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Neuroglycan C Is a Novel Midkine Receptor Involved in Process Elongation of Oligodendroglial Precursor-like Cells

2006; Elsevier BV; Volume: 281; Issue: 41 Linguagem: Inglês

10.1074/jbc.m602228200

ISSN

1083-351X

Autores

Keiko Ichihara-Tanaka, Atsuhiko Oohira, Martin G. Rumsby, Takashi Muramatsu,

Tópico(s)

Fibroblast Growth Factor Research

Resumo

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation. Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation. Midkine (MK), 2The abbreviations used are: MK, midkine; mMK, mouse MK; PTN, pleiotrophin; CS, chondroitin sulfate; CS-E, chondroitin sulfate E; NGC, neuroglycan C; EGF, epidermal growth factor; PG, proteoglycan; AB, acidic amino acid cluster box; HEK293T, human embryonic kidney 293T; FN, fibronectin; FITC, fluorescence isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; PBS; phosphate-buffered saline; TBS-T, Tris-buffered saline containing Tween 20; CHAPS, 3-[(3-cholamidopropyl)dimethylammomio]propanesulfonic acid; GST, glutathione S-transferase; HBS, Hepes-buffered saline; mH, Myc-His; HA, hemagglutinin. 2The abbreviations used are: MK, midkine; mMK, mouse MK; PTN, pleiotrophin; CS, chondroitin sulfate; CS-E, chondroitin sulfate E; NGC, neuroglycan C; EGF, epidermal growth factor; PG, proteoglycan; AB, acidic amino acid cluster box; HEK293T, human embryonic kidney 293T; FN, fibronectin; FITC, fluorescence isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; PBS; phosphate-buffered saline; TBS-T, Tris-buffered saline containing Tween 20; CHAPS, 3-[(3-cholamidopropyl)dimethylammomio]propanesulfonic acid; GST, glutathione S-transferase; HBS, Hepes-buffered saline; mH, Myc-His; HA, hemagglutinin. a heparin-binding growth factor, is strongly expressed at the mid-embryonic stage of development and is preferentially expressed in the kidney after birth (1Muramatsu T. J. Biochem. (Tokyo). 2002; 132: 359-371Crossref PubMed Scopus (330) Google Scholar, 2Kadomatsu K. Muramatsu T. Cancer Lett. 2004; 204: 127-143Crossref PubMed Scopus (283) Google Scholar). MK is a 13-kDa protein with about 50% sequence identity to pleiotrophin (PTN), which is also called heparin-binding growth-associated molecule (3Rauvala H. EMBO J. 1989; 8: 2933-2941Crossref PubMed Scopus (352) Google Scholar, 4Li Y.S. Milner P.G. Chauhan A.K. Watson M.A. Hoffman R.M. Kodner C.M. Milbrandt J. Duel T.F. Science. 1990; 250: 1690-1694Crossref PubMed Scopus (452) Google Scholar). MK promotes neurite outgrowth and migration in neuronal cells isolated from the embryonic brain (5Muramatsu H. Shirahama H. Yonezawa S. Maruta H. Muramatsu T. Dev. Biol. 1993; 159: 392-402Crossref PubMed Scopus (234) Google Scholar, 6Kaneda N. Talukder A.H. Nishiyama H. Koizumi S. Muramatsu T. J. Biochem. (Tokyo). 1996; 119: 1150-1156Crossref PubMed Scopus (81) Google Scholar, 7Maeda N. Ichihara-Tanaka K. Kimura T. Kadomatsu K. Muramatsu T. Noda M. J. Biol. Chem. 1999; 274: 12474-12479Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and prevents apoptosis (8Owada K. Sanjyo N. Kobayashi T. Mizusawa H. Muramatsu H. Muramatsu T. Michikawa M. J. Neurochem. 