Phosphacan Short Isoform, a Novel Non-proteoglycan Variant of Phosphacan/Receptor Protein Tyrosine Phosphatase-β, Interacts with Neuronal Receptors and Promotes Neurite Outgrowth
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m211721200
ISSN1083-351X
AutoresJeremy Garwood, Nicolas Heck, Frank Reichardt, Andréas Faissner,
Tópico(s)Protein Tyrosine Phosphatases
ResumoPhosphacan, one of the principal proteoglycans in the extracellular matrix of the central nervous system, is implicated in neuron-glia interactions associated with neuronal differentiation and myelination. We report here the identification of a novel truncated form of phosphacan, phosphacan short isoform (PSI), that corresponds to the N-terminal carbonic anhydrase- and fibronectin type III-like domains and half of the spacer region. The novel cDNA transcript was isolated by screening of a neonatal brain cDNA expression library using a polyclonal antibody raised against phosphacan. Expression of this transcript in vivo was confirmed by Northern blot hybridization. Analysis of brain protein extracts reveals the presence of a 90-kDa glycosylated protein in the phosphate-buffered saline-insoluble 100,000 × g fraction that reacts with antisera against both phosphacan and a recombinant PSI protein and that has the predicted N-terminal sequence. This protein is post-translationally modified with oligosaccharides, including the HNK-1 epitope, but, unlike phosphacan, it is not a proteoglycan. The expression of the PSI protein varies during central nervous system development in a fashion similar to that observed for phosphacan, being first detected around embryonic day 16 and then showing a dramatic increase in expression to plateau around the second week post-natal. Both the native and recombinant PSI protein can interact with the Ig cell adhesion molecules, F3/contactin and L1, and in neurite outgrowth assays, the PSI protein can promote outgrowth of cortical neurons when used as a coated substrate. Hence, the identification of this novel isoform of phosphacan/receptor protein tyrosine phosphatase-β provides a new component in cell-cell and cell-extracellular matrix signaling events in which these proteins have been implicated. Phosphacan, one of the principal proteoglycans in the extracellular matrix of the central nervous system, is implicated in neuron-glia interactions associated with neuronal differentiation and myelination. We report here the identification of a novel truncated form of phosphacan, phosphacan short isoform (PSI), that corresponds to the N-terminal carbonic anhydrase- and fibronectin type III-like domains and half of the spacer region. The novel cDNA transcript was isolated by screening of a neonatal brain cDNA expression library using a polyclonal antibody raised against phosphacan. Expression of this transcript in vivo was confirmed by Northern blot hybridization. Analysis of brain protein extracts reveals the presence of a 90-kDa glycosylated protein in the phosphate-buffered saline-insoluble 100,000 × g fraction that reacts with antisera against both phosphacan and a recombinant PSI protein and that has the predicted N-terminal sequence. This protein is post-translationally modified with oligosaccharides, including the HNK-1 epitope, but, unlike phosphacan, it is not a proteoglycan. The expression of the PSI protein varies during central nervous system development in a fashion similar to that observed for phosphacan, being first detected around embryonic day 16 and then showing a dramatic increase in expression to plateau around the second week post-natal. Both the native and recombinant PSI protein can interact with the Ig cell adhesion molecules, F3/contactin and L1, and in neurite outgrowth assays, the PSI protein can promote outgrowth of cortical