouse neurons and neural precursor cells (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ,
      • Kimura I.
      • Konishi M.
      • Miyake A.
      • Fujimoto M.
      • Itoh N.
      ). First, we examined the neurotrophic activity of neudesin-hemin in mouse neuroblastoma cells (Neuro2a cells) by conducting a MTT assay. Neudesin-hemin significantly enhanced the survival of Neuro2a cells in a dose-dependent manner. However, Neuro2a cells were not affected by recombinant neudesin (Fig. 2, A and B), different from primary cultured mouse neurons and neural precursor cells (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ,
      • Kimura I.
      • Konishi M.
      • Miyake A.
      • Fujimoto M.
      • Itoh N.
      ). We also examined the mitogenic activity of neudesin-hemin in Neuro2a cells by conducting a BrdU assay. We found that neudesin-hemin exhibited more significant mitogenic activity than recombinant neudesin (Fig. 2C). Moreover, to confirm the survival-promoting activity of neudesin-hemin, we performed a TUNEL assay. Neudesin-hemin, but not recombinant neudesin, significantly decreased the number of TUNEL-positive apoptotic cells (Fig. 2, D and E). Because the neurotrophic activity of neudesin in primary cultured neurons and neural precursor cells is mediated through the MAPK and PI-3K pathways as described previously (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ,
      • Kimura I.
      • Konishi M.
      • Miyake A.
      • Fujimoto M.
      • Itoh N.
      ), we examined the effect of neudesin-hemin on the phosphorylation of ERK1/2 and Akt also in Neuro2a cells. The phosphorylation of ERK1/2 and Akt was remarkably promoted by neudesin-hemin, but not by recombinant neudesin (Fig. 2F).
      Figure thumbnail gr2
      FIGURE 2Association with hemin enhances neurotrophic activity of neudesin in Neuro2a cells. A, Neuro2a cells were cultured for 1 day in serum-free medium and then treated with neudesin-hemin (0-50 ng/ml) for 3 days. The effect of neudesin-hemin on cell survival was examined using the MTT assay. Results are means ± S.E. for five independent wells. B, Neuro2a cells were cultured for 1 day and then treated with recombinant neudesin (20 ng/ml), neudesin-hemin (20 ng/ml), or hemin (200 nm) in serum-free medium for 3 days. The effect of neudesin-hemin on cell survival was examined using the MTT assay. Results are means ± S.E. for five independent wells. C, mitogenic activities of recombinant neudesin (20 ng/ml), neudesin-hemin (20 ng/ml) and hemin (200 nm) were examined by determining the incorporation of BrdU. Results are means ± S.E. for five independent wells. D, TUNEL analysis of Neuro2A cells in the absence or presence of neudesin-hemin (20 ng/ml). Green signals indicate TUNEL-positive cells. Blue signals indicate cell nuclei counterstained with TOPRO3. Scale bar, 100 μm. E, effect of recombinant neudesin (20 ng/ml), neudeesin-hemin (20 ng/ml) and hemin (200 nm) on anti-apoptosis was quantified by counting TUNEL-positive cells. Results are means ± S.E. for five different fields from four independent slides. F, effect of recombinant neudesin (20 ng/ml), neudesin-hemin (20 ng/ml), and hemin (200 nm) on the phosphorylation of ERK1/2 and Akt in Neuro2A cells. Neuro2A cells were cultured for 30 min in the absence or presence of recombinant neudesin (20 ng/ml), neudesin-hemin (20 ng/ml), or hemin (200 nm). ERK1/2, phosphorylated ERK1/2, Akt, and phosphorylated Akt in Neuro2A cells were detected by Western blotting using rabbit antibodies against ERK1/2, phospho-ERK1/2, Akt, and phospho-Akt, respectively. The density of phosphorylated ERK1/2 (upper panel) and phosphorylated Akt (lower panel) were quantified, and values are means ± S.E. from four independent experiments. *, p < 0.05; **, p < 0.005.