1999; 73: 2084-2092PubMed Google Scholar, 9Sakaguchi N. Muramatsu H. Ichihara-Tanaka K. Maeda N. Noda M. Yamamoto T. Michikawa M. Ikematsu S. Sakuma S. Muramatsu T. Neurosci. Res. 2003; 45: 219-224Crossref PubMed Scopus (61) Google Scholar). MK also promotes the migration of inflammatory leukocytes (10Horiba M. Kadomatsu K. Nakamura E. Muramatsu H. Ikematsu S. Sakuma S. Hayashi K. Yuzawa Y. Matsuo S. Kuzuya M. Kaname T. Hirai M. Saito H. Muramatsu T. J. Clin. Investig. 2000; 105: 489-495Crossref PubMed Scopus (173) Google Scholar) and thereby plays a key role in the etiology of neointima formation (10Horiba M. Kadomatsu K. Nakamura E. Muramatsu H. Ikematsu S. Sakuma S. Hayashi K. Yuzawa Y. Matsuo S. Kuzuya M. Kaname T. Hirai M. Saito H. Muramatsu T. J. Clin. Investig. 2000; 105: 489-495Crossref PubMed Scopus (173) Google Scholar), renal injury (11Sato W. Kadomatsu K. Yuzawa Y. Muramatsu H. Hotta N. Matsuo S. Muramatsu T. J. Immunol. 2001; 167: 3463-3469Crossref PubMed Scopus (162) Google Scholar, 12Kawai H. Sato W. Yuzawa Y. Kosugi T. Matsuo S. Takei Y. Kadomatsu K. Muramatsu T. Am. J. Pathol. 2004; 165: 1603-1612Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), rheumatoid arthritis (13Maruyama K. Muramatsu H. Ishiguro N. Muramatsu T. Arthritis Rheum. 2004; 50: 1420-1429Crossref PubMed Scopus (88) Google Scholar), and intraperitoneal adhesion after surgery (14Inoh K. Muramatsu H. Ochiai K. Torii S. Muramatsu T. Biochem. Biophys. Res. Commun. 2004; 317: 108-113Crossref PubMed Scopus (33) Google Scholar). So far, protein-tyrosine phosphatase ζ (7Maeda N. Ichihara-Tanaka K. Kimura T. Kadomatsu K. Muramatsu T. Noda M. J. Biol. Chem. 1999; 274: 12474-12479Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 15Qi M. Ikematsu S. Maeda N. Ichihara-Tanaka K. Sakuma S. Noda M. Muramatsu T. Kadomatsu K. J. Biol. Chem. 2001; 276: 15868-15875Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), anaplastic lymphoma kinase (16Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey J. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar), low density-lipoprotein receptor-related protein-1 (17Muramatsu H. Zou K. Sakaguchi N. Ikematsu S. Sakuma S. Muramatsu T. Biochem. Biophys. Res. Commun. 2000; 270: 936-941Crossref PubMed Scopus (131) Google Scholar), low density lipoprotein receptor-related protein 6 (9Sakaguchi N. Muramatsu H. Ichihara-Tanaka K. Maeda N. Noda M. Yamamoto T. Michikawa M. Ikematsu S. Sakuma S. Muramatsu T. Neurosci. Res. 2003; 45: 219-224Crossref PubMed Scopus (61) Google Scholar), and integrin α4β1 and α6β1 (18Muramatsu H. Zou P. Suzuki H. Oda Y. Chen G.Y. Sakaguchi N. Sakuma S. Maeda N. Noda M. Takada Y. Muramatsu T. J. Cell Sci. 2004; 117: 5405-5415Crossref PubMed Scopus (108) Google Scholar) have been identified as MK receptors. These molecules cooperate during MK signaling, possibly as a receptor complex (18Muramatsu H. Zou P. Suzuki H. Oda Y. Chen G.Y. Sakaguchi N. Sakuma S. Maeda N. Noda M. Takada Y. Muramatsu T. J. Cell Sci. 2004; 117: 5405-5415Crossref PubMed Scopus (108) Google Scholar). However, not all the molecules serving as MK receptors have been clarified. Importantly, we do not know whether a function specific to a class of cells such as neurite extension requires another receptor to transmit the specific signal or not. Here, we report that a cell surface molecule is specifically expressed in the nervous system as a component of the MK receptor in cells extending processes.When undifferentiated CG-4 cells, an oligodendrocyte precursor cell line, are cultured on dishes coated with MK or PTN, they quickly extend processes and adopt bipolar shapes (19Rumsby M. Suggitt F. Haynes L. Hughson E. Kidd D. McNulty S. Glia. 1999; 26: 361-367Crossref PubMed Scopus (19) Google Scholar, 20Rumsby M. Ichihara-Tanaka K. Kimura T. Scott M. Haynes L. Muramatsu T. Neurosci. Res. Commun. 