neurons when used as a coated substrate. Hence, the identification of this novel isoform of phosphacan/receptor protein tyrosine phosphatase-β provides a new component in cell-cell and cell-extracellular matrix signaling events in which these proteins have been implicated. Differentiation and morphogenesis of neural tissues involve a diversity of interactions between neural cells and their environment (1Garwood J. Heck N. Rigato F. Faissner A. Walz W. The Neuronal Microenvironment. Humana Press, Totowa, NJ2002: 109-158Google Scholar). Although the organization of the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; CNS, central nervous system; CSPG, chondroitin sulfate proteoglycans; RPTP, receptor protein tyrosine phosphatase; CA, carbonic anhydrase-like; GAG, glycosaminoglycan; TN, tenascin; CAM, cell adhesion molecule; FNIII, fibronectin type III-like; mAb, monoclonal antibody; pAb, polyclonal antibody; GST, glutathione S-transferase; PSI, phosphacan short isoform; ORF, open reading frame; UTR, untranslated region; P, post-natal day; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; E, embryonic day; ChABC, chondroitinase ABC; CS, chondroitin sulfate. in the vertebrate central nervous system (CNS) is not well understood it is marked by the relative abundance of chondroitin sulfate proteoglycans (CSPGs) and hyaluronan (2Maleski M. Hockfield S. Glia. 1997; 20: 193-202Google Scholar, 3Rauch U. Cell Tissue Res. 1997; 290: 349-356Google Scholar). Previously we have characterized DSD-1-PG, one of the more abundant of the soluble CSPGs in the post-natal brain, showing this to be the mouse homolog of phosphacan and demonstrating that it can have opposing effects upon neurite outgrowth according to the neuronal lineage (4Faissner A. Clement A. Lochter A. Streit A. Mandl C. Schachner M. J. Cell Biol. 1994; 126: 783-799Google Scholar, 5Garwood J. Schnadelbach O. Clement A. Schutte K. Bach A. Faissner A. J. Neurosci. 1999; 19: 3888-3899Google Scholar). Although phosphacan occurs in the CNS as a large CSPG (>800 kDa) (4Faissner A. Clement A. Lochter A. Streit A. Mandl C. Schachner M. J. Cell Biol. 1994; 126: 783-799Google Scholar); it is in fact a secreted splice variant of an even larger transmembrane receptor protein tyrosine phosphatase (RPTP), RPTP-β (6Maurel P. Rauch U. Flad M. Margolis R.K. Margolis R.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2512-2516Google Scholar), also known as PTP-ζ (-zeta) (7Krueger N.X. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7417-7421Google Scholar). Hence phosphacan corresponds to the entire extracellular part of the long RPTP-β receptor (the relative structures of the proteins are illustrated in Fig. 1). These proteins are characterized by a carbonic anhydrase-like (CA) domain at their extracellular N terminus. A third splice variant, the short RPTP-β receptor form, contains the same intracellular phosphatase domains as the long receptor, but, unlike phosphacan and the long RPTP-β, it is distinguished by the absence of an 850-amino acid highly glycosylated sequence, the IS region, which possesses many of the predicted glycosaminoglycan (GAG) attachment sites. In the CNS, phosphacan and the RPTP-β receptors have punctual spatiotemporal expression patterns that suggest potential roles for these proteins in various developmental processes, including cell migration (8Maeda N. Nishiwaki T. Shintani T. Hamanaka H. Noda M. J. Biol. Chem. 1996; 271: 21446-21452Google Scholar), differentiation (9Meyer-Puttlitz B. Junker E. Margolis R.U. Margolis R.K. J. Comp. Neurol. 