Journal of Neuroscience Research 33:639-648 (1992)
Rapid Communication L2/HNK=1Carbohydrate and Protein-Protein Interactions Mediate the Homophilic Binding of the Neural Adhesion Molecule PO L.S. Griffith, B. Schmitz, and M. Schachner Department of Neurobiology, Swiss Federal Institute of Technology, Honggerberg, Zurich, Switzerland
The neural adhesion molecule PO, the most abundant glycoprotein in peripheral myelin of mammals, is a member of the immunoglobulin superfamily and expresses the L2/HNK-1 and L3 oligosaccharides at a single N-glycosylation site. It acts in both homophilic and heterophilic binding mechanisms. To investigate the molecular requirements for homophilic interaction, we have used PO from human sciatic nerve and the extracellular domain of PO expressed in bacteria to determine binding of PO to PO in solid phase and bead aggregation assays. The binding of PO to PO could be partially inhibited in both assays by antibodies to the L2/HNK-1 epitope and by the L2/HNK-l carbohydrate, but not by L3 antibodies or other carbohydrates. Inhibition of binding was also seen with polyclonal antibodies reacting with the protein backbone of PO. These observations indicate that both carbohydrate and protein structures are involved in the binding of PO to PO and that PO acts as a presenter of and a receptor for a functionally important carbohydrate. 0 1992 Witey-Liss, Inc. Key words: L2/HNK-1 carbohydrate, PO, homophilic binding INTRODUCTION The functional importance of Schwann cells in myelination and growth of axons has been well documented (for reviews, see: Abbott, 1991; Duncan, 1990; Martini, 1992). PO, the major glycoprotein of myelin in the peripheral nervous system of mammals (Ishaque et al., 1980), has been implicated in these processes. It is involved, either directly or indirectly in the formation of myelin during development and in the maintenance of the integrity of myelin in the adult, as observed in mice deficient in the PO gene (Giese et al., 1992). Functional studies in vitro indicate that PO mediates homophilic adhesion to PO expressing Schwann cells or PO transfected 0 1992 Wiley-Liss, Inc.
fibroblasts (Filbin et al., 1990; Schneider-Schaulies et al., 1990), accumulates at sites of contact in PO transfected fibroblasts (D’Urso et al., 1990), and increases neurite outgrowth in a heterophilic binding mechanism (Schneider-Schaulies et al., 1990). The detailed molecular mechanisms which underlie these binding mechanisms have, however, remained largely unknown. PO is a member of the immunoglobulin superfamily and contains a single V-type immunoglobulin-like domain (Lemke and Axel, 1985; Lemke et al., 1988; Uyemura et al., 1987). Its single N-glycosylation site exhibits considerable microheterogeneity in carbohydrate composition (Sakamoto et al., 1987; Uyemura and Kitamura, 1991; Field et al., 1992; Burger et al., 1992a,b). Amongst these oligosaccharides, the functionally important L2/HNK-1 and L3 glycans have been detected (Bollensen and Schachner, 1987). Both carbohydrate structures are expressed by many neural recognition molecules and have been recognized as mediators in cell interactions by several observations: The L2/HNK- 1 carbohydrate structure, a sulfated glucuronic acid attached to lactosamine residues (Chou et al., 1986; Ariga et al., 1987), is involved in adhesion of neural cells to each other and to laminin (Keilhauer et al., 1985; Kiinemund et al., 1988; Liu et al., 1992). It is involved in migration of neural crest cells (Bronner-Fraser, 1987) and promotes neurite outgrowth (Riopelle et al., 1985; Dow et al., 1988). Certain types of neurons respond to this carbohydrate by increased neurite outgrowth, whereas others are insensitive to it (Martini et al., 1992). The oligomannosidic L3 carbohydrate mediates neural cell adhesion and neurite outgrowth (Kiicherer et al., 1987; Fahrig et al., 1990; Schmitz et al., submitted) and is
Received September 1, 1992; accepted September 23, 1992. Address reprint requests to Melitta Schachner, Dept. of Neurobiology, Swiss Federal Institute of Technology, Honggerberg, 8093 Zurich, Switzerland.
