ARTICLE |
Correspondence to: Volker Herzog, Institut für Zellbiologie, Ulrich-Haberland-Str. 61a, D-53121 Bonn, Germany.
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Summary |
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The secretory N-terminal domain of the Alzheimer amyloid precursor protein (sAPP) evokes specific responses in cells on binding to their surfaces. Because APP is expressed in a large variety of cell types, the localization of sAPP binding requires detection techniques that selectively recognize sAPP as a ligand. For this purpose, we prepared antibodies against recombinant sAPP695 (sAPPrec) previously expressed in E. coli. Such antibodies were found to distinguish between sAPPrec and cellular APP or sAPP, as shown by immunocytochemistry and by immunoblot. In addition, they allowed the selective localization of bound sAPPrec on cell surfaces without any signal from cellular APP or sAPP. Saturation of sAPPrec binding to cell surfaces, as determined radiometrically, was reached at 10 nM [125I]-sAPPrec. Binding was specific because it was almost completely inhibited by a 100-fold excess of unlabeled sAPPrec. This specificity of binding was confirmed by surface plasmon resonance spectroscopy. Binding of sAPPrec to cell surfaces occurred in patches and was dependent on the state of cell differentiation. The sAPPrec used in this study contains heparin binding sites, but enzymatic removal of cell surface associated heparin did not affect sAPPrec binding. Aldehyde fixation of cells strongly inhibited their ability to bind sAPPrec. The data point to a fixation-sensitive sAPPrec binding protein which is detectable in the form of patches and therefore is part of assembled cell surface microdomains. (J Histochem Cytochem 47:373382, 1999)
Key Words: Alzheimer amyloid precursor protein, sAPPrec binding, cell surface, immunocytochemistry, microdomains
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Introduction |
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The Alzheimer amyloid-ß PRECURSOR PROTEIN (APP) is ubiquitously expressed although the level of its expression varies widely in different cell types (
The existence of a membrane receptor for sAPP has been postulated from the detection of sAPP signal transduction mechanisms observed in a variety of cell types (
Direct light or electron microscopic cellular localization of receptor molecules is usually performed by the use of antibodies directed against this receptor (for review see
The aim of this study was to develop suitable techniques for the cytochemical detection of cell surface-bound sAPP. For this purpose, recombinant sAPP (sAPPrec) containing a His tag was expressed in E. coli, purified, and biotinylated, or was used as an antigen. To exclude that cell surface binding of sAPPrec might be mediated by the KPI sequence (see above), we used sAPPrec derived from APP 695 lacking this domain. The detection techniques, which involved either streptavidin or antibodies against sAPPrec or against its His tag, were able to efficiently discriminate between sAPPrec and cellular APP. Our results indicate that anti-sAPPrec antibodies provided the most sensitive detection technique for cell surface-bound sAPPrec compared to both of the other techniques. We show that binding of sAPPrec occurs in the form of microdomains and that it is saturable and specific.
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Materials and Methods |
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Materials and Cells
FRTL-5 (
Cell Culture
FRTL-5 cells were cultured at 37C with 5% CO2/95% air in F-12 medium (Coons' modification; Seromed, Berlin, Germany) supplemented with 5% calf serum, TSH (1 milliU/ml), insulin (10 µg/ml), hydrocortisone (10 µM), Gly-His-Lys (10 ng/ml), somatostatin (10 ng/ml), transferrin (5 µg/ml), penicillin (50 U/ml), and streptomycin (50 U/ml). B104 or PC 12 cells were cultured at 37C and 5% CO2/95% air in Dulbecco's minimal essential medium (DMEM; Gibco, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS). Differentiation of B104 cells was induced by culturing cells for 3 days on collagen-coated coverslips in serum-free DMEM containing 1 mM db-cAMP (Sigma).
Light Microscopic Detection of Endogenous APP in FRTL-5, B104, and PC12 Cells
To localize APP, FRTL-5, B104, or PC12 cells were seeded on coverslips coated with collagen. Cells were fixed in paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked for 30 min with 3% BSA in PBS, and labeled with a rabbit serum against the C-terminal region of APP diluted in PBS with 0.3% BSA (PBSA). For immunofluorescence detection, the cells were incubated with DTAF-labeled goat anti-rabbit IgG (Dianova; Hamburg, Germany) diluted 1:50 in PBSA for 60 min at 37C and were viewed with a TCS 4D Leica confocal laser scanning microscope (Leica; Bensheim, Germany).