1996; 366: 44-54Google Scholar), synaptogenesis (10Haunso A. Celio M.R. Margolis R.K. Menoud P.A. Brain Res. 1999; 834: 219-222Google Scholar), synaptic function (11Murai K.K. Misner D. Ranscht B. Curr. Biol. 2002; 12: 181-190Google Scholar), and myelination (12Harroch S. Palmeri M. Rosenbluth J. Custer A. Okigaki M. Shrager P. Blum M. Buxbaum J.D. Schlessinger J. Mol. Cell. Biol. 2000; 20: 7706-7715Google Scholar) (13Garwood J. Rigato F. Heck N. Faissner A. Restor. Neurol. Neurosci. 2001; 19: 51-64Google Scholar), and phosphacan is also up-regulated upon wounding and regeneration in the CNS (14Barker R.A. Dunnett S.B. Faissner A. Fawcett J.W. Exp. Neurol. 1996; 141: 79-93Google Scholar, 15Laywell E.D. Steindler D.A. Ann. N. Y. Acad. Sci. 1991; 633: 122-141Google Scholar, 16Deller T. Haas C.A. Naumann T. Joester A. Faissner A. Frotscher M. Neuroscience. 1997; 81: 829-846Google Scholar). In vitro functional studies have provided evidence for such roles in the development and maintenance of the CNS, and there have been a number of reports of the effects of phosphacan on process outgrowth from neuronal cultures. These have shown that the proteoglycan can either promote or inhibit neurite outgrowth dependent upon the neuronal type tested and the conditions under which it is presented. For example, we have shown previously that phosphacan has outgrowth-promoting properties on mesencephalic and hippocampal neurons (4Faissner A. Clement A. Lochter A. Streit A. Mandl C. Schachner M. J. Cell Biol. 1994; 126: 783-799Google Scholar), whereas it is inhibitory for laminin-promoted outgrowth from neonatal dorsal root ganglion explants (5Garwood J. Schnadelbach O. Clement A. Schutte K. Bach A. Faissner A. J. Neurosci. 1999; 19: 3888-3899Google Scholar). Biochemical studies have demonstrated a number of potential binding partners for phosphacan, both in the ECM, such as the tenascins, TN-C (17Milev P. Meyer-Puttlitz B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1995; 270: 24650-24653Google Scholar, 18Milev P. Fischer D. Haring M. Schulthess T. Margolis R.K. Chiquet-Ehrismann R. Margolis R.U. J. Biol. Chem. 1997; 272: 15501-15509Google Scholar) and TN-R (19Milev P. Chiba A. Haring M. Rauvala H. Schachner M. Ranscht B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 6998-7005Google Scholar), and on the cell surface, such as the immunoglobulin cell adhesion molecules (IgCAMs), F3/contactin (20Peles E. Nativ M. Campbell P.L. Sakurai T. Martinez R. Lev S. Clary D.O. Schilling J. Barnea G. Plowman G.D. et al.Cell. 1995; 82: 251-260Google Scholar), and NrCAM (21Sakurai T. Lustig M. Nativ M. Hemperly J.J. Schlessinger J. Peles E. Grumet M. J. Cell Biol. 1997; 136: 907-918Google Scholar). In addition, a number of growth factors can bind to phosphacan, including bFGF (22Milev P. Monnerie H. Popp S. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 21439-21442Google Scholar) and HB-GAM (19Milev P. Chiba A. Haring M. Rauvala H. Schachner M. Ranscht B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 6998-7005Google Scholar). Interaction sites on phosphacan have been characterized at three levels (13Garwood J. Rigato F. Heck N. Faissner A. Restor. Neurol. Neurosci. 2001; 19: 51-64Google Scholar). First, there are the long negatively charged CS GAG polymer chains, which can bind, for example, to TAG-1/Axonin-1 (23Milev P. Maurel P. Haring M. Margolis R.K. Margolis R.U. J. Biol. Chem. 1996; 271: 15716-15723Google Scholar), and the cytokines, amphoterin (19Milev P. Chiba A. Haring M. Rauvala H. Schachner M. Ranscht B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 6998-7005Google Scholar), midkine (24Maeda N. Ichihara-Tanaka K. Kimura T. Kadomatsu K. Muramatsu T. Noda M. J. Biol. Chem. 1999; 274: 12474-12479Google Scholar), and HB-GAM/pleiotrophin (19Milev P. Chiba A. Haring M. Rauvala H. Schachner M. Ranscht B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 6998-7005Google Scholar, 25Maeda N. Noda M. J. Cell Biol. 1998; 142: 203-216Google Scholar). Second, on the core glycoprotein, there are other classes of glycosylation, represented by oligosaccharides carrying epitopes such as HNK-1 (sulfated glucuronic acid) and Lewis-X (sialyl-Lewis-X) (5Garwood J. Schnadelbach O. Clement A. Schutte K. Bach A. Faissner A. J. Neurosci. 