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required for the molecular association between L1 and NCAM (Horstkorte et al., submitted; Kadmon et al., 1990a,b). Based on the observation that PO expresses these two carbohydrate structures and that both the protein backbone and complex-type carbohydrates have been implicated in the homophilic binding of PO (Filbin and Tennekoon, 1991;Schneider-Schaulies et a1. , 1990), we set out to investigate the contributions of carbohydrates and the protein backbone in the homophilic interaction of PO. Here we report that the binding of PO to PO depends on the L2/HNK- 1 complex-type carbohydrate structure and on protein-protein interactions, indicating that the single immunoglobulin-like domain of PO is both a presenter of and receptor for the L2/HNK-1 carbohydrate.
MATERIALS AND METHODS Antigens and Antibodies PO was isolated from human sciatic nerve (designated human PO) and the bacterially expressed extracellular domain of rat PO (designated POED) was prepared as described (Kitamura et al., 1976; Poduslo, 1990; Schneider-Schaulies et al., 1990). NCAM and L2 glycoprotein (L2/HNK- 1 positive glycoprotein fraction from detergent extracts of adult mouse brain after removal of NCAM, MAG, and L1) were immunoaffinity purified as described (Kruse et al., 1985). L2 glycolipid was isolated from human sciatic nerve by the procedure of Chou et al. (1986) with the modifications described by Hall et al. (1992) and was a gift of Heike Hall, Department of Neurobiology, Swiss Federal Institute of Technology. The antibodies used in this study were polyclonal rabbit antiserum against POED (Schneider-Schaulies et al., 1990), rat monoclonal antibody H28 (IgG) reacting with an extracellular epitope of mouse NCAM expressed on all isoforms (Hirn et al., 1981), rat monoclonal antibody 412 (IgG) against the L2/HNK-l carbohydrate epitope (Kruse et al., 1984), and the rat monoclonal antibody 492 (IgM) directed against the L3 carbohydrate epitope (Kiicherer et al., 1987). Fab fragments of antibody 412 were prepared as described (Porter, 1959) and were a gift of Heike Hall. The concentration of antibodies in the polyclonal antiserum against POED was estimated by Coomassie blue staining of slab gels after SDS-PAGE (Laemmli, 1970) and comparison of corresponding bands in the antiserum with defined quantities of IgGs determined according to Bradford (1976). The antiserum contained approximately 15 mg/ml IgG. Horseradish peroxidase (HRP) coupled goat anti-rabbit and goat anti-rat antibodies (Dianova, Hamburg, Germany) were used as secondary antibodies.
Biotinylation of Proteins Human PO (0.5 mg or 1.0 mg/ml) or bacterial POED (0.5 or 1.0 mg/ml) were biotinylated using the Pierce NHS-LC biotin kit according to the manufacturer’s instructions. Centricon 10 concentrators (molecular weight cut-off at 10,000 Daltons; Amicon) were used to remove the biotin from biotinylated PO. The biotinylated protein solution was diluted to a concentration of 1 mg/ ml in phosphate buffered saline, pH 7.3 (PBS) and stored at 4°C until use. No degradation of biotinylated PO could be detected by Coomassie blue staining of gels after SDS-PAGE. The reactivity of the biotinylated protein with the polyclonal PO antibody was the same as with the non-biotinylated protein as determined by enzyme-linked immunosorbent assay (ELISA). The efficiency of biotinylation was tested by reactivity with HRP-coupled streptavidin (Sigma). Binding Assays A. Protein-protein binding. Binding of PO to PO was measured by a solid phase binding assay. Ninetysix-well microtiter plates (Nunc, MaxiSorp) were coated overnight at 4°C with PO or POED at a concentration of 5 kgiml unless otherwise stated. After washing the plates with PBS and blocking with 1 % fat free bovine serum albumin (BSA; Sigma, catalog number 7030) in PBS for 1 hr at room temperature, the plates were again thoroughly washed with PBS. The coated PO was then incubated with biotinylated POED or with biotinylated human PO for 2 hr at room temperature. Unless otherwise stated, the concentration of the biotinylated proteins was 5 kg/ ml. The plates were then washed three times with PBS, incubated with HRP-coupled streptavidin for a further 2 hr at room temperature and again washed with PBS before incubation with ABTS (2,2’-azino-di[3-ethylbenzthiazoline sulfonate (6)]) (Boehringer-Mannheim) in 0.1 M acetate buffer pH 4.2 for 20-30 min at room temperature. The enzymatic reaction was terminated by addition of 0.6% aqueous SDS. The optical density (OD) of the colour reaction product was determined at 405 nm in a Titertek-Multiscan Plus ELISA plate reader (Flow Laboratories). The ability of compounds to compete with the binding of PO to PO was measured by incubating them with the substrate-coated protein for 30 to 45 min at room temperature. Unbound compounds were removed by washing with PBS before the addition of biotinylated proteins. In some experiments, the compounds were added to the soluble biotinylated PO and pre-incubated for 30 to 45 min before the addition of substrate-coated PO. No significant differences in the effects were observed between the two procedures. The following compounds were added (concentrations in brackets): poly-
