| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 280, Issue 13, 12494-12502, April 1, 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Brain Research Institute, University of Zürich and Department of Biology, ETH Zürich, CH-8057 Zürich, Switzerland and the ¶Laboratoire de Signalisations Moléculaires et Neurodégénérescence, EA 3433, Université Louis Pasteur, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg Cedex, France
Received for publication, October 18, 2004 , and in revised form, January 6, 2005.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The C-terminal reticulon domain (about 180 amino acids) for these proteins is evolutionarily conserved and can be found in all eukaryotes (11). In contrast, the N-terminal regions are of very different lengths for the different reticulons and show less conservation (12). 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-
peptide (13). 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, 3). In between the two transmembrane domains, the 66-amino acid loop has been found to bind the Nogo receptor subunit, NgR (14). Another important region for the neurite outgrowth inhibitory function is located in the Nogo-A-specific region in the middle of the protein (15). 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). 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, 3, 8, 15, 16). 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). 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). 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 (18–20). 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). Many other proteins with ER retention/retrieval signals are located on the cell surface; for example, the glutamate receptors AMPA (22) and NMDA (23), a kainate receptor subunit (24), GABAB receptors (25), and a subunit of a calcium channel (26).
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.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
20 (amino acids 544–725), and NiG6 (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 and 27. 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. 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 40x/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 -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 -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--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 x 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 x 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 x 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.
|
|
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
-synuclein (29), 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); 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 known to have endogenous Nogo-A (27), and this can clearly be seen in the Western blot (Fig. 1B). The Nogo-A surface staining was punctate and was not uniform around the perimeter of the cells. In particular, the spots were often, but not always, localized to regions where the cells were contacting one another through long extended segments. An antibody against -tubulin and also non-immune rabbit serum showed no detectable staining, confirming the intactness of the cells and the specificity of the Nogo-A staining (data not shown). Intracellular staining for Nogo-A was extremely bright and reticular as would be expected (Fig. 3).
|
|
Immature skeletal muscle is known to express Nogo-A (6, 31). We, therefore, studied immature C2C12 myoblasts and found that Nogo-A can be detected on the plasma membrane of these cells using a Nogo-A-specific antibody (Fig. 2D). As with the other two cell types, myoblasts also showed a punctate staining, although it was more evenly distributed around the cell. In conclusion, endogenous Nogo-A can be detected on the surface of different cell types and this staining has a punctate, non-uniform pattern.
Immunfluorescence Studies Show Nogo-A on the Surface of Neurons in Rat Tissue—Nogo-A staining was also detected on the surface of DRG neurons, and on motoneuron fibers at the presynaptic site of the neuromuscular junction in the diaphragm of adult rats. Fig. 4 shows intracellular, reticular staining (see asterisk) as well as strong staining of the surface of the DRG neuron (see arrowheads). The surface staining on the DRG neuron from tissue covers much more surface and is not punctate as the surface staining of DRG neurons that were grown in culture (compare Fig. 4 to Fig. 2C). The diaphragm of adult rats was rapidly dissected, washed thoroughly and incubated in blocking buffer with Rb173A antibody or anti--tubulin antibody followed by secondary antibody and anti-bungarotoxin (Btx). This resulted in strong staining for Nogo-A at the neuromuscular junctions (Fig. 4). No detergent or fixative was used during the staining of this floating, intact diaphragm, and no -tubulin staining was detected. However, when the same tissue was fixed and permeablized, -tubulin staining was detected. Therefore, the tissue in the experiment had been intact and was not leaky confirming that the Nogo-A staining is surface Nogo-A. Nogo-A was presynaptic as it did not overlap with bungarotoxin, a marker for the postsynaptic acetylcholine receptors. Therefore, the surface staining of Nogo-A is not only found in cell culture, but can also be detected in animal tissue in vivo.