1999; 19: 3888-3899Google Scholar), which have also been implicated in IgCAM interactions (17Milev P. Meyer-Puttlitz B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1995; 270: 24650-24653Google Scholar). Third, there is the primary protein sequence, which includes the CA, and a fibronectin type III-like (FNIII) domain, but the remaining 80% of the molecule bears no strong homology to other characterized protein data base structures. Here we report the identification and characterization of a novel short isoform of phosphacan. This new isoform corresponds to the first third of phosphacan, and although it is glycosylated, it is not a proteoglycan. It is not as readily extracted from brain tissue as phosphacan, perhaps because of stronger interactions with the cell surface. A recombinant protein corresponding to the new isoform sequence can, like phosphacan, also promote neurite outgrowth from cortical neurons. Hence regulated expression of this protein may introduce an extra level of complexity to the proposed functional interactions of phosphacan and the RPTP-β receptor forms during CNS development and regeneration. Antibodies—Monoclonal antibody (mAb) 473HD is a rat IgM antibody directed against the chondroitin sulfate DSD-1 epitope (4Faissner A. Clement A. Lochter A. Streit A. Mandl C. Schachner M. J. Cell Biol. 1994; 126: 783-799Google Scholar, 26Clement A.M. Nadanaka S. Masayama K. Mandl C. Sugahara K. Faissner A. J. Biol. Chem. 1998; 273: 28444-28453Google Scholar), 324 is a rat IgG against the cell adhesion molecule L1 (27Rathjen F.G. Schachner M. EMBO J. 1984; 3: 1-10Google Scholar), and 412 is a rat IgG recognizing both sulfated and non-sulfated epitopes of the HNK-1 carbohydrate (28Kruse J. Mailhammer R. Wernecke H. Faissner A. Sommer I. Goridis C. Schachner M. Nature. 1984; 311: 153-155Google Scholar). Mouse mAb 4–121 against chick F11 cross-reacts with mouse F3/contactin (29Brummendorf T. Hubert M. Treubert U. Leuschner R. Tarnok A. Rathjen F.G. Neuron. 1993; 10: 711-727Google Scholar) and was provided by F. Rathjen (Max Delbruck Center for Molecular Medicine, Berlin, Germany). 3F8 is a mouse mAb against rat phosphacan (9Meyer-Puttlitz B. Junker E. Margolis R.U. Margolis R.K. J. Comp. Neurol. 1996; 366: 44-54Google Scholar, 30Rauch U. Gao P. Janetzko A. Flaccus A. Hilgenberg L. Tekotte H. Margolis R.K. Margolis R.U. J. Biol. Chem. 1991; 266: 14785-14801Google Scholar) that cross-reacts with mouse phosphacan (5Garwood J. Schnadelbach O. Clement A. Schutte K. Bach A. Faissner A. J. Neurosci. 1999; 19: 3888-3899Google Scholar), and it is available from the Developmental Studies Hybridoma Bank (Department of Biological Sciences, University of Iowa, Iowa City, IA 52242). Polyclonal sera were all from rabbit. KAF13 was raised against the purified DSD-1-PG (4Faissner A. Clement A. Lochter A. Streit A. Mandl C. Schachner M. J. Cell Biol. 1994; 126: 783-799Google Scholar) and has been used to clone all forms of RPTP-β from mouse brain expression libraries (5Garwood J. Schnadelbach O. Clement A. Schutte K. Bach A. Faissner A. J. Neurosci. 1999; 19: 3888-3899Google Scholar), KAF14 was raised against purified TN-C from post-natal mouse brains (31Faissner A. Kruse J. Neuron. 