2001; 28: 31-39Crossref Scopus (6) Google Scholar). Protein-tyrosine phosphatase ζ is considered to be an MK receptor in CG-4 cells since chondroitin sulfate E (CS-E) inhibited its attachment (20Rumsby M. Ichihara-Tanaka K. Kimura T. Scott M. Haynes L. Muramatsu T. Neurosci. Res. Commun. 2001; 28: 31-39Crossref Scopus (6) Google Scholar). On the other hand, preliminary experiments showed that an unknown cell surface protein of 110-120 kDa was mainly cross-linked by 125I-labeled MK in CG-4 cells (data not shown). Thus, we thought that other MK receptors might be expressed in CG-4 cells, due to the complex nature of the MK receptor (7Maeda N. Ichihara-Tanaka K. Kimura T. Kadomatsu K. Muramatsu T. Noda M. J. Biol. Chem. 1999; 274: 12474-12479Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 16Stoica G.E. Kuo A. Aigner A. Sunitha I. Souttou B. Malerczyk C. Caughey J. Wen D. Karavanov A. Riegel A.T. Wellstein A. J. Biol. Chem. 2001; 276: 16772-16779Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 17Muramatsu H. Zou K. Sakaguchi N. Ikematsu S. Sakuma S. Muramatsu T. Biochem. Biophys. Res. Commun. 2000; 270: 936-941Crossref PubMed Scopus (131) Google Scholar, 18Muramatsu H. Zou P. Suzuki H. Oda Y. Chen G.Y. Sakaguchi N. Sakuma S. Maeda N. Noda M. Takada Y. Muramatsu T. J. Cell Sci. 2004; 117: 5405-5415Crossref PubMed Scopus (108) Google Scholar). We noticed that neuroglycan C (NGC), a membrane-bound chondroitin-sulfated proteoglycan, is specifically expressed in the nervous system. NGC, also called chicken acidic leucine-rich epidermal growth factor (EGF)-like domain-containing brain protein, is a developmentally regulated part-time proteoglycan (21Yasuda Y. Tokita Y. Aono S. Matsui F. Ono T. Sonta S. Watanabe E. Nakanishi Y. Oohira A. Neurosci. Res. 1998; 32: 313-322Crossref PubMed Scopus (36) Google Scholar, 22Oohira A. Shuo T. Tokita Y. Nakanishi K. Aono S. Glycoconj. J. 2004; 21: 53-57Crossref PubMed Scopus (16) Google Scholar, 23Aono S. Tokita Y. Shuo T. Yamauchi S. Matsui F. Nakanishi K. Hirano K. Sano M. Oohira A. J. Biol. Chem. 2004; 279: 46536-46541Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and is expressed as isoforms NGC-I, -II, and -III (22Oohira A. Shuo T. Tokita Y. Nakanishi K. Aono S. Glycoconj. J. 2004; 21: 53-57Crossref PubMed Scopus (16) Google Scholar, 23Aono S. Tokita Y. Shuo T. Yamauchi S. Matsui F. Nakanishi K. Hirano K. Sano M. Oohira A. J. Biol. Chem. 2004; 279: 46536-46541Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 24Yamauchi S. Tokita Y. Aono S. Matsui F. Shuo T. Ito H. Kato K. Kasahara K. Oohira A. J. Biol. Chem. 2002; 277: 20583-22059Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Recently a new isoform, NGC-IV, was identified (25Aono S. Tokita Y. Yasuda Y. Hirano K. Yamauchi S. Shuo T. Matsui F. Keino H. Kashiwai A. Kawamura N. Shimada A. Kishikawa M. Asai M. Oohira A. J. Neurosci. Res. 2006; 83: 110-118Crossref PubMed Scopus (16) Google Scholar). NGC contains the CS-E structure in the proteoglycan (PG) form (26Shuo T. Aono