|
|
Nogo-A, -B, and -C Interact and Form a High Molecular Mass Complex—We next wanted to discover binding partners of Nogo-A to see if these could give us any clues as to how the protein traffics to the cell surface, and if there could be an intracellular function for this large pool of Nogo-A remaining in the ER. CHO cells, either wild-type (CHO-WT) or stably overexpressing rat Nogo-Amyc (CHO-NogoA), were labeled with [35S]methionine and -cysteine, and the lysate was immunoprecipitated with different Nogo-specific and control antibodies. As shown in Fig. 6, all Nogo antibodies precipitated large amounts of Nogo-A from the stable CHO-Nogo-A cells, whereas the mouse IgG and rabbit serum did not. Small, but detectable, amounts of endogenous Nogo-A could be detected in the CHO-WT cells and this Nogo-A was not immunoprecipitated with the Myc antibody. Endogenous Nogo-B was pulled-down by the Rb1 antibody in both cell types. Interestingly, not many proteins were detected in every lane from the Nogo-A immunoprecipitated samples that were not nonspecific proteins found also in the negative control lanes: Only two proteins, one slightly above 56 kDa and one slightly below 29 kDa, were detected. These potential Nogo-A interactors were pulled down by all of the Nogo-A-specific antibodies (11C7 + 7B12, Rb173A, Rb173B, and S544). The protein at around 56 kDa is most likely Nogo-B, since it ran at exactly the same molecular mass as the band that is seen in the lane where antibody Rb1 was used for immunoprecipitation of Nogo-A and Nogo-B. The 
29-kDa protein would be around the right size for Nogo-C and, as was shown by further immunoprecipitations with Nogo-C recognizing antibodies and by immunoprecipitations combined with Western blots, is most likely Nogo-C (see below). This 29-kDa protein was also immunoprecipitated by the Rb1 antibody in CHO-WT cells. This experiment suggests that Nogo-A interacts with Nogo-B and Nogo-C, independently of species preference because rat Nogo-A pulls down endogenous hamster Nogo-B and Nogo-C.
|
-tubulin antibody immunoprecipitates contained many of these nonspecific proteins. However, a specific strong band representing -tubulin was clearly detected in the -tubulin antibody lanes in both gels slightly above the 56-kDa marker, and for both controls in both gels, Nogo-A, -B, and -C were not detected (Fig. 7, A and B).
|
The experiments with radiolabeling did not definitively show that the two lower molecular mass proteins that co-immunoprecipitate with Nogo-A are Nogo-B and Nogo-C. Therefore, to determine whether the interacting proteins were really Nogo-B and Nogo-C, immunoprecipitation was combined with Western blotting. CHO Nogo-A cells, 3T3 cells, the CG4 oligodendrocyte line (undifferentiated or differentiated) and NGF-primed or unprimed PC12 cells were lysed with CHAPS or octyl--glucoside as detergents, immunoprecipitated with the Nogo-A-specific S544 antibody and Nogo proteins were detected with the Nogo-A/B-specific antiserum Rb1. In all four cell types, Nogo-A co-immunoprecipitated Nogo-B (Fig. 8). The detergent in the lysis buffer made a large difference in the amount of Nogo-B that could be co-immunoprecipitated. The interaction of the two Nogo proteins was retained in the relatively weak detergent CHAPS, but the stronger octyl--glucoside detergent disrupted most of this interaction.
To test whether the lowest molecular mass protein that was immunoprecipitated after radiolabeling was Nogo-C, immunoprecipitations with the sheep Nogo-A-specific antibody S544 were done with 3T3 cells expressing Nogo-A with a FLAG tag on the N terminus and Nogo-C with a Myc tag on the C terminus. A large amount of Nogo-B and a very small amount of Nogo-C co-immunoprecipitated with Nogo-A when the S544 antibody was used (Fig. 9). When the immunoprecipitation was done with an antibody against the FLAG tag, Nogo-A co-immunoprecipitated Nogo-B, but Nogo-C was not present. When the Myc antibody was used, Nogo-B co-immunoprecipitated with the Myc-tagged Nogo-C; however, Nogo-A was not present. The reason that Nogo-A and Nogo-C did not co-immunoprecipitate when the N- and C-terminal tags were used for the precipitations may be caused by the low level of Nogo-C in the complex in this cell line making the protein level of either Nogo-A or Nogo-C undetectable by Western blot after the immunoprecipitation. However, the lack of the co-immunoprecipitation could also be caused by interference of the Nogo-A, -B, -C complex interaction because the antibodies binding the tags interfere with the stability of the complex. In either case, this result shows that the interaction of Nogo-A, -B, and -C is most likely not caused by a nonspecific aggregation of the three proteins, but more likely by a specific formation of a distinct complex.
To test whether Nogo-A can interact with itself, endogenous Nogo-A from mouse was immunoprecipitated from 3T3 cells transfected with Myc-tagged rat Nogo-A. It is possible to detect the species-specific proteins because of their slightly different molecular masses; mouse Nogo-A runs at 210 kDa whereas rat Nogo-A migrates closer to 200 kDa. As shown in Fig. 10, the endogenous mouse Nogo-A did not co-immunoprecipitate the transfected rat Nogo-A. Previous experiments demonstrated that the Myc antibody successfully co-immunoprecipitated rat Nogo-A with endogenous hamster Nogo-B and Nogo-C (Fig. 5), and another experiment showed that endogenous mouse Nogo-A co-immunoprecipitated transfected rat Nogo-C (Fig. 10). Therefore, it is unlikely that the rat Nogo-A protein does not pull-down the mouse Nogo-A protein because of a species difference. This result shows that the interaction between the Nogo-A, -B, and -C proteins is specific and is not due, for example, to aggregation by the common C-terminal domain.