      Association with Hemin Enhances Neurotrophic Activity of Neudesin in Primary Cultured Cortical Neurons–Recombinant neudesin shows neurotrophic activity in primary cultured mouse neurons (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ). Therefore, we compared the activity of neudesin-hemin with that of recombinant neudesin in primary cultured mouse cerebral cortical neurons. Neudesin-hemin exhibited a more significant neuroprotective effect in primary neurons than did recombinant neudesin. Under the same conditions, hemin alone did not exhibit any significant effect (Fig. 3, A and B). In the TUNEL assay, neudesin-hemin significantly decreased the number of TUNEL-positive apoptotic cells (Fig. 3, C and D), but the activity of neudesin-hemin was not greater than that of recombinant neudesin. Therefore, we examined the effect of neudesin-hemin or recombinant neudesin over a shorter period (30 min of incubation), on the phosphorylation of ERK1/2 and Akt, in primary cultured neurons. Neudesin-hemin promoted the phosphorylation of ERK1/2 and Akt more remarkably than did recombinant neudesin (Fig. 3E). As shown in (Fig. 3, F and G) neudesinΔHBD without the ability to bind heme did not exhibit the neurotrophic activity and did not promote the phosphorylation of ERK1/2. These results suggest that the ability to bind heme is essential for the activity of neudesin.
      Figure thumbnail gr3
      FIGURE 3Association with hemin enhances neurotrophic activity of neudesin in primary cultured cortical neurons. A, mouse cerebral cortical neurons were cultured in the absence or presence of recombinant neudesin (10 ng/ml), neudesin-hemin (10 ng/ml), hemin (100 nm), or HS (10%). After culture for 4 days, cells were double-stained with anti-MAP2 and anti-GFAP antibodies. Green and red signals indicate MAP2-positive neurons and GFAP-positive astrocytes, respectively. Blue signals indicate cell nuclei counterstained with TOPRO3. Scale bar, 100 μm. B, effect of recombinant neudesin, neudesin-hemin, and hemin on neurotrophic activity was qualified by counting MAP2-positive neurons. Results are means ± S.E. for five different fields from four independent slides. C, TUNEL analysis of cortical neurons treated with or without neudesin-hemin (10 ng/ml). After culture for 4 days, cells were double-stained with anti-MAP2 antibodies and TUNEL. Green and red signals indicate TUNEL-positive cells and MAP2-positive neurons, respectively. Blue signals indicate cell nuclei counterstained with TOPRO3. Scale bar, 100 μm. D, effect of recombinant neudesin (10 ng/ml), neudesin-hemin (10 ng/ml), and hemin (100 nm) on anti-apoptosis was qualified by counting MAP2 and TUNEL double-positive neurons. Results are means ± S.E. for five different fields from four independent slides. *, p < 0.05; **, p < 0.005. E, effect of neudesin, neudesin-hemin, hemin, and FGF2 on the phosphorylation of ERK1/2 and Akt in primary cultured neurons. Primary cultured neurons were cultured for 30 min in the absence or presence of neudesin (10 ng/ml), neudesin-hemin (10 ng/ml), hemin (100 nm), or FGF2 (10 ng/ml). ERK1/2, phosphorylated ERK1/2, Akt, and phosphorylated Akt in primary cultured mouse cerebral cortical neurons were detected by Western blotting using rabbit antibodies against ERK1/2, phospho-ERK1/2, Akt, and phospho-Akt, respectively. F, the effect of neudesin-hemin and neudesin HBD on neurotrophic activity was qualified by counting MAP2-positive neurons. Results are means ± S.E. for five different fields from four independent slides. G, the effect of neudesinhemin and neudesin HBD on the phosphorylation of ERK1/2 in primary cultured neurons is shown. Primary cultured neurons were cultured for 30 min in the absence or presence of neudesin-hemin (10 ng/ml) or neudesin HBD (10 ng/ml). ERK1/2 and phosphorylated ERK1/2 in primary cultured mouse cerebral cortical neurons were detected by Western blotting using rabbit antibodies against ERK1/2 and phospho-ERK1/2, respectively.
      Activity of Neudesin-Hemin Requires Fe(III)–We next examined whether the activities of neudesin are dependent on the amount of hemin-bound neudesin. First, we prepared a series of neudesin-hemin samples by changing the ratio of hemin to neudesin. Then, we examined the phosphorylation of ERK1/2 induced by a series of neudesin-hemin preparations. The phosphorylation of ERK1/2 was dependent on the hemin added in a ratio-dependent manner under a fixed concentration of recombinant neudesin (Fig. 4A).