S. Matsui F. Tokita Y. Maeda H. Shimada K. Oohira A. Glycoconj. J. 2004; 20: 267-278Crossref PubMed Scopus (28) Google Scholar), to which MK preferentially binds, and the acidic amino acid cluster box (AB) in the middle of the ectodomain (Fig. 1A). Interestingly, NGC lacking chondroitin sulfate (CS) is reported to be a 120-kDa glycoprotein (21Yasuda Y. Tokita Y. Aono S. Matsui F. Ono T. Sonta S. Watanabe E. Nakanishi Y. Oohira A. Neurosci. Res. 1998; 32: 313-322Crossref PubMed Scopus (36) Google Scholar). Here, we demonstrated that NGC-I and NGC-III were expressed in CG-4 cells and that they were non-PG forms. Then, we examined the interaction between MK and NGC in CG-4 cells and found that NGC mediates process elongation on MK-coated dishes but not cell adhesion. Furthermore, we investigated the roles of NGC-I and NGC-III using cDNA transfection and overexpression.EXPERIMENTAL PROCEDURESCells and Cell Culture—CG-4 cells were maintained on cell culture dishes coated with poly-d-lysine at 5 μg/ml in sterile water. The cells were cultured in Dulbecco's modified Eagle's medium/N1 basal medium supplemented with 30% B104 rat neuroblastoma-conditioned medium (CG-4 medium) to maintain a bipolar phenotype (27Louis J.C. Magal E. Muir D. Manthorpe M. Varon S. J. Neurosci. Res. 1992; 31: 193-204Crossref PubMed Scopus (360) Google Scholar).Reagents and Chemicals—Recombinant mouse MK was expressed using a baculovirus system and purified with a heparin-Sepharose column (28Akhter S. Ichihara-Tanaka K. Kojima S. Muramatsu H. Inui T. Kimura T. Kaneda N. Talukder A.H. Kadomatsu K. Inagaki F. Muramatsu T. J. Biochem. (Tokyo). 1998; 123: 1127-1136Crossref PubMed Scopus (37) Google Scholar). Human plasma fibronectin (FN) was purified as described (29Ichihara-Tanaka K. Titani K. Sekiguchi K. J. Biol. Chem. 1990; 265: 401-407Abstract Full Text PDF PubMed Google Scholar). Protease-free chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4) was purchased from Seikagaku Corp. (Tokyo, Japan). Rhodamine-phalloidin and secondary antibodies labeled with fluorescence isothiocyanate (FITC), TRITC, or horseradish peroxidase were purchased from Sigma. Lipofectamine Plus reagent was provided by Invitrogen.Immunofluorescence Microscopy—Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS, and blocked with 3% bovine serum albumin in PBS. For the detection of NGC, cells were incubated with either monoclonal anti-NGC antibody C5 (30Watanabe E. Maeda N. Matsui F. Kushima Y. Noda M. Oohira A. J. Biol. Chem. 1995; 270: 26876-26882Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) or polyclonal rabbit anti-NGC antiserum (30Watanabe E. Maeda N. Matsui F. Kushima Y. Noda M. Oohira A. J. Biol. Chem. 1995; 270: 26876-26882Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). For the staining of tubulin, cells were treated as described (31Trinczek B. Brajenovic M. Ebneth A. Drewes G. J. Biol. Chem. 2004; 279: 5915-5923Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and incubated with monoclonal anti-β-tubulin antibody (clone2-28-33, Sigma). Cells were also stained by monoclonal anti-Myc tag antibody (clone 9E10, Upstate Biotechnology, Lake Placid, NY). After reacting with secondary antibodies, cells were mounted in 50% glycerol/PBS containing 2.6% 1,4-diazabicyclo[2.2.2]octane and examined with a laser scanning confocal imaging