If the three Nogo proteins form a discrete complex, then they should co-elute after filtration on a size exclusion column. As shown in Fig. 11, Nogo proteins were found in high molecular mass complexes when the lysate from 3T3 cells transfected with Nogo-C were run on a Superdex 200 column. Fractions 32–35 had very high molecular mass complexes of 560 kDa and contained all three Nogo proteins confirming that these proteins interact together. However, Nogo-A alone could be found in fractions 20–25 with much larger complexes of 670 kDa. The discrete pattern of the Nogo proteins found in these fractions underline the specificity of the interactions, and again shows that the proteins are not aggregating together. Nogo-A in the very high molecular mass fractions was not complexed to Nogo-B and Nogo-C, and therefore, is probably interacting with other proteins.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
As shown by immunofluorescence with a variety of antibodies as well as by cell surface biotinylations the plasma membranes of cultured oligodendrocytes, neurons, fibroblasts, and myoblasts have Nogo-A with the large N terminus region facing the extracellular space. Therefore, the name reticulon and the early studies that found reticulons to be localized to the ER because of the ER retention signal give a wrong impression: Nogos are also plasma membrane molecules, which, however, does not exclude a second, intracellular function. Many myelin proteins have ER retention signals but are located on the cell surface. Potassium channels, GABA receptors, AMPA receptors, NMDA receptors, kainate receptors, and glutamate receptors are functional complexes only when located on the plasma membrane; however, they have large intracellular ER pools, and only a fraction of the total cellular amount of these proteins are found on the cell surface (22–26).
The present results also show that the large N-terminal region of Nogo-A is located outside cells. In addition, the N-terminal region of Nogo-B was recently shown exposed on the surface of endothelial and smooth muscle cells (8). Therefore, some initial studies depicting the topology of Nogo-A with the N terminus facing the cytoplasm give an erroneous or incomplete interpretation of the topology of Nogo-A on the surface of the cell (3, 32). Nogo-A topology is not entirely clear. When the plasma membrane is damaged by incubation with detergents such as saponin or digitonin, under conditions where the ER membrane is left intact, N-terminal Nogo-A is strongly reticular indicating that the N terminus of Nogo-A is in the cytoplasm when the proteins is in the ER (15). Therefore, the N-terminal region of Nogo-A should also be intracellular when the protein is on the plasma membrane. How does Nogo-A have the N-terminal extracellular when it seems to be cytoplasmic in the ER? One possibility is that the protein is able to adopt more than one topology. One topological form has the N terminus in the cytoplasm and the other has the N terminus extracellular. Many other proteins are known to adopt multiple topologies, such as: P-glycoprotein, ductin, cytochrome P-450s, microsomal epoxide hydrolase (review in Ref. 33), the prion protein (34), hepatitis B, and C virus envelope proteins (35–37), lactose permease (38, 39), and phenylalanine permease (40). Phospholipid composition was found to direct the topology of phenylalanine permease. Other than this hint, there are no other possible mechanisms known for how these proteins are able to achieve multiple topologies.
The Nogo proteins have no signal sequence and may belong, therefore, to the growing group of unconventional proteins that are either secreted or brought to the plasma membrane by alternative trafficking pathways. These unconventional proteins include the secreted interleukin-1, many members of the FGF family and CNTF. There are also transmembrane proteins that are transported unconventionally that include the ATP-binding cassette (ABC) transport superfamily proteins, cystic fibrosis transmembrane protein (CFTR) and P-glycoprotein (41, 42). The translocation through the membrane for CFTR and PgP occurs in an unusual manner (43–45). How proteins traffic in a nonclassical pathway to the cell surface is largely unknown.
The presence of Nogo on the plasma membrane has important biological significance. Nogo-A on the surface of oligodendrocytes and myelin in the adult CNS inhibits neurite growth and regeneration (3, 46–48) and restricts CNS plasticity (47). The exposed N-terminal region of Nogo-B is important for regulating vascular remodeling by influencing endothelial and smooth muscle cell migration and by promoting cell adhesion (8). The N-terminal region of Nogo-A and -B must interact with receptors for these processes to occur. There are three different known receptor binding regions on Nogo (15): (i) The 66 amino acids between the two large transmembrane domains at the C terminus of all Nogo proteins bind the Nogo-66 receptor, NgR, inhibiting neurite outgrowth (14, 32). (ii) There is a Nogo-A-specific region that has a high affinity binding site for a yet unidentified receptor that not only inhibits neurite outgrowth but also the spreading of fibroblasts (15). Active Nogo-A fragments bind with high affinity to brain membranes and live 3T3 cells. (iii) There is a region at the common N-terminal region of Nogo-A and -B that inhibits fibroblast spreading (15), and alters endothelial and smooth muscle migration and adhesion (8) and binds to an unknown receptor. Many signaling molecules are known to have multisubunit receptor complexes; this is probably also the case for Nogo.
