Nogo-A, -B, and -C Are Found on the Cell Surface and Interact Together in Many Different Cell Types
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m411827200
ISSN1083-351X
AutoresDana A. Dodd, Barbara Niederoest, Stefan Bloechlinger, Luc Dupuis, Jean‐Philippe Loeffler, Martin E. Schwab,
Tópico(s)Calpain Protease Function and Regulation
ResumoNogo-A, -B, and -C are generated from the Nogo/RTN-4 gene and share a highly conserved C-terminal domain. They lack an N-terminal signal sequence and are predominantly localized to the endoplasmic reticulum (ER). We found the N terminus of endogenous Nogo-A exposed on the surface of fibroblasts, DRG neurons, and myoblasts. Surface-expressed Nogo-A was also present on presynaptic terminals of the neuromuscular junction and on DRG neurons in vivo. Surface biotinylations confirmed the presence of all Nogo isoforms on the surface. To search for proteins that interact with Nogo-A and suggest a function for the large intracellular pool of Nogo-A, immunoprecipitations were performed. Surprisingly, the most predominant proteins that interact with Nogo-A are Nogo-B and Nogo-C as seen with radiolabeled lysates and as confirmed by Western blotting in multiple cell lines. Nogo-A, -B, and -C share a 180-amino acid C-terminal domain with two highly conserved hydrophobic stretches that could form a channel or transporter in the ER and/or on the cell surface. Nogo-A, -B, and -C are generated from the Nogo/RTN-4 gene and share a highly conserved C-terminal domain. They lack an N-terminal signal sequence and are predominantly localized to the endoplasmic reticulum (ER). We found the N terminus of endogenous Nogo-A exposed on the surface of fibroblasts, DRG neurons, and myoblasts. Surface-expressed Nogo-A was also present on presynaptic terminals of the neuromuscular junction and on DRG neurons in vivo. Surface biotinylations confirmed the presence of all Nogo isoforms on the surface. To search for proteins that interact with Nogo-A and suggest a function for the large intracellular pool of Nogo-A, immunoprecipitations were performed. Surprisingly, the most predominant proteins that interact with Nogo-A are Nogo-B and Nogo-C as seen with radiolabeled lysates and as confirmed by Western blotting in multiple cell lines. Nogo-A, -B, and -C share a 180-amino acid C-terminal domain with two highly conserved hydrophobic stretches that could form a channel or transporter in the ER and/or on the cell surface. Reticulons are a family of proteins that share a common C-terminal domain and are particularly abundant in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; HRP, horseradish peroxidase; WT, wild type; mAb, monoclonal antibody; DABCO, 1,4-diazabicyclo[2.2.2]octane; DRG, dorsal root ganglion; PFA, paraformaldehyde. (1.Oertle T. Schwab M.E. Trends Cell Biol. 2003; 13: 187-194Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The most recognized reticulon, Nogo-A, is a potent neurite outgrowth inhibitor of high molecular mass (200 kDa) (2.Chen M.S. Huber A.B. van der Haar M.E. Frank M. Schnell L. Spillmann A.A. Christ F. Schwab M.E. Nature. 2000; 403: 434-439Crossref PubMed Scopus (334) Google Scholar, 3.GrandPre T. Nakamura F. Vartanian T. Strittmatter S.M. Nature. 2000; 403: 439-444Crossref PubMed Scopus (1024) Google Scholar, 4.Prinjha R. Moore S.E. Vinson M. Blake S. Morrow R. Christie G. Michalovich D. Simmons D.L. Walsh F.S. Nature. 2000; 403: 383-384Crossref PubMed Scopus (549) Google Scholar) mainly expressed in the nervous system in oligodendrocytes and myelin, and in some neuronal populations (5.Huber A.B. Weinmann O. Brosamle C. Oertle T. Schwab M.E. J. Neurosci. 