      Figure thumbnail gr4
      FIGURE 4Activity of neudesin-hemin requires Fe(III). A, activity of neudesin increases proportionally with the amount of added hemin. After recombinant neudesin (10 μg) and hemin (0, 50, 100, 200, 250, or 500 μm) were incubated at 24 °C for 30 min, neudesin-hemin was isolated by gel filtration. Primary neurons were cultured for 30 min in the presence of a series of neudesin-hemin complexes (10 ng/ml each). ERK1/2 and phosphorylated ERK1/2 were detected by Western blotting using rabbit antibodies against ERK1/2 and phospho-ERK1/2, respectively. B, activity of neudesin requires Fe(III)-protoporphyrin IX, but not Fe(II)-protoporphyrin IX. Primary cultured neurons were cultured for 30 min in the presence of neudesin-hemin (10 ng/ml), neudesin-reduced hemin (10 ng/ml), hemin (100 nm), or reduced hemin (100 nm). Neudesin-reduced hemin and reduced hemin were produced by reducing Fe(III) to Fe(II) with sodium dithionite. ERK1/2 and phosphorylated ERK1/2 were detected by Western blotting using rabbit antibodies against ERK1/2 and phospho-ERK1/2, respectively. C, activity of neudesin requires iron-chelated PP IX. Primary cultured neurons were cultured for 30 min in the presence of recombinant neudesin (10 ng/ml), neudesin-hemin (10 ng/ml) or neudesin-PP IX (10 ng/ml). ERK1/2 and phosphorylated ERK1/2 were detected by Western blotting using rabbit antibodies against ERK1/2 and phospho-ERK1/2, respectively. The density of phosphorylated ERK1/2 was quantified, and values are means ± S.E. from four independent experiments. *, p < 0.05.
      Cytochrome b5 has Fe(III)-protoporphyrin IX as hemin in a normal state. We examined whether the activity of neudesin is dependent on hemin[Fe(III)-protoporphyrin IX] or reduced hemin [Fe(II)-protoporphyrin IX] by reducing Fe(III) to Fe(II) with sodium dithionite. Neudesin-hemin containing Fe(III), but not Fe(II), promoted the phosphorylation IX of ERK1/2. Free Fe(II)-protoporphyrin IX, but not Fe(III)-protoporphyrin, promoted the phosphorylation of ERK1/2 in primary cultured neurons (Fig. 4B). Neudesin-PP IX (protoporphyrin IX-bound neudesin) little promoted the phosphorylation of ERK1/2, similar to recombinant neudesin (Fig. 4C).
      Inhibition of Endogenous Neudesin Decreases Cell Survival and Proliferation in Neuro2a Cells–To elucidate the neurotrophic activity of endogenous neudesin, we examined the effect of neudesin RNAi in Neuro2a. The RT-PCR experiment revealed that the first target neudesin siRNA, but not the control siRNA, suppressed neudesin expression (Fig. 5A). We also found that the first target nedesin siRNA significantly decreased cell survival and cell proliferation in Neuro2a cells in the MTT assay and BrdU assay, respectively (Fig. 5, B and C). The first target neudesin siRNA significantly increased the number of TUNEL-positive cells in the Neuro2a population (Fig. 5, D and E). Likewise, the phosphorylation of ERK1/2 or Akt was remarkably suppressed by the first target neudesin siRNA (Fig. 5F). The second and third target neudesin siRNA also gave the same results as the first one (supplemental Fig. S1).