system (Bio-Rad).SDS-PAGE and Western Blotting—SDS-PAGE was performed according to Laemmli (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206057) Google Scholar) after reduction with 2-mercaptoethanol. Then, proteins were electrotransferred onto a polyvinylidene difluoride membrane (Millipore, Billerca, MA) according to Towbin et al. (33Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44709) Google Scholar). The membrane was incubated with 20 mm Tris-HCl, pH 7.4, containing 150 mm NaCl and 0.1% Tween 20 (TBS-T) with 5% nonfat milk for blocking and incubated with a primary antibody in TBS-T, namely monoclonal anti-NGC C5, monoclonal anti-FLAG M2 antibody (Sigma), monoclonal anti-Myc tag antibody, or monoclonal anti-HA tag antibody (Roche Diagnostics). After extensive washing with TBS-T, membranes were incubated with a horseradish peroxidase-labeled secondary antibody diluted with TBS-T followed by ECL detection reagents (Amersham Biosciences) according to the instruction manual. Signals were visualized on Fuji x-ray film.Isolation of a PG-rich Fraction from Postnatal 10-day Rat Brains and Chondroitinase ABC Treatment—Membrane-bound proteoglycans were prepared from a PBS-insoluble fraction of 10-day-old (P10) Sprague-Dawley rat brains as described previously (30Watanabe E. Maeda N. Matsui F. Kushima Y. Noda M. Oohira A. J. Biol. Chem. 1995; 270: 26876-26882Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To remove chondroitin sulfate chains, samples were digested with chondroitinase ABC at 50 milliunits/ml in 100 mm Tris-HCl, pH 7.4, containing 30 mm sodium acetate and 5 mm EDTA (chondroitinase ABC buffer) for 2 h at 37 °C.Construction of NGC Expression Vectors for Mammalian Cells—Rat NGC cDNA (GenBank™ accession numbers NM_019284 and NM_133652) was amplified by PCR with specific primers for NGC, 5′-GGGATCCGCGCAATGGGCCGAGCTG-3′, named primer F1, and 5′-TCATCAGGTCAGGTTATTCTGGAGA-3′ (R1650) using oligo(dT) primed CG-4 cDNA as a template. PCR products were subcloned into pGEM-T Easy (Promega Corp., Madison, WI). To construct a Myc-His-tagged NGC-I vector and a Myc-His-tagged NGC-III vector, another PCR using cDNA encoding either NGC-I or NGC-III as a template with primers 5′-GGGATCCCTGGGACGGGACAGCCCACCAGTCGATGG-3′ (F958BM) and 5′-ACTAGTGGTCAGGTTATTCTGGAGACA-3′ (R1650Sp) was performed, generating 692-and 773-bp fragments. They were subcloned into pGEM-T Easy and digested with KpnI and SpeI to give rise to cDNA fragments encoding the cytoplasmic domain of NGC-I or NGC-III. Each of the resulting fragment was ligated into pcDNA3.1/Myc-His(+) cleaved with BamHI and XbaI together with a 1100-bp BamHI-KpnI fragment from NGC-I cDNA, a sequence common to NGC-I and NGC-III.Domain-specific cDNA clones were obtained by the following PCR: for the full-sized extracellular domain tagged with a FLAG epitope (CSAE-FLAG), the primer F1 and 5′-CTACTACTTGTCATCGTCGTCCTTGTAATCGAAGTCGGTGATGATGGACTC-3′ (RFL1280); for the chondroitin sulfate attachment domain (CS domain), F1 and 5′-GACTAGTCTCTAGAGTGCCCTGAAG-3′ (R635); for the signal sequence, F1 and 5′-AGGTACCAAGGCCTCCCGTGCCGGTACAGCCC-3′ (R120); for the acidic amino acid cluster box domain (AB domain), 5′-GGATATCTAGAGACACAACCAGC-3′ (F625) and 5′-GTTCGAACGTGGCCAGGTCCTTGCCTGG-3′ (R1090); for the EGF domain, 5′-GAGGCCTCTGGGACGGGACAGCCCACCAGTCG-3′ (F959ST) and