2002; 22: 3553-3567Crossref PubMed Google Scholar, 6.Hunt D. Coffin R.S. Prinjha R.K. Campbell G. Anderson P.N. Mol. Cell Neurosci. 2003; 24: 1083-1102Crossref PubMed Scopus (101) Google Scholar, 7.Tozaki H. Kawasaki T. Takagi Y. Hirata T. Brain Res. Mol. Brain Res. 2002; 104: 111-119Crossref PubMed Scopus (47) Google Scholar). Nogo-B (55 kDa), a splice variant of Nogo-A, is ubiquitously expressed in many tissues; it has been suggested to function in vascular remodeling (8.Acevedo L. Yu J. Erdjument-Bromage H. Miao R.Q. Kim J.E. Fulton D. Tempst P. Strittmatter S.M. Sessa W.C. Nat. Med. 2004; 10: 382-388Crossref PubMed Scopus (204) Google Scholar) and in apoptosis (9.Li Q. Qi B. Oka K. Shimakage M. Yoshioka N. Inoue H. Hakura A. Kodama K. Stanbridge E.J. Yutsudo M. Oncogene. 2001; 20: 3929-3936Crossref PubMed Scopus (101) Google Scholar), but its function is far from clear. An alternate promoter is used for expressing the smallest RTN-4 form, Nogo-C (25 kDa), a protein highly expressed in differentiated muscle fiber, although there is some expression also in brain, in particular in adult Purkinje cells (5.Huber A.B. Weinmann O. Brosamle C. Oertle T. Schwab M.E. J. Neurosci. 2002; 22: 3553-3567Crossref PubMed Google Scholar, 10.Dupuis L. Gonzalez de Aguilar J.L. di Scala F. Rene F. de Tapia M. Pradat P.F. Lacomblez L. Seihlan D. Prinjha R. Walsh F.S. Meininger V. Loeffler J.P. Neurobiol. Dis. 2002; 10: 358-365Crossref PubMed Scopus (146) Google Scholar). As with the related RTN-1 to RTN-3, there is no known function for Nogo-C. The C-terminal reticulon domain (about 180 amino acids) for these proteins is evolutionarily conserved and can be found in all eukaryotes (11.Oertle T. Klinger M. Stuermer C.A. Schwab M.E. FASEB J. 2003; 17: 1238-1247Crossref PubMed Scopus (146) Google Scholar). In contrast, the N-terminal regions are of very different lengths for the different reticulons and show less conservation (12.Di Scala F. Dupuis L. Gaiddon C. De Tapia M. Jokic N. Gonzalez De Aguilar J.L. Raul J.S. Ludes B. Loeffler J.P. Biochem. J. 2004; 385: 125-134Crossref Scopus (37) Google Scholar). For instance, the N-terminal regions of the three Nogo proteins can only be found in higher vertebrates. A recent study shows that reticulons can modulate the activity of BACE1 and affect the secretion of amyloid-β peptide (13.He W. Lu Y. Qahwash I. Hu X.Y. Chang A. Yan R. Nat. Med. 2004; 10: 959-965Crossref PubMed Scopus (244) Google Scholar). However, solid data are sparse, and any common functional role for the C-terminal region of reticulons has not yet been discovered. All reticulons have two large hydrophobic domains near the C terminus of the protein. In the case of rat Nogo proteins these presumed transmembrane regions are 35 and 36 amino acids each, respectively, long enough to span the membrane twice (2.Chen M.S. Huber A.B. van der Haar M.E. Frank M. Schnell L. Spillmann A.A. Christ F. Schwab M.E. Nature. 2000; 403: 434-439Crossref PubMed Scopus (334) Google Scholar, 3.GrandPre T. Nakamura F. Vartanian T. Strittmatter S.M. Nature. 2000; 403: 439-444Crossref PubMed Scopus (1024) Google Scholar). In between the two transmembrane domains, the 66-amino acid loop has been found to bind the Nogo receptor subunit, NgR (14.Fournier A.E. GrandPre T. Strittmatter S.M. Nature. 2001; 409: 341-346Crossref PubMed Scopus (976) Google Scholar). Another important region for the neurite outgrowth inhibitory function is located in the Nogo-A-specific region in the middle of the protein (15.Oertle T. van der Haar M.E. Bandtlow C.E. Robeva A. Burfeind P. Buss A. Huber A.B. Simonen M. Schnell L. Brosamle C. Kaupmann K. Vallon R. Schwab M.E. J. Neurosci. 