Protein- and Carbohydrate-Dependent PO-PO Binding
clonal PO antibodies (5 pg/ml), monoclonal antibody H28 (50 pgiml), Fab fragments of monoclonal L2 antibody (25 pgiml), monoclonal L3 antibody (50 pg/ml), human PO (5 pg/ml), POED ( 5 pg/ml), NCAM (5 pgi ml), L2 glycoprotein (5 pg/ml), ganglioside GDlb (5 pgiml), L2 glycolipid (5 pg/ml), and sulfatide ( 5 pgl ml) . B. Protein-glycolipid binding. Sulfatide (Sigma), ganglioside GDlb (Sigma), and L2 glycolipid were added as ethanolic solutions (30 pmoliwell) to 96-well microtiter plates and the ethanol was evaporated at room temperature. After washing, blocking with 1% fat free bovine serum albumin in PBS for 1 hr and washing again, glycolipids were incubated with human PO or POED (5 pgiml) in PBS and, after a further washing step, with polyclonal PO antibodies followed by HRPcoupled secondary antibodies. Development of the HRP reaction product was performed as described in A). All incubation steps were carried out for 2 hr at room temperature. The determinations in A and B were performed in at least duplicate samples. Values were corrected for non-specific binding by subtracting the background values (obtained in the absence of biotinylated PO) from the experimental values. The degree of inhibition (% inhibition) of PO to PO binding in the presence of additives was determined in relation to the background-corrected values in the absence of additives. The statistical significance of differences between values was determined by one way ANOVA and Newman-Keuls multiple comparison among means on non-normalized data. Differences were reported as significant when P was at least < 0.05.
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bated at a 1 :100 dilution in Isoton solution (Coulter Electronics) for 3 hr at room temperature with gentle shaking (120 rpm on a rotary shaker) and aggregation was then determined on a Coulter Counter Multisizer with a Coulter sampling stand (Coulter Electronics) by measuring partial volumes (Kadmon et al., 1990a). When aggregation was measured in the presence of additives, these were added to the Isoton solution at the same time as the beads. The concentrations of additives were the same as in the solid phase assay. As only few uncoated beads (less than 2%) showed aggregate sizes greater than 4.5 pm, this particle size was taken as the cut-off value for non-specific aggregation (Kadmon et al. , 1990a). The experimental values for aggregation were taken as the percentage of partial volumes greater than 4.5 pm. Values are from at least three experiments performed at least in triplicate. The statistical significance of differences between values was determined by one way ANOVA and Newman-Keuls multiple comparison among means on non-normalized data. Differences were reported as significant when P was at least < 0.05.
RESULTS Determination of the Contribution of the Protein Backbone to Homophilic Binding of PO To determine whether the hornophilic binding of PO was mediated by its protein backbone, the non-glycosylated, extracellular domain of PO from the rat expressed in bacteria (POED) was used. Binding was determined by a solid phase binding assay, in which POED was substrate-coated into microtiter plates and biotinylated POED was used as probe for homophilic binding. POED bound well to POED in this assay (Fig. 1). Polyclonal PO antibodies preincubated with substrate-coated POED before addition of biotinylated POED resulted in a complete inhibition of the binding of POED to POED (Fig. 1). Identical results were obtained, when the biotinylated POED was preincubated with the antibodies before the antigen-antibody mixture was added to substrate-coated POED (not shown). Fab fragments of monoclonal L2 antibody 412 did not interfere with binding (Fig. 1). With the recombinant proteins expressed in bacteria not being glycosylated, these experiments indicate that PO is able to interact with itself via the protein backbone.