RFL1280. The BamHI-EcoRI fragment encoding CSAE-FLAG, the BamHI-EcoRV fragment encoding the signal sequence, and the StuI-EcoRI fragment encoding the FLAG-tagged EGF domain was ligated into pcDNA3.1(+) cleaved with BamHI and EcoRI. The BamHI-SpeI fragment encoding the CS domain, BamHI-EcoRV fragment encoding the signal sequence, and StuI-SpeI fragment encoding A domain cDNA were ligated into pcDNA3.1/Myc-His(+) cleaved with BamHI and XbaI.Expression of Recombinant Glutathione S-Transferase (GST)-NGC Fusion Protein—The PCR product generated with the primers F1 and R1090 was subcloned into pGEM-T Easy vector and digested by StuI and NotI. The resulting fragment was ligated into pGEX 5X-2 (Amersham Biosciences) cleaved with SmaI and NotI and introduced into Escherichia coli XL1 blue F′. The GST-NGC fusion protein and GST protein were induced by 0.4 mm isopropyl-1-thio-β-d-galactopyranoside and isolated on a glutathione-Sepharose column, and the eluates were further purified by passing through a DEAE-Sepharose column.Expression of Recombinant Mouse MK Tagged with HA (mMK-HA) in Baculovirus System—To generate a transfer vector encoding mouse MK with an HA epitope, PCR was performed using mouse MK cDNA as a template, with primers, 5′-GCCGGATCCATGCAGCACCGAGGCTTCTTC-3′ and 5′-ACTAGCATAATCAGGAACATCATAGTCCTTTCCTTTTCCTTT-3′. The PCR product was subcloned into pGEM-T Easy vector, digested by BamHI and EcoRI, and ligated into pVL1393 cleaved with BamHI and EcoRI. Using the transfer vector, mMK-HA was produced and purified as in the case of mMK (28Akhter S. Ichihara-Tanaka K. Kojima S. Muramatsu H. Inui T. Kimura T. Kaneda N. Talukder A.H. Kadomatsu K. Inagaki F. Muramatsu T. J. Biochem. (Tokyo). 1998; 123: 1127-1136Crossref PubMed Scopus (37) Google Scholar).MK Binding Assay of NGC—MK affinity column chromatography was performed as described (9Sakaguchi N. Muramatsu H. Ichihara-Tanaka K. Maeda N. Noda M. Yamamoto T. Michikawa M. Ikematsu S. Sakuma S. Muramatsu T. Neurosci. Res. 2003; 45: 219-224Crossref PubMed Scopus (61) Google Scholar). Eluted fractions were analyzed by Western blotting using anti-NGC antibody or tag-specific antibodies. The binding of GST-NGC fusion protein to MK was evaluated using the BIAcore 3000 system (BIAcore, AB), using BIAevaluation software (BIAcore).Cross-linking of NGC—HEK293T cells overexpressing NGC were washed with ice-cold HBS(+), which contained 1 mm CaCl2 and 1 mm MgCl2 in HBS (10 mm Hepes-NaOH, pH 7.2, containing 150 mm NaCl), three times, and incubated with HBS(+) containing mMK-HA at 1 μg/ml for 2 h at 4 °C. After being washed with 0.1% bovine serum albumin in HBS(+), cells were treated with 0.27 mm disuccinimidyl suberate (Pierce) for 15 min at 4 °C, and 1 m Tris-HCl, pH 7.4 was added to 20 mm as a final concentration. Then, cells were lysed in lysis buffer, which contained 0.6% CHAPS, 0.1% sodium deoxycholate, 5 μg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, and 1 mm pepstatin A in HBS.Assay for Process Extension—Each well of a Falcon 1147 plate (BD Biosciences) was incubated with a coating solution for 2 h at 27 °C and washed with sterile water three times. CG-4 cells were trypsinized, suspended with 0.1% soybean trypsin inhibitor in PBS, washed with PBS two times, and seeded at 4 × 104 cells/well in CG-4 medium. CG-4 cells were incubated with a reagent for 