2003; 23: 5393-5406Crossref PubMed Google Scholar). For this domain to be inhibitory, it naturally must be displayed outside the oligodendrocyte to bind and activate a receptor on the surface of neurons or fibroblasts. Specific high affinity binding of this domain to brain membranes or to live fibroblasts has been shown (15.Oertle T. van der Haar M.E. Bandtlow C.E. Robeva A. Burfeind P. Buss A. Huber A.B. Simonen M. Schnell L. Brosamle C. Kaupmann K. Vallon R. Schwab M.E. J. Neurosci. 2003; 23: 5393-5406Crossref PubMed Google Scholar). However, the localization and topology of Nogo-A on plasma membranes have not been clear up to now. Other characteristics of reticulons include a di-lysine ER retention/retrieval signal at the extreme C terminus and a lack of a signal sequence at the N terminus. All the studied proteins of this family display a reticular pattern that co-localizes with ER markers, at least for a large part of the protein localization, when tested by indirect immunofluorescence (1.Oertle T. Schwab M.E. Trends Cell Biol. 2003; 13: 187-194Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 3.GrandPre T. Nakamura F. Vartanian T. Strittmatter S.M. Nature. 2000; 403: 439-444Crossref PubMed Scopus (1024) Google Scholar, 8.Acevedo L. Yu J. Erdjument-Bromage H. Miao R.Q. Kim J.E. Fulton D. Tempst P. Strittmatter S.M. Sessa W.C. Nat. Med. 2004; 10: 382-388Crossref PubMed Scopus (204) Google Scholar, 15.Oertle T. van der Haar M.E. Bandtlow C.E. Robeva A. Burfeind P. Buss A. Huber A.B. Simonen M. Schnell L. Brosamle C. Kaupmann K. Vallon R. Schwab M.E. J. Neurosci. 2003; 23: 5393-5406Crossref PubMed Google Scholar, 16.van de Velde H.J. Roebroek A.J. Senden N.H. Ramaekers F.C. Van de Ven W.J. J. Cell Sci. 1994; 107: 2403-2416Crossref PubMed Google Scholar). When the second transmembrane domain of Nogo-A was deleted the protein showed a partial cytoplasmic staining; therefore, this region could be critical for insertion into the ER (1.Oertle T. Schwab M.E. Trends Cell Biol. 2003; 13: 187-194Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Many membrane-bound proteins that do not contain a signal sequence use one of their hydrophobic domains and regions around it for signaling entry into the ER (17.Goder V. Spiess M. FEBS Lett. 2001; 504: 87-93Crossref PubMed Scopus (139) Google Scholar). In addition, other myelin proteins (MAL and PMP-22) have putative di-lysine ER retention/retrieval signals, yet can be found on the cell surface. Surprisingly, even proteins that are well known ER markers such as calreticulin and calnexin can be found in low amounts on the plasma membrane (18.Okazaki Y. Ohno H. Takase K. Ochiai T. Saito T. J. Biol. Chem. 2000; 275: 35751-35758Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 19.Johnson S. Michalak M. Opas M. Eggleton P. Trends Cell Biol. 2001; 11: 122-129Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 20.White T.K. Zhu Q. Tanzer M.L. J. Biol. Chem. 1995; 270: 15926-15929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Furthermore, many studies with potassium channels demonstrate that the ER retention signals are masked once the individual subunits form a functional channel, whereby the bulk of the total individual potassium channel subunits remain in the ER with only a very low amount of functional channel expressed on the cell surface (21.Ma D. Jan L.Y. Curr. Opin Neurobiol. 2002; 12: 287-292Crossref PubMed Scopus (153) Google Scholar). Many other proteins with ER retention/retrieval signals are located on the cell surface; for example, the glutamate receptors AMPA (22.Greger I.H. Khatri L. Ziff E.B. Neuron. 2002; 34: 759-772Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) and NMDA (23.Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (103) Google Scholar), a kainate receptor subunit (24.Ren Z. Riley N.J. Garcia E.P. Sanders J.M. Swanson G.T. Marshall J. J. Neurosci. 2003; 23: 6608-6616Crossref PubMed Google Scholar), GABAB receptors (25.Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Neuron. 2000; 27: 97-106Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), and a subunit of a calcium channel (26.Bichet D. Cornet V. Geib S. Carlier E. Volsen S. Hoshi T. Mori Y. De Waard M. Neuron. 2000; 25: 177-190Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Here we show that not only is Nogo-A found on the surface of multiple cell types, but that Nogo-B and Nogo-C can also be detected on the plasma membrane. In addition, all immunofluorescent studies for Nogo-A were performed with antibodies that are specific for the Nogo-A-specific, N-terminal, and middle portion of this large protein, meaning that different cell types have the ability for expressing this region of Nogo-A on the cell surface facing the extracellular space. Immunoprecipitations of cell lysates done with protein radiolabeling demonstrate that in different cell types the predominant proteins that interact specifically with Nogo-A are Nogo-B and Nogo-C. Both Western blotting after immunoprecipitation and size exclusion chromatography confirm the existence of a high molecular mass complex that includes all three Nogo proteins. Antibodies—Most antibodies are described in Ref. 15.Oertle T. van der Haar M.E. Bandtlow C.E. Robeva A. Burfeind P. Buss A. Huber A.B. Simonen M. Schnell L. Brosamle C. Kaupmann K. Vallon R. Schwab M.E. J. Neurosci. 2003; 23: 5393-5406Crossref PubMed Google Scholar, although some of the names have been changed. These are the former names with the new names: Bianca = Rb1, Laura = Rb173A, Florina = Rb173B. The sheep antibody was made by immunization with two bacterially produced purified peptides from rat Nogo-A, NiGΔ20 (amino acids 544–725), and NiGΔ6 (amino acids 763–975). For the production of AR2, one 15-amino acid peptide, AKIQAKIPGLKRKAE, was chosen from murine Nogo-A sequence according to the hydrophilicity/hydrophobicity profile and antigenicity prediction analyses. This peptide was located in the C terminus of the reticular homology domain and did not show potential cross reactivity with other reticulons (data not shown). After synthesis and purification by standard protocols, peptides were conjugated to keyhole limpet hemocyanin and injected into two specific pathogen-free rabbits. Immunization was boosted with three subsequent injections at 14, 28, and 56 days. Terminal bleeds were affinity purified against the immunizing peptide and stored as frozen aliquots. The polyclonal antisera were found to react specifically with Nogo extracts was confirmed by the elimination of staining in the presence of excess immunizing peptide (data not shown). Cells and Cultures—Production and maintenance of cell lines is described in Refs. 15.Oertle T. van der Haar M.E. Bandtlow C.E. Robeva A. Burfeind P. Buss A. Huber A.B. Simonen M. Schnell L. Brosamle C. Kaupmann K. Vallon R. Schwab M.E. J. Neurosci. 2003; 23: 5393-5406Crossref PubMed Google Scholar and 27.Oertle T. Huber C. van der Putten H. Schwab M.E. J. Mol. Biol. 2003; 325: 299-323Crossref PubMed Scopus (105) Google Scholar. In more detail, 3T3 cells were grown in Dulbecco's modified Eagle's medium with high glucose (Invitrogen, Life Technologies, Inc.) containing 10% fetal bovine serum (Invitrogen, Life Technologies, Inc.) and gentamicin (Invitrogen, Life Technologies, Inc.). The cells were always split the day before an experiment and kept in an incubator at 37 °C with 5% CO2. A description of the brain oligodendrocyte culture