Coulter Counter Analysis Preparation of protein-coated beads. Human PO and NCAM in PBS containing 0.1% deoxycholate were coated onto latex beads (3 p m in diameter; Sigma) according to Kadmon et al. (1990a). Briefly, 75 pl of a 10% suspension of beads were added to 900 p1 of the protein solutions (all at 200 pg/ml) and dialyzed against two changes of PBS overnight at 4°C. The beads were pelleted by centrifugation and the supernatants collected for determination of remaining protein by the Bradford assay (Bradford, 1976). The coupling efficiency was determined by measurement of protein concentrations in the solutions before and after incubation with the beads. For PO, the coupling efficiency was 95% and for NCAM Determination of the Involvement of Carbohydrates 25% of the protein offered in solution. After coating, the in Homophilic Binding of PO To determine whether in addition to protein-protein beads were blocked with 1% fat free BSA in PBS for 1 hour at room temperature, washed three times with PBS, binding, also carbohydrates may mediate binding besuspended in 1 ml 1% BSA in PBS and stored at 4°C tween PO molecules, PO from human sciatic nerve caruntil use. All experiments were performed within 2 rying the L2/HNK- 1, L3 and other oligosaccharides (Field et al., 1992) was substrate-coated into microtiter weeks after coating the beads. Analysis of bead aggregation. Beads were incu- plates and binding was probed with biotinylated POED.
Griffith et al.
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._
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0 control
+ anti PO
+ anti L2
Fig. 1. Determination of binding of POED to POED in the absence (control) or presence of polyclonal PO antibodies ( + anti PO) and Fab fragments of monoclonal L2 antibody 412 ( + anti L2). Antibodies were preincubated with substrate-coated POED before addition of biotinylated POED. Binding of POED to POED in the absence of antibodies was set to 100%. Bars represent mean values 2 SEM from three independent experiments carried out in triplicate. Hatched bars are control and value not different from the control, while the black bar is significantly different from the control at P
Rapid Communication L2/HNK=1Carbohydrate and Protein-Protein Interactions Mediate the Homophilic Binding of the Neural Adhesion Molecule PO L.S. Griffith, B. Schmitz, and M. Schachner Department of Neurobiology, Swiss Federal Institute of Technology, Honggerberg, Zurich, Switzerland
The neural adhesion molecule PO, the most abundant glycoprotein in peripheral myelin of mammals, is a member of the immunoglobulin superfamily and expresses the L2/HNK-1 and L3 oligosaccharides at a single N-glycosylation site. It acts in both homophilic and heterophilic binding mechanisms. To investigate the molecular requirements for homophilic interaction, we have used PO from human sciatic nerve and the extracellular domain of PO expressed in bacteria to determine binding of PO to PO in solid phase and bead aggregation assays. The binding of PO to PO could be partially inhibited in both assays by antibodies to the L2/HNK-1 epitope and by the L2/HNK-l carbohydrate, but not by L3 antibodies or other carbohydrates. Inhibition of binding was also seen with polyclonal antibodies reacting with the protein backbone of PO. These observations indicate that both carbohydrate and protein structures are involved in the binding of PO to PO and that PO acts as a presenter of and a receptor for a functionally important carbohydrate. 0 1992 Witey-Liss, Inc. Key words: L2/HNK-1 carbohydrate, PO, homophilic binding INTRODUCTION The functional importance of Schwann cells in myelination and growth of axons has been well documented (for reviews, see: Abbott, 1991; Duncan, 1990; Martini, 1992). PO, the major glycoprotein of myelin in the peripheral nervous system of mammals (Ishaque et al., 1980), has been implicated in these processes. It is involved, either directly or indirectly in the formation of myelin during development and in the maintenance of the integrity of myelin in the adult, as observed in mice deficient in the PO gene (Giese et al., 1992). Functional studies in vitro indicate that PO mediates homophilic adhesion to PO expressing Schwann cells or PO transfected 0 1992 Wiley-Liss, Inc.