      Figure thumbnail gr5
      FIGURE 5Inhibition of endogenous neudesin decreases cell survival and proliferation in Neuro2a cells. A, RT-PCR analysis of the knockdown of endogenous neudesin by neudesin siRNA in Neuro2a cells. After being treated with neudesin siRNA or control siRNA, Neuro2a cells were cultured for 1 day. 18S was used as a loading control. B, after being treated with neudesin siRNA or control siRNA, Neuro2a cells were cultured for 5 days in N2-supplemented DF. The inhibitory effect of neudesin siRNA on cell survival was examined by MTT assay. Results are means ± S.E. for five independent wells. C, inhibitory effect of neudesin siRNA on the mitogenic activity was examined by determining the incorporation of BrdU. Results are means ± S.E. for five independent wells. D, after being treated with neudesin siRNA or control siRNA, Neuro2a cells were cultured for 3 days in N2-supplemented DF. Cell survival was examined using the TUNEL method. Green signals indicate TUNEL-positive cells. Blue signals indicate cell nuclei counterstained with TOPRO3. Scale bar, 100 μm. E, inhibitory effect of neudesin siRNA on anti-apoptosis was qualified by counting TUNEL-positive cells. Results are means ± S.E. for five different fields from four independent slides. **, p < 0.005. F, inhibitory effect of neudesin siRNA on the phosphorylation of ERK1/2 and Akt in Neuro2a cells. After being treated with control siRNA or neudesin siRNA, Neuro2a cells were cultured for 2 or 3 days in N2-supplemented DF. ERK1/2, phosphorylated ERK1/2, Akt, and phosphorylated Akt in Neuro2a cells were detected by Western blotting using rabbit antibodies against ERK1/2, phospho-ERK1/2, Akt, and phospho-Akt, respectively.
      Detection of Endogenous Mouse Neudesin–Last, we attempted to detect endogenous neudesin in the culture medium of Neuro2a cells by using anti-neudesin antibodies. A major band of 15.6 kDa was detected in the culture medium (Fig. 6A). The observed molecular weight was consistent with the putative molecular weight of mature neudesin. A few heme-binding proteins such as hemopexin have been purified using heme affinity chromatography (
      • Hvidberg V.
      • Maniecki M.B.
      • Jacobsen C.
      • Hojrup P.
      • Moller H.J.
      • Moestrup S.K.
      ). To examine whether endogenous neudesin is associated with hemin, we performed heme-affinity chromatography. Almost all the endogenous neudesin was recovered in the pass-through fraction, whereas the recombinant neudesin was not recovered in the pass-through fraction at all (Fig. 6B). These results mean that the endogenous neudesin produced by Neuro2a cells binds hemin.
      Figure thumbnail gr6
      FIGURE 6Detection of endogenous mouse neudesin. A, Neuro2a cells secreted neudesin. The culture medium and the lysate of Neuro2A cells were subjected to the detection of neudesin using Western blotting. Neudesin was detected by Western blotting with anti-neudesin antibodies (15.6 kDa). B, heme-affinity chromatography, endogenous neudesin in the culture medium of Neuro2a cells was not trapped on hemin-agarose. The culture medium and recombinant neudesin (10 μg) were charged on hemin-agarose and then eluted with 0.2 m sodium acetate (pH 4.0). Pass-through and eluted fractions were subjected to SDS-PAGE followed by Western blotting to detect endogenous neudesin and recombinant neudesin with anti-neudesin antibodies (15.6 kDa and 20 kDa).

      DISCUSSION

      Neudesin, a novel secreted neurotrophic factor, belongs to the membrane-associated progesterone receptor (MAPR) family, a subfamily of the cytochrome b5 family, which possesses a cytochrome b5-like heme/steroid-binding domain (
      • Mifsud W.
      • Bateman A.
      ). It was reported in 1996 that MAPRs, discovered as progesterone-binding proteins (
      • Meyer C.
      • Schmid R.
      • Scriba P.C.
      • Wehling M.
      ,
      • Selmin O.
      • Lucier G.W.
      • Clark G.C.
      • Tritscher A.M.
      • Vanden Heuvel J.P.
      • Gastel J.A.
      • Walker N.J.
      • Sutter T.R.
      • Bell D.A.
      ,
      • Labombarda F.
      • Gonzalez S.L.
      • Deniselle M.C.
      • Vinson G.P.
      • Schumacher M.
      • De Nicola A.F.
      • Guennoun R.
      ), contain a cytochrome b5-like heme/steroid-binding domain, suggesting that MAPRs interact also with heme (
      • Min L.
      • Takemori H.
      • Nonaka Y.
      • Katoh Y.
      • Doi J.
      • Horike N.
      • Osamu H.
      • Raza F.S.
      • Vinson G.P.
      • Okamoto M.
      ). In fact, the adrenal inner zone antigen (IZA) purified as a heme-binding protein was shown to be identical with a MAPR, 25-Dx (
      • Min L.
      • Strushkevich N.V.
      • Harnastai I.N.
      • Iwamoto H.
      • Gilep A.A.
      • Takemori H.
      • Usanov S.A.
      • Nonaka Y.