is in Ref. 28.van der Haar M.E. Visser H.W. de Vries H. Hoekstra D. J. Neurosci. Res. 1998; 51: 371-381Crossref PubMed Scopus (15) Google Scholar. Dissociated DRG neuron cultures were made from the trypsin- and collagenase-treated, triturated DRGs of newborn rats that were plated on poly-l-lysine (PLL) and laminincoated coverslips. The DRG neurons were kept in L15 medium with l-glutamine (Sigma) and supplemented with N1 additives (Sigma), 100 ng/ml NGF (2.5 S NGF, purified from male mouse salivary glands) and gentamicin (Invitrogen, Life Technologies, Inc.). C2C12 myoblast cells were grown in Dulbecco's modified Eagle's medium with high glucose (Invitrogen, Life Technologies, Inc.), 10% fetal bovine serum, and chicken embryo extract. Immunofluorescent Staining—For surface staining: Cells were either plated onto PLL-coated glass coverslips (3T3 and DRG) or onto chamber slides (C2C12 myoblasts) using the medium described above. The cells were washed once with room temperature PBS, then were placed either on ice (3T3 and C2C12 myoblasts) or between 10 and 14 °C (DRG), and washed once with cold PBS containing 1 mm CaCl2 and 0.5 mm MgCl2 (Ca/Mg-PBS). Cold primary antibody, diluted in blocking buffer (either 2% goat serum, 0.2% fish skin gelatin in Ca/Mg-PBS or 10% fetal bovine serum in Ca/Mg-PBS), was added to live cells on ice for 30 min. The cells were washed three times with cold Ca/Mg-PBS. Fixation was done with cold 4% PFA, and the cells were placed at room temperature for 10–30 min. The cells were then washed three times with PBS. The secondary antibody diluted in blocking buffer was added to the cells for 30 min at room temperature. The cells were washed three times with PBS and mounted on slides with Mowiol (10% Mowiol 4–88 (w/v) (Calbiochem) was dissolved in 100 mm Tris, pH 8.5. with 25% glycerol (w/v) and 0.1% 1,4-diazabicyclo[2.2.2]octane (DABCO) was added as an anti-bleaching reagent). For intracellular staining: cells were washed with PBS, fixed with 4% PFA for 15 min, permeablilized with 0.3% Triton X-100 for 5–10 min, washed three times with PBS, and then blocked for 30 min in blocking buffer (2% goat serum, 0.2% fish skin gelatin in PBS). Primary antibody was added for 30 min in blocking buffer. Subsequently, cells were washed three times with PBS and incubated for 30 min in secondary goat anti-mouse or anti-rabbit Cy3 (1:3000) in blocking buffer. Coverslips were washed three times with PBS and mounted on slides with Mowiol containing 0.1% DABCO. Images were acquired on a Leica type DM RE microscope using the confocal laser scanning system TCS-SL from Leica. An HCX PL APO 40×/1.25 Ph 3 objective was used. Immunohistochemistry—For the DRGs: an adult rat was injected with NEMBUTAL (Abbott Laboratories), and perfused transcardially with Ringer's solution followed by 4% PFA in 0.1 m phosphate buffer with 5% sucrose. The lumbar DRGs were removed and put in the same fixative for 2 h at 4 °C. After fixation the DRGs were incubated in 0.1 m phosphate buffer with 30% sucrose for 2 days. Embedding was done in Tissue Tek (OCT compound; Zoeterwoude, the Netherlands) that was subsequently frozen in isopentane at –40 °C. 20-μm serial cryostat sections were mounted on Superfrost-Plus slides (Menzel-Glaeser; Germany) and frozen at –20 °C. After washing the slides in PBS, they were left in blocking buffer (5% bovine serum albumin, 0.2% Triton X-100 in PBS) for 1 h. Then, the slides were incubated with Rb173A antibody (1:1000) in blocking buffer overnight at 4 °C. Slides were washed three times with PBS and incubated in anti-rabbit Cy3 antibody (1:500) (Jackson ImmunoResearch) in blocking buffer for 1 h at room temperature. The slides were then