fibroblasts (Filbin et al., 1990; Schneider-Schaulies et al., 1990), accumulates at sites of contact in PO transfected fibroblasts (D’Urso et al., 1990), and increases neurite outgrowth in a heterophilic binding mechanism (Schneider-Schaulies et al., 1990). The detailed molecular mechanisms which underlie these binding mechanisms have, however, remained largely unknown. PO is a member of the immunoglobulin superfamily and contains a single V-type immunoglobulin-like domain (Lemke and Axel, 1985; Lemke et al., 1988; Uyemura et al., 1987). Its single N-glycosylation site exhibits considerable microheterogeneity in carbohydrate composition (Sakamoto et al., 1987; Uyemura and Kitamura, 1991; Field et al., 1992; Burger et al., 1992a,b). Amongst these oligosaccharides, the functionally important L2/HNK-1 and L3 glycans have been detected (Bollensen and Schachner, 1987). Both carbohydrate structures are expressed by many neural recognition molecules and have been recognized as mediators in cell interactions by several observations: The L2/HNK- 1 carbohydrate structure, a sulfated glucuronic acid attached to lactosamine residues (Chou et al., 1986; Ariga et al., 1987), is involved in adhesion of neural cells to each other and to laminin (Keilhauer et al., 1985; Kiinemund et al., 1988; Liu et al., 1992). It is involved in migration of neural crest cells (Bronner-Fraser, 1987) and promotes neurite outgrowth (Riopelle et al., 1985; Dow et al., 1988). Certain types of neurons respond to this carbohydrate by increased neurite outgrowth, whereas others are insensitive to it (Martini et al., 1992). The oligomannosidic L3 carbohydrate mediates neural cell adhesion and neurite outgrowth (Kiicherer et al., 1987; Fahrig et al., 1990; Schmitz et al., submitted) and is
Received September 1, 1992; accepted September 23, 1992. Address reprint requests to Melitta Schachner, Dept. of Neurobiology, Swiss Federal Institute of Technology, Honggerberg, 8093 Zurich, Switzerland.
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required for the molecular association between L1 and NCAM (Horstkorte et al., submitted; Kadmon et al., 1990a,b). Based on the observation that PO expresses these two carbohydrate structures and that both the protein backbone and complex-type carbohydrates have been implicated in the homophilic binding of PO (Filbin and Tennekoon, 1991;Schneider-Schaulies et a1. , 1990), we set out to investigate the contributions of carbohydrates and the protein backbone in the homophilic interaction of PO. Here we report that the binding of PO to PO depends on the L2/HNK- 1 complex-type carbohydrate structure and on protein-protein interactions, indicating that the single immunoglobulin-like domain of PO is both a presenter of and receptor for the L2/HNK-1 carbohydrate.
MATERIALS AND METHODS Antigens and Antibodies PO was isolated from human sciatic nerve (designated human PO) and the bacterially expressed extracellular domain of rat PO (designated POED) was prepared as described (Kitamura et al., 1976; Poduslo, 1990; Schneider-Schaulies et al., 1990). NCAM and L2 glycoprotein (L2/HNK- 1 positive glycoprotein fraction from detergent extracts of adult mouse brain after removal of NCAM, MAG, and L1) were immunoaffinity purified as described (Kruse et al., 1985). L2 glycolipid was isolated from human sciatic nerve by the procedure of Chou et al. (1986) with the modifications described by Hall et al. (1992) and was a gift of Heike Hall, Department of Neurobiology, Swiss Federal Institute of Technology. The antibodies used in this study were polyclonal rabbit antiserum against POED (Schneider-Schaulies et al., 1990), rat monoclonal antibody H28 (IgG) reacting with an extracellular epitope of mouse NCAM expressed on all isoforms (Hirn et al., 1981), rat monoclonal antibody 412 (IgG) against the L2/HNK-l carbohydrate epitope (Kruse et al., 1984), and the rat monoclonal antibody 492 (IgM) directed against the L3 carbohydrate epitope (Kiicherer et al., 1987). Fab fragments of antibody 412 were prepared as described (Porter, 1959) and were a gift of Heike Hall. The concentration of antibodies in the polyclonal antiserum against POED was estimated by Coomassie blue staining of slab gels after SDS-PAGE (Laemmli, 1970) and comparison of corresponding bands in the antiserum with defined quantities of IgGs determined according to Bradford (1976). The antiserum contained approximately 15 mg/ml IgG. Horseradish peroxidase (HRP) coupled goat anti-rabbit and goat anti-rat antibodies (Dianova, Hamburg, Germany) were used as secondary antibodies.