      • Hori H.
      • Vinson G.P.
      • Okamoto M.
      ).
      These reports led us to examine whether neudesin can bind steroids, in particular progesterone, and/or heme. First, we tested the binding of several steroids including progesterone to neudesin by dot blotting, but found that neudesin could bind no steroids at all (data not shown). Next, we attempted to check the hemin binding to neudesin by measuring hemin-specific absorbance. Hemin and reduced hemin show specific peaks of absorbance at 402 and 420 nm, respectively. Purified mouse recombinant neudesin seemed to bind hemin that was added exogenously, while neudesinΔHBD, a mutant lacking the heme-binding domain, only minimally bound exogenous hemin. Furthermore, we found that recombinant neudesin itself bound hemin because it was tinged with faint green and a 20-kDa band was slightly detected after SDS-PAGE following the peroxidase reaction. Compared with this, the peroxidase reaction on the gel for SDS-PAGE performed after incubation with hemin revealed that recombinant neudesin could bind exogenous hemin extensively. In most cases where heme-binding proteins are produced in a baculovirus/insect cell expression system, the addition of exogenous hemin to the culture medium is necessary for full biological activity because insect cells cannot provide enough hemin for the mass production of heme-binding proteins (
      • Seo H.G.
      • Fujii J.
      • Soejima H.
      • Niikawa N.
      • Taniguchi N.
      ). This was also the case with neudesin.
      The functional activity of neudesin-hemin was examined using Neuro2a cells. Neudesin-hemin, but not recombinant neudesin or hemin alone, showed neurotrophic and neuroprotective activities. Phosphorylation of ERK1/2 and Akt was remarkably promoted also only by neudesin-hemin, not by recombinant neudesin or hemin alone. The iron in hemin seemed to be necessary for the activity, because neudesin-protoporphyrin IX was little able to enhance the phosphorylation of ERK1/2 and Akt (Fig. 4C). Furthermore, Fe(III), but not Fe(II), in hemin was required for the activity of the neudesinhemin complex because the complex-induced enhancement of phosphorylation of ERK1/2 and Akt disappeared with the reduction of iron in hemin to Fe(II) by the sodium dithionite (Fig. 4B). It is not plausible that hemin dissociated from the complex bears the activity of neudesin because hemin alone has no effect on the phosphorylation of ERK1/2 and Akt although reduced hemin alone showed some activity. Nor is it plausible that neudesin protects neuronal cells from the cytotoxic effect of free hemin because hemin alone has no effect on the neuronal cell survival (Figs. 2 and 3).
      Recombinant neudesin did not have any significant effect on cell survival, cell proliferation, or the phosphorylation of ERK1/2 and Akt in Neuro2a cells. In contrast, in primary cultured neurons, recombinant neudesin exhibited significant neurotrophic activity and weak enhancement of the phosphorylation of ERK1/2 and Akt (
      • Kimura I.
      • Yoshioka M.
      • Konishi M.
      • Miyake A.
      • Itoh N.
      ). Because neudesinΔHBD did not exhibit neurotrophic activity in primary cultured neurons, heme may be indispensable for the neurotrophic activity of neudesin in primary neurons. Primary cultured neurons may be sensitive enough to respond to a very small amount of neudesin-hemin that was included in the recombinant neudesin as mentioned above. Another possibility is that hemin, which is released from dying primary cultured neurons in a time-dependent manner, may be associated with hemin-free neudesin during incubation. This is consistent with the result that, in primary cultured neurons, neudesin had only a weak effect on the phosphorylation of ERK1/2 and Akt which was examined with 30 min of incubation, while neudesin had a significant effect on the neurotrophic activity examined with 4 days of incubation.
      As described above, exogenous neudesin seems to require hemin for its biological activity. Then, does endogenous neudesin contain hemin? In other words, does endogenous neudesin have biological activity? To answer this question, we examined the inhibition of endogenous neudesin in Neuro2a cells with RNAi. Neudesin siRNA, but not control siRNA, sufficiently reduced cellular viability. The results suggest that endogenous neudesin is involved in the cellular function and contains hemin, because Neuro2a have no response to recombinant neudesin. Furthermore, we detected endogenous neudesin of 15.6 kDa, a molecular weight predictive of mature neudesin, by using anti-neudesin peptide antibodies. When the culture medium of Neuro2a cells was applied to a heme-affinity column, by which hemopexin, a plasma heme-binding protein, was isolated from human serum (
      • Hvidberg V.