1990; 5: 627-637Google Scholar), polyclonal F3/contactin-1367 was raised against amino acids 37–50 (peptide KGFG-PIFE-EQPINT) of F3/contactin coupled to keyhole limpet hemocyanin (32Koch T. Brugger T. Bach A. Gennarini G. Trotter J. Glia. 1997; 19: 199-212Google Scholar), and anti-PSI was raised against the purified recombinant GST-PSI protein described below using standard immunization protocols (4Faissner A. Clement A. Lochter A. Streit A. Mandl C. Schachner M. J. Cell Biol. 1994; 126: 783-799Google Scholar). Antibody Screening of cDNA Expression Libraries—A mouse brain cDNA expression library was screened using the polyclonal antibody, KAF13, at 1 μg/ml as described previously (5Garwood J. Schnadelbach O. Clement A. Schutte K. Bach A. Faissner A. J. Neurosci. 1999; 19: 3888-3899Google Scholar). The library used was BALB/c neonatal whole brain oligo(dT) and random-primed Uni-ZAP XR l (Stratagene). Screening 3 × 106 recombinant phages, yielded four positive clones, corresponding to bases 1144–1509 and 1032–3591 of phosphacan/long RPTP-β and 1186–7850 of short RPTP-β. The fourth cDNA clone contained the truncated ORF corresponding to a shorter form of phosphacan. This begins at base 1276 and ends with a termination codon at 1792. This is followed by a 3′-UTR of 1988 bases. A Marathon cDNA amplification kit (Clontech, Heidelberg, Germany) was used to generate P0 and P7 total mouse brain cDNA libraries from 1 μg of poly(A)+ RNA. Using PCR amplification of this cDNA, bands from the ORF to 3′-UTR were cloned and sequenced (primers were as follows: 1 (sense), 5′-ATG CGA ATC CTG CAG AGC TTC CTC; 1700 (sense), 5′-GCC TCC TTA AAC AGT GGC T; 1891 (antisense), 5′-CTA TCC AGC TGA AGA GTC ATC GGC; 2120 (antisense), 5′-ACT TAG AAC TGG TGC GGA C). Northern Blot Analysis—Poly(A)+ RNA was prepared from 1 g of mouse brain from different developmental stages using a Fast-Track 2.0 mRNA isolation kit (Invitrogen). It was separated on formaldehyde/1% agarose gels and then blotted by capillary action onto Hybond nylon membrane (Amersham Biosciences). Blots were pre-hybridized for 1 h and then hybridized overnight at 50 °C for DNA probes and at 65 °C for riboprobes. Prehybridization was in hybridization buffer without probe (7% SDS, 50% deionized formamide, 5× SSC, 50 mm sodium phosphate, pH 7, 0.1% N-lauroyl sarcosine, 2% blocking reagent (Roche Applied Science)). Washing for DNA probes was in three changes of wash buffer (40 mm sodium phosphate, pH 7.2, 1% SDS, 1 mm EDTA) at 68 °C for 40 min, and for riboprobes, at 65 °C, 2 × 5 min in 2× SSC (where 1× SSC = 0.15 m NaCl, 15 mm sodium citrate, pH 7), and in 0.1× SSC, 3 × 15 min. Signals were revealed with Hyperfilm (Amersham Biosciences) and amplifying screens at –80 °C. The DNA probe for the 5′-ORF of all RPTP-β isoforms (1–1785; see Fig. 1) was labeled with [32P]dCTP by random priming. Because of homology in the 3′-UTR with the complementary 28 S rRNA sequence, it was not possible to employ a DNA probe without a strong cross-reaction with rRNA, because sequences from the sense strand hybridize to 28 S rRNA. Hence, it was necessary to employ an antisense riboprobe that only hybridized to the sense strand of the PSI transcript. This corresponded to bases 2653–3784 of the 3′-UTR of the PSI transcript and was synthesized with incorporation of radioactive α-35S-UTP (Amersham Biosciences) using T7 RNA polymerase (MBI Fermentas GmbH, St Leon Rot, Germany) and the linearized pBluescript II plasmid (Stratagene) containing the PSI cDNA. A riboprobe against β-actin was used as an indicator of relative RNA concentrations (bases 739–970). Tissue Fractionation—P7 mouse brains were homogenized in different buffers to test the efficiency of different extraction conditions. Protease inhibitors were used throughout (1 mm phenylmethylsulfonyl fluoride, 1 μm trypsin inhibitor, 0.1 μm Aprotinin, 1 μm pepstatin, 5 μm leupeptin, 1 μm antipain, 1 mm benzamidine, 1 mm EDTA). Initially, 20 brains were homogenized in PBS on ice with a mechanical dounce. Nuclei and insoluble debris were removed by