10 min and seeded on the MK-coated wells. After 30 min, photographs of five different areas in each well were taken. Based on the photographs, the percentage of cells with processes was quantified. NGC-overexpressing B104 cells and mock-transfected cells were incubated in Dulbecco's modified Eagle's medium overnight, trypsinized, suspended with 0.1% soybean trypsin inhibitor in PBS, washed with PBS two times, seeded on the wells of Falcon 1147 plates coated with mMK, and also analyzed for process extension.RESULTSCG-4 Cells Express NGC-I and NGC-III as a Non-PG Form—First, we examined the expression of NGC in bipolar CG-4 cells by immunofluorescence microscopy using monoclonal anti-NGC antibody C5 (Fig. 2A). NGC was located in the cell processes and the basal parts of these processes. Then, to study whether NGC expressed in CG-4 cells carries chondroitin sulfate chains, NGC was immunoprecipitated from lysate of CG-4 cells with polyclonal anti-NGC antibodies, incubated with chondroitinase ABC or with buffer alone, and analyzed by Western blotting (Fig. 2B). NGC from CG-4 cells behaved as a 100-kDa band on the SDS-PAGE gel irrespective of chondroitinase ABC treatment, whereas NGC from P10 rat brain was 180 kDa before chondroitinase treatment and 100 kDa after the treatment. These results indicate that CG-4 cells expressed NGC and that the non-PG form was predominant.FIGURE 2Expression of NGC in CG-4 cells. A, CG-4 cells seeded on a poly-d-lysine-coated substrate were visualized either by phase contrast microscopy (left panel) or by immunofluorescence microscopy with monoclonal anti-NGC C5 followed by FITC-labeled goat anti-mouse IgG (right panel). Arrow, basal area of process. Asterisk, extending edge of process. Bar, 50 μm. B, Western blot analysis of NGC expressed in CG-4 cells and P10 rat brain. CG-4 cells were lysed in the lysis buffer. NGC was isolated by immunoprecipitation with polyclonal anti-NGC antibodies associated with protein A-Sepharose from the lysate of CG-4 cells and the PG-rich fraction of P10 rat brain. Half of each precipitate was treated with chondroitinase ABC (CHase)(+), and the other half was incubated with buffer alone (-). These samples were analyzed by immunoblotting using anti-NGC antibody C5. An arrow represents the position of NGC lacking chondroitin sulfate chains.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Four isoforms due to alternative splicing were reported in NGC (25Aono S. Tokita Y. Yasuda Y. Hirano K. Yamauchi S. Shuo T. Matsui F. Keino H. Kashiwai A. Kawamura N. Shimada A. Kishikawa M. Asai M. Oohira A. J. Neurosci. Res. 2006; 83: 110-118Crossref PubMed Scopus (16) Google Scholar). To know what isoforms were expressed in CG-4 cells, we performed reverse transcription-PCR using primers specific for the rat NGC sequence and single-stranded cDNA prepared from CG-4 cells as a template and isolated cDNA encoding the open reading frame of NGC. PCR products were subcloned into pGEM-T Easy, and the DNA sequences of 12 randomly chosen clones were analyzed. Nine of the clones were NGC-I, and the others were NGC-III. They all had the same amino acid sequence in the ectodomain but a different 27-amino acid insert in the cytoplasmic part as already reported. We could not detect the PCR product of NGC-II using primers specific for NGC-II.Binding of Various Forms of