washed three times with PBS and mounted with Mowiol containing 0.1% DABCO. For the diaphragm: an adult rat was decapitated and the diaphragm was immediately and thoroughly washed with PBS and cut in half. One half of the diaphragm was incubated with Rb173A (1:1000) antibody and the other half with β-tubulin (1:500) (Chemicon) in PBS containing 5% bovine serum albumin (blocking buffer) for 5 days at 4 °C. The floating tissue was then washed with PBS at 4 °C for 4 days and subsequently incubated with anti-rabbit or anti-mouse Alexa546 (1: 300)(Molecular Probes) and anti-bungarotoxin Alexa 488 (1:1000) (Molecular Probes) in blocking buffer for 2 days at 4 °C. The tissue was then washed for 2 days with PBS. Confocal imaging was done with the floating tissue placed between a glass slide and a glass coverslip. The microscope and imaging process was as described in the above section. The day after imaging, the same non-permeabilized half of the diaphragm that had been incubated with anti β-tubulin antibody was then fixed with 4% PFA in PBS, pH 7.4 for 20 min at 4 °C, washed with PBS, permeablilized with 0.2% Triton X-100 in blocking buffer for 1 h and incubated with anti-β-tubulin antibody (1:500) for 1 h. The tissue was washed with PBS and incubated with goat anti-mouse Alexa 546 (1:300) in permeabilization blocking buffer for 1 h at room temperature. Confocal imaging was done as described above. Surface Biotinylation—3T3 cells were plated the day before the experiment. Plates were placed on ice and washed five times with cold PBS containing 1 mm CaCl2 and 0.5 mm MgCl2 (Ca/Mg-PBS). A solution of cold 0.2 mm Sulfo-NHS-LC-LC biotin (Pierce) in Ca/Mg-PBS was incubated on the cells for 1 h. After washing three times with cold Ca/Mg-PBS the biotin was quenched for 10 min with cold Ca/Mg-PBS containing 20 mm glycine. Cells were lysed in either radioimmune precipitation assay buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) or RSB-Nonidet P-40 buffer (10 mm Tris pH 7.5, 10 mm NaCl, 1.5 mm MgCl2, 1% Nonidet P-40) containing 1 mm phenylmethylsulfonyl fluoride and protease inhibitor mixture (Roche Applied Science). The cells were scraped from the plate and centrifuged for 10 min at 10,000 × g. The supernatant was incubated either with streptavidin gel (Pierce) or with preclearing goat anti-mouse IgG and IgM antibody (Jackson ImmunoResearch) for 2 h rotating at 4 °C. The samples incubated with streptavidin gel were then washed five times with cold Ca/Mg-PBS, aspirated with a 27-gauge needle, and SDS loading buffer was added. They were boiled for 5 min before the supernatant was loaded on a 10% SDS-PAGE. The Western blot was probed with Rb1 antibody (1:25,000). The samples incubated with preclearing antibody had protein G beads (Pierce) added, and were rotated for 2 h at 4 °C. The supernatant was moved to a new tube and they were precleared again with Sheep IgG (Sigma) and rabbit serum (Vector Laboratories, Inc.) for 2 h followed by 2 h of incubation with protein G beads. The supernatant was moved to a new tube and incubated with the antibodies of interest while rotating overnight at 4 °C. Protein G beads were then added for 2 h at 4 °C. The beads were then washed one time with RSB-Nonidet P-40 buffer, three times with Buffer A (10 mm Tris, pH 7.5, 150 mm NaCl, 2 mm EDTA, 0.2% Nonidet P-40), two times with Buffer B (10 mm Tris, pH 7.5, 500 mm NaCl, 2 mm EDTA, 0.2% Nonidet P-40) and one time with Buffer C (10 mm Tris, pH 7.5). The beads were then aspirated with a 27-gauge needle, SDS loading buffer was added, they were boiled for 5 min, and loaded on a 14% SDS-PAGE. The blot was probed with neutravidin-HRP (1:20,000) (Pierce). 