We show that Nogo proteins are preferred mutual interaction partners, regardless of whether the proteins are endogenous or overexpressed, and this interaction occurs in fibroblasts, CHO cells, oligodendrocytes, and PC12 cells. Because Nogo-A does not interact with itself, the interaction of Nogo-A, -B and -C is specific and most likely not due to aggregation. We predict that there would be a certain stoichiometry for the Nogo complex. That the three proteins co-elute in a limited number of fractions from a gel filtration column implies that the proteins interact to form a complex of a precise molecular mass. Nogo-A was found, in addition, in fractions from the gel filtration column with very high molecular mass devoid of Nogo-B and Nogo-C, suggesting that there must be Nogo-A-containing complexes with additional proteins. Work is currently being done to find other Nogo-A-interacting proteins.
The staining pattern for Nogo-A on the cell surface is punctate in all three cell types studied. Similarly, Acevedo et al. (8) found Nogo-B surface staining also to be punctate on the surface of endothelial cells. Many receptor complexes cluster on the plasma membrane in a punctate pattern, and their localization is often caused by specific plasma membrane microdomains. The discovery of Nogo-B as an inhibitor of vascular remodeling was based on its enrichment in caveolae and/or lipid rafts (8). Clustering of Nogo with other neurite outgrowth inhibitors on the plasma membrane, such as myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) might be important for transmitting an optimal neurite outgrowth inhibitory signal to the neuron. One reason that Nogo-A antibody treatment in vivo is so successful may be that antibody binding to surface Nogo-A not only blocks the interaction of Nogo-A with the neuronal receptor(s), but the other inhibitory proteins in the same microdomain would also lose their binding to the neuron by steric hindrance or by endocytosis of the entire inhibitory domain into the oligodendrocyte.
What could the function for the Nogo-A, -B, -C complex be? The hydrophobic regions at the C termini of the proteins are highly conserved evolutionarily, even among other reticulons. The two transmembrane domains that are 35 and 36 amino acids, respectively could be traversing the membrane once or looping through the membrane twice. Protein complexes that form by interactions among proteins that are spliced variants and have conserved transmembrane domains include ion channels, pores, and transporters (49, 50). The N-terminal regions of both Nogo-A and -B have a putative calcium binding site (27). Therefore, we speculate that Nogo proteins could form a channel or transporter that may regulate calcium. The variable length N termini of reticulon subunits is also a property frequently found in the spliced variant subunits that form ion channel complexes. If the Nogo complex is operating as a channel, it could be functional in the ER, in another intracellular organelle and/or on the plasma membrane. Indeed, it may be that reticulons could form a family of channels where the different N-terminal regions of the proteins would alter the channel function or operation. This could be why different reticulons are up- or down-regulated during different periods of development and in different cell types. For example, there is a down-regulation of Nogo-A in Purkinje cells and in skeletal muscle during differentiation (5). In the muscle of patients with ALS there is a down-regulation of Nogo-C while there is a concomitant up-regulation of Nogo-A (10). For RTN-3, the expression for different spliced variants is highly regulated during development (12). Also, in the Nogo-A knock-out mouse, we found an up-regulation of Nogo-B (51).
In conclusion, our results suggest that Nogo proteins are multifunctional proteins. There is most likely an evolutionarily "old" function that developed from the ancient, highly conserved reticulon domain. Reticulons can create protein complexes with the different splice variants as subunits, and these most likely function in the ER and possibly also on the cell surface. The evolutionarily "new" function for Nogo would be from the more newly evolved N-terminal domain. The N terminus of Nogo-A and -B contains an active site that influences cell migration and in the middle region of Nogo-A is a site that inhibits neurite growth. These active regions of Nogo, when presented outside of the cell, are involved in the regulation of nervous system plasticity and repair and in cell motility and migration of non-neuronal cells.
|
FOOTNOTES |
|---|
To whom correspondence should be addressed: Brain Research Institute, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Tel.: 41-1-635-3262; Fax: 41-1-635-3303; E-mail: dodd{at}hifo.unizh.ch.
1 The 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.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