      • Maniecki M.B.
      • Jacobsen C.
      • Hojrup P.
      • Moller H.J.
      • Moestrup S.K.
      ), the neudesin immunoreactivity was detected in the pass-through fraction, strongly suggesting that endogenous neudesin is already associated with hemin.
      A lot of proteins require heme for their biological activity as a prosthetic group, such as guanylate cyclase concerned with cGMP synthesis (
      • Denninger J.W.
      • Marletta M.A.
      ), NPAS2 concerned with circadian rhythm (
      • Dioum E.M.
      • Rutter J.
      • Tuckerman J.R.
      • Gonzalez G.
      • Gilles-Gonzalez M.A.
      • McKnight S.L.
      ), DGCR8 concerned with microRNA synthesis (
      • Faller M.
      • Matsunaga M.
      • Yin S.
      • Loo J.A.
      • Guo F.
      ), nitricoxide synthases concerned with NO synthesis (
      • Sono M.
      • Stuehr D.J.
      • Ikeda-Saito M.
      • Dawson J.H.
      ), and cytochrome b concerned with steroidogenesis (
      • Denninger J.W.
      • Marletta M.A.
      ). However, all are intracellular proteins. Although hemopexin is the only such protein that is extracellular, its function other than as a carrier of heme has not been reported. Neudesin may be the first extracellular heme-binding protein known to be involved in intercellular signal transduction. The attempt to detect its molecular target, a receptor associated with neudesin, is now under investigation.
      In conclusion, we report that neudesin is a heme-binding protein and its neurotrophic activity requires the attachment of hemin [Fe(III)-protoporphyrin IX] to its heme-binding domain. In addition, neudesin is the first novel extracellular heme-binding protein with intrinsic signal transducing activity. Elucidation of the precise function of neudesin may provide new insights into the physiological role of heme-binding proteins.

      Supplementary Material

      References

        • Balla G.
        • Jacob H.S.
        • Balla J.
        • Rosenberg M.
        • Nath K.
        • Apple F.
        • Eaton J.W.
        • Vercellotti G.M.
        J. Biol. Chem. 1992; 267: 18148-18153
        • Balla G.
        • Jacob H.S.
        • Eaton J.W.
        • Belcher J.D.
        • Vercellotti G.M.
        Arterioscler. Thromb. 1991; 11: 1700-1711
        • Smith M.A.
        • Hirai K.
        • Hsiao K.
        • Pappolla M.A.
        • Harris P.L.
        • Siedlak S.L.
        • Tabaton M.
        • Perry G.
        J. Neurochem. 1998; 70: 2212-2215
        • Hentze M.W.
        • Muckenthaler M.U.
        • Andrews N.C.
        Cell. 2004; 117: 285-297
        • Hrkal Z.
        • Vodrazka Z.
        • Kalousek I.
        Eur. J. Biochem. 1974; 43: 73-78
        • Camejo G.
        • Halberg C.
        • Manschik-Lundin A.
        • Hurt-Camejo E.
        • Rosengren B.
        • Olsson H.
        • Hansson G.I.
        • Forsberg G.B.
        • Ylhen B.
        J. Lipid Res. 1998; 39: 755-766
        • Jeney V.
        • Balla J.
        • Yachie A.
        • Varga Z.
        • Vercellotti G.M.
        • Eaton J.W.
        • Balla G.
        Blood. 2002; 3: 879-887
        • Burmester T.
        • Weich B.
        • Reinhardt S.
        • Hankeln T.
        Nature. 2000; 407: 520-523
        • Black S.M.
        • Harikrishna J.A.
        • Szklarz G.D.
        • Miller W.L.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7247-7251
        • Denninger J.W.
        • Marletta M.A.
        Biochim. Biophys. Acta. 1999; 1411: 334-350
        • Faller M.
        • Matsunaga M.
        • Yin S.
        • Loo J.A.
        • Guo F.
        Nat. Struct. Mol. Biol. 2007; 14: 23-29
        • Kimura I.
        • Yoshioka M.
        • Konishi M.
        • Miyake A.
        • Itoh N.