35S-labeling and Immunoprecipitations—With radioactivity: CHO-NogoA and 3T3 cells were plated the day before the experiment. Primary oligodendrocytes were grown in differentiation medium for 5 days prior to the experiment. Cells were washed with warmed medium and incubated on a rocker at 37 °C for 1 or 2 h with a solution containing DME without methionine and cysteine (Invitrogen) and with 450 μCi/ml of Trans35S-LABEL (ICN). All subsequent steps were done on ice or at 4 °C. The cells were washed with PBS and lysed with CHAPS buffer (50 mm NaH2PO4, pH 8, 150 mm NaCl, 0.5% CHAPS) with 1 mm phenylmethylsulfonyl fluoride and protease inhibitor mixture (Roche Applied Science) for 30 min. The cells were scraped, and the solution was centrifuged for 10 min at 10,000 × g. The supernatant was put into a new tube, and a preclearing goat anti-mouse IgG and IgM antibody (Jackson ImmunoResearch) was added. These tubes were rotated for 2 h, then protein G beads were added, and the samples were again rotated for 2 h. This preclearing step was repeated. Subsequently, the antibodies of interest were added, and the samples were rotated overnight. Protein G beads were added to each sample, and the tubes were rotated for 2 h, washed as detailed in the surface biotinylation protocol, aspirated with a 27-gauge needle, incubated with SDS loading buffer and run on a 14% SDS-PAGE. The gel was subsequently dried for 1 h, and then imaged on a Phosphorimager (the gel with CHO samples) or were put on film (the gels with 3T3 and oligodendrocyte samples). Without radioactivity: cells were plated the day before the experiment. The experiment in Fig. 8 did not involve any transfection, but for Figs. 9 and 10 cells were transiently transfected with the indicated plasmids using Lipofectamine Plus (Invitrogen). The day after transfection cells were washed, incubated with RSB-Nonidet P-40 lysis buffer (10 mm Tris, pH 7.5, 10 mm NaCl, 1.5 mm MgCl2, 1% Nonidet P-40 containing 1 mm phenylmethylsulfonyl fluoride and protease inhibitor mixture (Roche Applied Science)), scraped, and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was put into a new tube, the antibodies of interest were added, and the samples were processed as detailed above in the radiolabeled samples.Fig. 9Nogo-B and Nogo-C interact with Nogo-A. Plasmids containing FLAG-tagged Nogo-A and Myc-tagged Nogo-C were transiently transfected into 3T3 cells. After 24 h cells were lysed, and proteins were immunoprecipitated with different antibodies. The control Ab was β-tubulin. Samples were run on 14% SDS-PAGE, Western blotted, and probed with the AR2 antibody that recognizes Nogo-A, -B, and -C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 10Transiently expressed Nogo-A does not co-immunoprecipitate with endogenous mouse Nogo-A. 3T3 cells were transfected with a plasmid that expresses rat Nogo-A containing a C-terminal Myc epitope. The cells were lysed, and the lysate was immunoprecipitated with the indicated antibodies. Samples were run on an SDS-PAGE, Western-blotted, and probed with 11C7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Size Exclusion Gel Filtration Column—Ten 150-mm plates were transfected with a plasmid containing rat Nogo-C using Lipofectamine Plus according to the manufacturer's description. After 24 h, the cells were scraped into cold PBS and centrifuged at 1000 × g for 10 min at 4 °C. The cells were lysed in 5 ml of ice-cold RSB-Nonidet P-40 lysis buffer (see above) and centrifuged for 10 min at 10,000 × g at 4 °C. 140 mm NaCl was added to the extract. A Superdex 200 26/60 column was equilibrated with extraction buffer (150 mm NaCl) on an FPLC system from Amersham Biosciences. 