Binding of Exogenously Added sAPPrec to the Surface of FRTL-5, B104, or PC12 Cells
To analyze the binding properties of sAPPrec, FRTL-5 or B104 cells were seeded on coverslips and incubated with sAPPrec. For specific detection of bound sAPPrec the rabbit anti-sAPPrec antibody (antiserum 3329) exclusively recognizing sAPPrec but not endogenous APP was used. Cells were blocked for 30 min with 3% BSA in PBS for 60 min at 4C and then incubated with 0.06, 0.1, 1, 10, or 100 nM sAPPrec, which was detected by immunofluorescence as described above. The concentration of 60 pM sAPPrec was included in our experiments because this concentration might be relevant for cells exposed to the bloodstream, in which this sAPP level has been reported to appear (
Control Experiments
Several control experiments for each labeling procedure were performed to assess the immunocytochemical specificity. Controls included the substitution of antiserum by rabbit or chicken preimmune serum to evaluate the specificity of the primary antibody. To evaluate nonspecific binding of the secondary fluorescence labeled antibodies, control experiments with omission of the primary antibody were performed. The inhibitory effect of free antigen was tested by adsorption of the specific antibodies to sAPPrec before immunolabeling. For this purpose, diluted rabbit or chicken antisera were preincubated overnight with an excess of purified sAPPrec and were then used for immunocytochemical detection.
Transmission Electron Microscopy
For transmission electron microscopy, cells were seeded on plastic coverslips and incubated with sAPPrec, followed by the primary antibody as described above. As secondary antibody, a gold-labeled goat anti-rabbit IgG (Dianova) was used, and the gold label (5 nm) was enhanced using a silver enhancement kit (IntenSE; Amersham, Freiburg, Germany). Cells were postfixed for 10 min with 1% osmium tetroxide, stained en bloc with 2% aqueous uranyl acetate, embedded in Epon 820 (Fluka; Deisenhofen, Germany), and mounted on plastic coverslips. Thin sections were stained with lead citrate (10 min) and examined with a Philips CM120 electron microscope (Philips Electron Optics; Eindhoven, Netherlands).
SDS-PAGE and Immunoblotting
FRTL-5 cells were cultured for 48 hr in serum-free F-12 medium. Medium was collected at 4C and concentrated with a SpeedVac evaporation device (SC100; Savant, Farmingdale, NY). Tenfold concentrated medium was boiled for 5 min in sample buffer and proteins were separated on a 12.5% reducing SDS gel (
Determination of sAPPrec Interaction with Cells
Radiometric Quantitation.
For quantitation of sAPPrec binding to cells, sAPPrec was iodinated with [125I]-NaI using iodobeads (Pierce; Oud-Beijer, The Netherlands) for 15 min at RT (-amino capronic acid) and incubated with ligand binding buffer containing 0.1 to 100 nM [125I]-sAPPrec for 90 min at 4C. For determination of nonspecific binding, this incubation medium was supplemented with a 100-fold excess of unlabeled sAPPrec. After washing in ligand binding buffer, the cells were lysed in 0.5 M perchloric acid. Aliquots were used to determine radioactivity (Gamma 5.500; Beckmann Instruments, Munich, Germany) and amount of total DNA (
Surface Plasmon Resonance Spectroscopy.
Proteinprotein and cellprotein interactions were analyzed by surface plasmon resonance (SPR) spectroscopy as described (
Statistical Analysis of Quantitative Data
Fluorescence intensity assays were performed by measuring a minimum of 40 cells per concentration. Fluorescence intensity assays were repeated three times in each experiment. The radiometric determinations were derived from two separate experiments and each point of concentration was completed in triplicate, i.e., data from three different wells were collected and analyzed statistically. Results were expressed as the mean ± SD. Data were analyzed for statistical differences by Student's unpaired t-test. p<0.05 was considered statistically significant.