NGC to MK—To evaluate the binding of NGC to MK, we performed MK affinity column chromatography using both native NGC and a recombinant NGC lacking glycosaminoglycan chains. First, the lysate of CG-4 cells was applied to an MK-Sepharose column and eluted at 0.2, 0.3, 0.4, 0.5, and 1 m NaCl. SDS-PAGE and subsequent Western blotting revealed that NGC was eluted with 0.5 m NaCl (Fig. 3A).FIGURE 3Various forms of NGC bind to MK. A, NGC in lysate of CG-4 cells binds to MK. CG-4 cells were lysed in the lysis buffer, applied to an MK-Sepharose column, eluted with increasingly higher concentrations of NaCl, and analyzed by Western blotting using anti-NGC C5. B, a PG-rich fraction of P10 rat brain was applied to an MK-Sepharose column after chondroitinase ABC (CHase) treatment (lower panel) or without enzyme treatment (upper panel) and eluted with increasingly higher concentrations of NaCl. The eluates were immunoprecipitated with polyclonal anti-NGC antibodies associated with protein A-Sepharose and analyzed by Western blotting with monoclonal anti-NGC C5. To obtain a clear band, immunoprecipitated samples without enzyme treatment were digested with chondroitinase ABC before SDS-PAGE. C, purified GST-NGC (50 ng) and GST protein (80 ng) were separated on a 10% gel and silver-stained. D, analysis of the binding of GST-NGC fusion protein (upper panel) and GST (lower panel) to immobilized MK using the BIAcore system, which was equilibrated with 10 mm Hepes-NaOH, pH 7.4, containing 150 mm NaCl and 0.005% Tween 20 as a running buffer at a flow rate of 15 μl/min. Recombinant mouse MK (5 μg/ml in 10 mm sodium acetate buffer, pH 4.5) was immobilized on the flow cells of the CM5 sensor chip for 7 min using an amine coupling kit (BIAcore) at a flow rate of 5 μl/min according to the instruction manual. The binding assay was performed at 25 °C with a constant flow rate of 15 μl/min in both association and dissociation phases. GST-NGC or GST in a series of concentrations ranging from 80 to 320 nm in the running buffer was injected into the flow cells, and the changes in resonance units (RU) were recorded. Rate constants were determined using BIAevaluation software (BIAcore). Arrow A indicates the beginning of the association phase initiated by injection of each of the sample; arrow B indicates the end of the sample injection or the beginning of the dissociation phase initiated with running buffer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Second, the PG-rich fraction from P10 rat brain was applied to an MK-Sepharose column before or after chondroitinase ABC treatment (Fig. 3B). Intact NGC was mainly eluted with 0.5 and 0.6 m NaCl, but minor fractions eluted with 0.7 and 1.0 m NaCl were present. After chondroitinase ABC treatment, NGC was mainly eluted with 0.5 m NaCl. These results showed that native NGC from P10 rat brain bound to MK more strongly than that lacking glycosaminoglycan chains. Third, to establish that NGC without glycosaminoglycan chains can bind to MK, we expressed GST-NGC and GST proteins in bacteria and purified them (Fig. 3C). The binding affinity of GST-NGC and GST proteins was analyzed by surface plasmon resonance spectroscopy (Fig. 3D). We confirmed that GST-NGC bound to MK, but GST did not. BIAevaluation software gave the Kd of GST-

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