5 ml of cell extract was separated with a flow of 1.8 ml/min, and after 40 min the fraction collector started to collect 1.8-ml fractions. 30 μl of fractions 20–45 were run on a 4–12% NuPage gel (Invitrogen) and analysed by Western blotting using the AR2 antibody. Fraction 20 corresponds to 36 ml after sample injection. Antibodies That Recognize Nogo—To study the cellular localization of Nogo-A and to discover proteins that interact with it, different antibodies generated against specific regions of the protein were used for immunofluorescent and immunoprecipitation experiments (Fig. 1A). Two rabbit polyclonal antiserum, a sheep polyclonal antisera, and two mouse mAbs specifically recognize Nogo-A and not the spliced variant Nogo-B or the small protein, Nogo-C, which uses an alternate promoter. The N-terminal 172 amino acids of Nogo-A are identical to the N terminus of Nogo-B, and therefore, one rabbit polyclonal anti-serum, Rb1, recognizes both Nogo-A and Nogo-B. The AR2 antibody, a rabbit polyclonal antiserum with the epitope in the common C-terminal region of the protein is able to recognize all three Nogo proteins. The Western blot shows that all antibodies are specific for the epitopes they should recognize (Fig. 1B). In all lysates investigated there were two Nogo-B bands, one at around the apparent molecular mass of 42 kDa and one that ran slightly lower (see the * in Fig. 1B). The lower band may represent a cleavage product or a modified form of Nogo-B. The predicted molecular mass for rat Nogo-A is 139 kDa and rat Nogo-B is 39 kDa (according to the website: ca.expasy.org/tools/protparam.html). However, on standard SDS-PAGE, Nogo-A migrates at around 200 kDa and Nogo-B around 55 kDa. The slower migration of Nogo-A has been noted before in the literature and was attributed to the high number of charged amino acids (3.GrandPre T. Nakamura F. Vartanian T. Strittmatter S.M. Nature. 2000; 403: 439-444Crossref PubMed Scopus (1024) Google Scholar). However, we have found that Nu-PAGE 4–12% gradient gels run with MOPS buffer (Invitrogen) show Nogo-A and Nogo-B running closer to their predicted molecular mass of 140 and 40 kDa, respectively. A difference of protein migration on a standard SDS-PAGE compared with the NuPAGE system has been described for α-synuclein (29.Moussa C.E. Wersinger C. Rusnak M. Tomita Y. Sidhu A. Neurosci. Lett. 2004; 371: 239-243Crossref PubMed Scopus (20) Google Scholar), a protein, similar to Nogo-A, which has many acidic residues. In all figures with gels or Western blots arrows point to the Nogo proteins, and the figure legends detail the gel type that was used. Nogo-A Is Found on the Surface of Live Cells by Immunofluorescence—Staining of the cell surface of live, cultured cells were done with 3T3 fibroblasts, DRG neurons, and C2C12 myoblasts. All cells were incubated with Nogo-A-specific antibodies either on a metal sheet on ice (for 3T3 fibroblasts and myoblasts) or at 10–14 °C temperatures (for DRG cultures that appeared to come off the coverslip when put directly on the ice-cold metal sheet). Protein internalization is blocked at temperature below 19 °C (30.Sipe D.M. Jesurum A. Murphy R.F. J. Biol. Chem. 1991; 266: 3469-3474Abstract Full Text PDF PubMed Google Scholar); therefore, our staining method should only show surface-bound antibody, and capping of the antibody should not occur, especially for the case of 3T3 cells and myoblasts that were kept at 0 °C. Either the Nogo-A-specific rabbit polyclonal antiserum Rb173A (Fig. 2A) or a combination of the two mouse mAbs, 11C7 and 7B12, (Fig. 2B) resulted in a weak but clear surface staining of mouse 3T3 fibroblasts. 3T3 cells are kn
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