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Results |
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Detection of Exogenously Added sAPPrec
Previous attempts to immunocytochemically visualize cell surface-bound sAPP failed because of the simultaneous recognition of the N-terminal region of APP by any of the available antibodies. Therefore, we developed antibodies against sAPPrec that were highly specific because no interference with endogenous APP in cell lysates or with secreted sAPP in culture media was observed (Figure 1). For immunocytochemical detection of endogenous APP, formaldehyde-fixed FRTL-5, B104, or PC 12 cells were permeabilized and incubated with an antibody against the C-terminal portion of APP. As shown in Figure 2 (insets in Figure 2A, Figure 2C and Figure 2E), the cells exhibited a strong perinuclear crescent-shaped staining pattern that has been shown to co-localize with staining of mannosidase II and therefore to be detectable mainly in the Golgi complex (
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For surface labeling and subsequent visualization, cells were incubated with sAPPrec at concentrations of 0.06100 nM. Application of the anti-sAPPrec antibody (antiserum 3329) resulted in the clear visualization of cell surface-bound sAPPrec by both fluorescence and transmission electron microscopy (Figure 2 and Figure 3). Labeling was observed on all free accessible cell surface domains, except for the domains adhering to the culture dish or the coverslips. The same result was obtained with the chicken anti sAPPrec antibody B1. Highest fluorescence intensity was observed at concentrations of 10 and 100 nM sAPPrec. Weakest but still detectable immunofluorescence was seen at 1 nM. It has been shown that the concentration of human sAPP in the circulation amounts to about 60 pM sAPP (
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In cells incubated at 4C with sAPPrec followed by detection with anti-sAPPrec antibodies, sAPPrec was found to be clustered (Figure 2) and to form patches of 0.51.0 µm in diameter on the cell surfaces (Figure 3). These patches were evenly distributed over the cell surfaces of FRTL-5, B 104, and PC12 cells, excluding the membrane areas attached to the culture dish. This characteristic cell surface labeling pattern differed significantly from the labeling pattern of endogenous APP, as shown by double immunofluorescence labeling experiments (Figure 2B, Figure 2D, and Figure 2F). In these double labeling experiments, the simultaneous visualization of cellular APP and of exogenously added sAPPrec was made possible by the use of rabbit antiserum 2189 directed against the APP C-terminus and the chicken antibody B1 against sAPPrec. The use of the chicken antiserum B1 alone was previously tested and was observed to provide the same immunocytochemical staining as the rabbit anti-sAPPrec antibody 3329. The localization of cell surface-bound sAPPrec, as analyzed by confocal laser scanning microscopy, was compared with the results obtained with an antibody against His tag or with streptavidinCy3. The results indicated that the antibodies against sAPPrec provided the most sensitive detection technique as compared to the antibody against His tag or the vizualization of biotinylated sAPPrec with streptavidin-Cy3 (results not shown).
The labeling density of cell surface-bound sAPPrec differed markedly with the state of differentiation of the cells. Increased formation of neurites and of cellcell interactions were taken as signs of cell differentiation. As shown in Figure 4 for B 104 cells, undifferentiated cells bound considerably more sAPPrec than fully mature ones.
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Immunocytochemical Control Experiments (Not Shown)
Immunocytochemical staining was totally absent when the rabbit or chicken preimmune serum from nonimmunized animals was used or after omission of the primary antibody. This shows that the immunostaining was due to the presence of the corresponding antibodies and that nonspecific staining by the secondary fluorescently labeled antibody was negligible. Immunostaining was almost completely blocked when chicken or rabbit antiserum was preadsorbed with purified sAPPrec, thus confirming the specificity of the immunostaining with chicken and rat antisera.
Characteristics of sAPPrec Binding
Cells were incubated at 4C with [125I]-sAPPrec at concentrations ranging from 0.1 nM to 100 nM. The results showed that saturation of binding appeared to be reached at 10 nM sAPPrec. A 100-fold excess of unlabeled sAPPrec showed almost complete inhibition of binding, thus indicating that sAPPrec binding was specific (Figure 5A).