        J. Neurosci. Res. 2005; 79: 287-294
        • Kimura I.
        • Konishi M.
        • Miyake A.
        • Fujimoto M.
        • Itoh N.
        J. Neurosci. Res. 2006; 83: 1415-1424
        • Sawada H.
        • Kawamura T.
        • Shimohama S.
        • Akaike A.
        • Kimura J.
        J. Neurosci. Res. 1996; 43: 503-510
        • Zhu L.J.
        • Altmann S.W.
        Anal. Biochem. 2005; 345: 102-109
        • Yamashita T.
        • Konishi M.
        • Miyake A.
        • Inui K.
        • Itoh N.
        J. Biol. Chem. 2002; 277: 28265-28270
        • Shinohara H.
        • Udagawa J.
        • Morishita R.
        • Ueda H.
        • Otani H.
        • Semba R.
        • Kato K.
        • Asano T.
        J. Biol. Chem. 2004; 279: 41141-41148
        • Thomas P.E.
        • Ryan D.
        • Levin W.
        Anal. Biochem. 1976; 75: 168-176
        • Druzhyna N.M.
        • Musiyenko S.I.
        • Wilson G.L.
        • LeDoux S.P.
        J. Biol. Chem. 2005; 280: 21673-21679
        • Hoshikawa M.
        • Yonamine A.
        • Konishi M.
        • Itoh N.
        Brain Res. Mol. Brain Res. 2002; 105: 60-66
        • Hvidberg V.
        • Maniecki M.B.
        • Jacobsen C.
        • Hojrup P.
        • Moller H.J.
        • Moestrup S.K.
        Blood. 2005; 106: 2572-2579
        • Mifsud W.
        • Bateman A.
        Genome Biol. 2002; 3: 12
        • Krebs C.J.
        • Jarvis E.D.
        • Chan J.
        • Lydon J.P.
        • Ogawa S.
        • Pfaff D.W.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 23: 12816-12821
        • Nakanishi N.
        • Takeuchi F.
        • Okamoto H.
        • Tamura A.
        • Hori H.
        • Tsubaki M.
        J. Biochem. 2006; 140: 561-571
        • Min L.
        • Strushkevich N.V.
        • Harnastai I.N.
        • Iwamoto H.
        • Gilep A.A.
        • Takemori H.
        • Usanov S.A.
        • Nonaka Y.
        • Hori H.
        • Vinson G.P.
        • Okamoto M.
        FEBS J. 2005; 22: 5832-5843
        • Taketani S.
        • Adachi Y.
        • Kohno H.
        • Ikehara S.
        • Tokunaga R.
        • Ishii T.
        J. Biol. Chem. 1998; 47: 31388-31394
        • Meyer C.
        • Schmid R.
        • Scriba P.C.
        • Wehling M.
        Eur. J. Biochem. 1996; 239: 726-731
        • Selmin O.
        • Lucier G.W.
        • Clark G.C.
        • Tritscher A.M.
        • Vanden Heuvel J.P.
        • Gastel J.A.
        • Walker N.J.
        • Sutter T.R.
        • Bell D.A.
        Carcinogenesis. 1996; 17: 2609-2615
        • Labombarda F.
        • Gonzalez S.L.
        • Deniselle M.C.
        • Vinson G.P.
        • Schumacher M.
        • De Nicola A.F.
        • Guennoun R.
        J. Neurochem. 2003; 87: 902-913
        • Min L.
        • Takemori H.
        • Nonaka Y.
        • Katoh Y.
        • Doi J.
        • Horike N.
        • Osamu H.
        • Raza F.S.
        • Vinson G.P.
        • Okamoto M.
        Mol. Cell Endocrinol. 2004; 215: 143-148
        • Seo H.G.
        • Fujii J.
        • Soejima H.
        • Niikawa N.
        • Taniguchi N.
        Biochem. Biophys. Res. Commun. 1995; 208: 10-18
        • Dioum E.M.
        • Rutter J.
        • Tuckerman J.R.
        • Gonzalez G.
        • Gilles-Gonzalez M.A.
        • McKnight S.L.
        Science. 2002; 298: 2385-2387
        • Sono M.
        • Stuehr D.J.
        • Ikeda-Saito M.
        • Dawson J.H.
        J. Biol. Chem. 1995; 270: 19943-19948