centrifugation at 600 × g for 20 min. Half of this supernatant was detergent-solubilized by addition of 1% Nonidet P-40 and mixing at 4 °C for 1 h. The remaining 600-g PBS supernatant was ultracentrifuged at 100,000 × g for 1 h at 4 °C to precipitate PBS-insoluble material including total cellular membranes. This PBS-insoluble pellet was subsequently resuspended in 1% Nonidet P-40/PBS. As an alternative to detergent-solubilization with Nonidet P-40, 10 brains were homogenized in 60 mmn-octyl-β-d-glucopyranoside, 50 mm Tris, pH 8, 50 mm sodium acetate and then centrifuged at 100,000 × g for 1 h at 4 °C. Finally, for urea extraction, 10 brains were homogenized in 8 m urea, 10 mm sodium acetate, pH 6, and then centrifuged at 100,000 × g for 1 h at 4 °C. Protein determination used the DC assay kit (Bio-Rad). Deglycosylation Studies—Protein fractions were incubated for 4 h at 37 °C in 40 mm Tris, pH 8, 40 mm sodium acetate with 0.4 units/100 μg peptide N-glycosidase F (EC 3.5.1.52; Roche Diagnostics), 2 milliunits/100 μg keratanase (endo-β-galactosidase, EC 3.2.1.103; Seikagaku, Kogyo, Tokyo), or 20 milliunits/100 μg chondroitin ABC lyase (EC 4.2.2.4; Roche Molecular Biochemicals). Partial Protein Purification and Protein Sequencing—50 P7 mouse brains were homogenized in 1% Nonidet P-40/PBS (10 mm KH2PO4, 10 mm Na2HPO4-2H2O, pH 7.4, 150 mm NaCl) in the presence of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1.5 μm antipain, 1 μmortho-phenanthroline, 1 μm pepstatin, 1 μm aprotinin, 1 μm leupeptin, 1 mm benzamidine, 2 mm EDTA). The homogenate was centrifuged at 20,000 × g for 15 min at 4 °C and then ammonium sulfate was added to the supernatant to a final concentration of 1.5 m (35% saturation). Precipitated proteins were removed by centrifugation at 20,000 × g for 1 h at 4 °C followed by filtration through 0.45-μm filter units. This supernatant was loaded onto a methyl HIC (hydrophobic interaction chromatography) column (Bio-Rad) in a Biologic Duo-Flow gradient chromatography system (Bio-Rad), equilibrated with 1.5 m AmSO4/PBS. The column was washed until the baseline was attained and then eluted with a linear gradient from 1.5 to 0 m AmSO4/PBS. The 90-kDa protein eluted at around 1 m AmSO4. The eluate was diluted by addition of 2× binding buffer (20 mm Tris, pH 7.4, 0.5 m NaCl, 1 mm MnCl2,1mm CaCl2) and then loaded onto a concanavalin A-Sepharose affinity chromatography column (Amersham Biosciences). The column was washed with 20 volumes of binding buffer before being eluted with elution buffer (0.5 m methyl-α-d-mannopyranoside, 20 mm Tris, pH 7.4, 0.5 m NaCl). Sample buffer was added to this eluate, which was then separated on 10% SDS-PAGE. The proteins were transferred by electroblotting onto ProBlott PVDF membrane (Applied Biosystems). Bands were revealed by Ponceau Red coloration, and the 90-kDa band was confirmed by alignment with an adjacent strip, which was revealed using immunodetection with pAbs KAF13 and anti-PSI. The N-terminal protein sequence of the 90-kDa KAF13-positive band was determined by automatic Edman degradation on an Applied Biosystems 473A microsequencer. Developmental Expression Blot—Whole brains from mice at different developmental stages were homogenized on ice in PBS with protease inhibitors. The homogenate was centrifuged at 600 × g for 20 min at 4 °C and then the supernatant was further centrifuged at 100,000 × g for 1 h at 4 °C. The pellet was resuspended in PBS/20 mmn-octyl-β-d-glucopyranoside with protease inhibitors. Preparation of Recombinant PSI Protein—The recombinant PSI protein was generated by expression in Escherichia coli of a plasmid vector containing the PSI cDNA sequence fused in-frame to GST. The PSI cDNA was amplified by PCR using primers 5′-ATG CGG ATC CTG CAG AGC TTC C (which introduces