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To ensure the specificity of sAPPrec binding to cell surfaces, B 104 cells were preincubated in suspension with different concentrations of sAPPrec at 4C. After this preincubation, their ability to bind to immobilized sAPPrec was monitored by surface plasmon resonance spectroscopy. Preincubation with 1 µM sAPPrec resulted in a decrease of binding by 60%. Binding was almost completely inhibited at 4 µM (Figure 5B) thus confirming that the sAPPrec binding was specific.
Influence of Heparinase I on sAPPrec Binding
Several growth factors such as FGF are known to bind to heparin (
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Sensitivity of sAPPrec Binding Towards Aldehyde Fixation
To further investigate the binding characteristics of sAPPrec to cell surfaces, FRTL-5 or PC 12 cells were fixed for 30 min with 4% paraformaldehyde before the incubation with sAPPrec was performed. Quantitation of bound sAPPrec was performed by densitometry as decribed in Materials and Methods. After fixation the binding of sAPPrec to cell surfaces was significantly reduced by 6075% as shown in Figure 7.
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Discussion |
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The binding of sAPP to cell surfaces is of particular importance because it is the first step in the cascade of signal transduction events that lead to epithelial proliferation or to distinct physiological responses in other cell types. However, the visualization of exogenously added sAPP and its binding on cells is greatly hampered due to the ubiquitous expression of APP. The indirect localization of binding sites reported here makes use of recombinant sAPP (sAPPrec) and of the following features of sAPPrec: It elicits signal transduction and proliferation in thyrocytes (
The anti-sAPPrec antibodies allowed selective localization of cell surface-bound sAPPrec in a variety of cell types in which highest fluorescence intensity appeared to be reached at 10 nM sAPPrec. Radiometric observations with iodinated sAPPrec showed that the binding of sAPPrec to its putative receptor is saturable reaching saturation at 10 nM sAPPrec. The specificity of binding was also shown by these radiometric determinations and confirmed by surface plasmon resonance spectroscopy in that binding of cells to immobilized sAPPrec was almost completely inhibited at 4 µM of free sAPPrec.
sAPP occurs in the circulation at concentrations of about 60 pM. However, platelets which are the main source of circulating sAPP may contribute up to 30 nM sAPP (
The efficiency of sAPPrec specific antibodies for immunocytochemical detection of sAPPrec was compared to a number of other procedures which involved biotinylated sAPPrec or sAPPrec carrying a His tag on its C-terminal portion. Both sAPPrec derivatives, which are also able to bind to cell surfaces and to induce physiological responses such as cell proliferation, can be recognized by a specific antibody against the His tag or by fluorescently labeled streptavidin, thereby offering additional possibilities for the selective visualization of sAPPrec. Our observations show that the antibodies against sAPPrec provide the most sensitive detection device as compared to both other techniques.
As also shown in this study, the putative receptor for sAPPrec is highly sensitive to aldehyde fixation because the ability of cells to bind sAPPrec is markedly reduced after this treatment. Because of the preferential reaction of aldehyde groups with lysine residues (
sAPPrec binding occurs in the form of patches evenly distributed on the free accessible cell surfaces. These patches are preformed, i.e., they are present at 4C and not induced by ligand binding. They appear to correspond to membrane rafts that have been implicated in participation in cellular processes such as sorting in polarized cells (
In conclusion, we present a method for selective visualization of cell surface-bound sAPPrec without interference by cellular APP or sAPP. The method provides the experimental basis to indirectly locate cell surface binding sites that appear to be assembled in cell surface microdomains.
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Acknowledgments |
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Supported by Deutsche Forschungsgemeinschaft (SFB 284), by a Nordrhein Westfalen research grant, and by Fonds der Chemischen Industrie.
We thank Drs K. Beyreuther and G. Multhaup for the monoclonal antibody 22C11 and W. Neumüller for discussions and critical reading of the manuscript. We are grateful to Ms Babette Baumann and Ms. Beate Wolf for technical assistance, to Ms Andrea Roth for typing the manuscript, and to Ms Ann Icking for assistance throughout the binding experiments.
Received for publication July 20, 1998; accepted October 20, 1998.
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