a BamHI restriction site just after the initiation codon by changing the "A" at the sixth base into a "G") and 5′-CTA TCC AGC TGA AGA GTC ATC GGC (which includes the termination code in the PSI transcript). The PCR cDNA product was purified from an agarose gel and digested with BamHI before being ligated into the plasmid vector, pGEX-5X-3 (Amersham Biosciences), which had been pre-digested with the restriction endonucleases, BamHI and SmaI. Competent E. coli cells (TOP10F′; Invitrogen) were transformed, and clones were selected. The correct insertion of the PSI cDNA sequence into the vector was verified by DNA sequencing of the entire construct. For recombinant protein expression, the plasmid-bearing bacterial clone was grown in LB broth supplemented with 50 μg/ml ampicillin at 37 °C to an A595 nm of 0.8, when protein expression was induced by addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.05 mm, and the culture was transferred to 24 °C for a further 3 h. Bacteria were pelleted and then lysed in PBS/1% Nonidet P-40 with 50 μg/ml lysozyme. The PSI-GST recombinant protein was purified by affinity chromatography using a HiTrap glutathione column (Amersham Biosciences). The GST control protein was produced in the same way using bacteria transformed with the same plasmid vector but without insert. GST Pull-down—Purified GST and GST-PSI protein was incubated with glutathione-agarose beads (2 μg/10 μl pre-swollen beads; Amersham Biosciences) in PBS/1 mm dithiothreitol/0.5% Nonidet P-40 for 1 h at room temperature with mixing and then washed three times with PBS. 20 μl of beads were incubated with the 1 mg (2 μg/μl) P7 PBS-insoluble 100,000 × g brain fraction in PBS, 1 mm dithiothreitol, 0.5% Nonidet P-40, 0.5% n-octyl-β-d-glucopyranoside, 1 mm EDTA, plus protease inhibitors at room temperature with mixing for 1 h, and then placed on ice for 30 min before washing with 3 × 1 ml of ice-cold PBS. The beads were finally boiled in SDS-PAGE sample buffer, and the proteins were separated on 10% SDS-PAGE gels before transfer onto PVDF membrane. Immunoprecipitation—Antibodies (2 μg) were added to 2 mg (2 μg/μl) of P7 mouse brain PBS-insoluble 100,000 × g fraction solubilized in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 40 mm glucose, 1% n-octyl-β-d-glucopyranoside, 0.3% polyoxyethylene sorbitan monolaurate (Tween 20) plus protease inhibitors and incubated at 4 °C with mixing overnight. Subsequently, 50 μl of 50% pre-swollen bead slurry was added for a further 1 h of mixing at 4 °C; protein A-Sepharose (Sigma) was used to precipitate the rabbit polyclonal antisera, and anti-rat Ig-Sepharose (Sigma) was employed for the rat antibodies. The beads were then precipitated by centrifugation and washed by three rounds of resuspension and precipitation in radioimmune precipitation assay buffer (Tris-buffered saline/1% Nonidet P-40/0.1% SDS/0.5% deoxycholate) before being boiled in SDS-PAGE sample buffer. Proteins were separated on 10% SDS-PAGE and transferred to PVDF membrane before immunodetection. For coimmunoprecipitation, the 100,000 × g PBS-insoluble mouse brain fraction solubilized in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 40 mm glucose, 1% n-octyl-β-d-glucopyranoside, 0.1% polyoxyethylene sorbitan monolaurate plus protease inhibitors was incubated with 2 μg of mAb 324 (anti-L1) or mAb 4–121 (anti-F3/contactin) overnight with mixing at 4 °C. A 50-μl 50% slurry of anti-mouse IgG-agarose (for anti-F3/contactin; Sigma-Aldrich) or anti-rat IgG-agarose (for anti-L1; Sigma-Aldrich) were then added and incubated with mixing for 2 h at 4 °C. The beads were precipitated by centrifugation and then resuspended and reprecipitated three times with 20 mm Tris, pH 7.4, before being boiled with SDS sample buffer. Eluted proteins were separated on 10% SDS-PAGE and electroblotted to PVDF membranes. Phosphacan Purification—Phosphacan was purified from detergent-free physiological saline-buffered brain lysates from post-natal day 7 to 14 mice as described previously (4Faissner A. Clement A. Lochter A. Streit A. Mandl C. Schachner M. J. Cell Biol. 1994; 126: 783-799Google Scholar) using a combination of affinity chromatography with the mAb, 473HD, bound to Sepharose resin and anion-exchange chromatography (4Faissner A. Clement A. Lochter A. Streit A. Mandl C. Schachner M. J. Cell Biol. 1994; 126: 783-799Google Scholar). It was quantitated using the protein assay (Bio-Rad) and by the determination of uronic acid equivalents (33Blumenkrantz N. Asboe-Hansen G. Anal. Biochem. 1973; 54: 484-489Google Scholar). Cell Culture—Neuronal cell cultures were established from embryonic day 17 (E17) mouse brains. The cerebral hemispheres were dissected in PBS, and the meninges were removed. Dissociation was achieved by addition of 0.25% trypsin at 37 °C for 10 min, followed by passage through a sieve with 48-μm pores. The resulting cell suspension was plated on coverslips at a density of 7,000 cells/cm2 in minimal Eagle's medium (Invitrogen) supplemented with the N2 mixture, namely 5 μg/ml insulin, 20 nm progesterone, 100 μm putrescine, 30 nm selenite (34Bottenstein J.E. Sato G.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 514-517Google Scholar), 1 mm pyruvate, 0.1% (w/v) ovalbumin, and 100 μg/ml transferrin (Sigma-Aldrich). The cultures were kept in a humidified atmosphere with 5% CO2 at 37 °C. Neurite Outgrowth Assays—E17 mouse cortical neurons were plated on supports coated with different substrates; glass coverslips were treated with 15 μg/ml poly-l-lysine in 0.1 m borate buffer, pH 8.2, for 1 h at 37 °C. The coverslips were then washed with water and dried. Purified phosphacan (50 μg/ml uronic acid equivalents) or recombinant proteins (50 μg/ml) were coated for 4 h at 37 °C. After coating, the coverslips were washed three times with PBS. After 24 h of culture, neurons were fixed with 4% paraformaldehyde for 15 min. After permeabilization with 0.1% Triton X-100 for 3 min and a blocking step with 3% bovine serum albumin in PBS, cells were stained with a mAb directed against the neuronal marker, β3-tubulin (mouse; Sigma; 2-h incubation in 3% bovine serum albumin/PBS) and then revealed with a cy3-conjugated antibody (goat anti-mouse-cy3; Jackson Immunoresearch Laboratories; 30-min incubation in 3% bovine serum albumin/PBS). The coverslips were then mounted on slides in Mowiol. Neurons were observed using a DMRB microscope (Leica), and pictures were taken with an axiocam camera (Zeiss). A first parameter considered was the fraction of process-bearing cells (with at least a process longer than one neuronal cell body) from at least 100 neurons per experiment, chosen at random. Subsequently the neurite lengths of the longest neurite on each process-bearing neuron was measured, and morphometric analysis was performed using the ImageTool software (University of Texas Health Science Center, San Antonio, TX). Three independent experiments were performed, and the data were analyzed with the Kyplot software (Kyence Inc.) using one-way analysis of variance statistics (α = 0.05), followed by a Tukey test. Antibody Screening of Brain cDNA Expression Libraries Yields a Novel Splice Variant of Phosphacan/RPTP-β—The cDNA corresponding to phosphacan was cloned previously (5Garwood J. Schnadelbach O. Clement A. Schutte K. Bach A. Faissner A. J. Neurosci. 1999; 19: 3888-3899Google Scholar) from a mouse cDNA expression library using KAF13, a pAb raised against the entire proteoglycan purified from post-natal mouse brain. In addition
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