Biotinylation of Proteins Human PO (0.5 mg or 1.0 mg/ml) or bacterial POED (0.5 or 1.0 mg/ml) were biotinylated using the Pierce NHS-LC biotin kit according to the manufacturer’s instructions. Centricon 10 concentrators (molecular weight cut-off at 10,000 Daltons; Amicon) were used to remove the biotin from biotinylated PO. The biotinylated protein solution was diluted to a concentration of 1 mg/ ml in phosphate buffered saline, pH 7.3 (PBS) and stored at 4°C until use. No degradation of biotinylated PO could be detected by Coomassie blue staining of gels after SDS-PAGE. The reactivity of the biotinylated protein with the polyclonal PO antibody was the same as with the non-biotinylated protein as determined by enzyme-linked immunosorbent assay (ELISA). The efficiency of biotinylation was tested by reactivity with HRP-coupled streptavidin (Sigma). Binding Assays A. Protein-protein binding. Binding of PO to PO was measured by a solid phase binding assay. Ninetysix-well microtiter plates (Nunc, MaxiSorp) were coated overnight at 4°C with PO or POED at a concentration of 5 kgiml unless otherwise stated. After washing the plates with PBS and blocking with 1 % fat free bovine serum albumin (BSA; Sigma, catalog number 7030) in PBS for 1 hr at room temperature, the plates were again thoroughly washed with PBS. The coated PO was then incubated with biotinylated POED or with biotinylated human PO for 2 hr at room temperature. Unless otherwise stated, the concentration of the biotinylated proteins was 5 kg/ ml. The plates were then washed three times with PBS, incubated with HRP-coupled streptavidin for a further 2 hr at room temperature and again washed with PBS before incubation with ABTS (2,2’-azino-di[3-ethylbenzthiazoline sulfonate (6)]) (Boehringer-Mannheim) in 0.1 M acetate buffer pH 4.2 for 20-30 min at room temperature. The enzymatic reaction was terminated by addition of 0.6% aqueous SDS. The optical density (OD) of the colour reaction product was determined at 405 nm in a Titertek-Multiscan Plus ELISA plate reader (Flow Laboratories). The ability of compounds to compete with the binding of PO to PO was measured by incubating them with the substrate-coated protein for 30 to 45 min at room temperature. Unbound compounds were removed by washing with PBS before the addition of biotinylated proteins. In some experiments, the compounds were added to the soluble biotinylated PO and pre-incubated for 30 to 45 min before the addition of substrate-coated PO. No significant differences in the effects were observed between the two procedures. The following compounds were added (concentrations in brackets): poly-
Protein- and Carbohydrate-Dependent PO-PO Binding
clonal PO antibodies (5 pg/ml), monoclonal antibody H28 (50 pgiml), Fab fragments of monoclonal L2 antibody (25 pgiml), monoclonal L3 antibody (50 pg/ml), human PO (5 pg/ml), POED ( 5 pg/ml), NCAM (5 pgi ml), L2 glycoprotein (5 pg/ml), ganglioside GDlb (5 pgiml), L2 glycolipid (5 pg/ml), and sulfatide ( 5 pgl ml) . B. Protein-glycolipid binding. Sulfatide (Sigma), ganglioside GDlb (Sigma), and L2 glycolipid were added as ethanolic solutions (30 pmoliwell) to 96-well microtiter plates and the ethanol was evaporated at room temperature. After washing, blocking with 1% fat free bovine serum albumin in PBS for 1 hr and washing again, glycolipids were incubated with human PO or POED (5 pgiml) in PBS and, after a further washing step, with polyclonal PO antibodies followed by HRPcoupled secondary antibodies. Development of the HRP reaction product was performed as described in A). All incubation steps were carried out for 2 hr at room temperature. The determinations in A and B were performed in at least duplicate samples. Values were corrected for non-specific binding by subtracting the background values (obtained in the absence of biotinylated PO) from the experimental values. The degree of inhibition (% inhibition) of PO to PO binding in the presence of additives was determined in relation to the background-corrected values in the absence of additives. The statistical significance of differences between values was determined by one way ANOVA and Newman-Keuls multiple comparison among means on non-normalized data. Differences were reported as significant when P was at least < 0.05.
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bated at a 1 :100 dilution in Isoton solution (Coulter Electronics) for 3 hr at room temperature with gentle shaking (120 rpm on a rotary shaker) and aggregation was then determined on a Coulter Counter Multisizer with a Coulter sampling stand (Coulter Electronics) by measuring partial volumes (Kadmon et al., 1990a). When aggregation was measured in the presence of additives, these were added to the Isoton solution at the same time as the beads. The concentrations of additives were the same as in the solid phase assay. As only few uncoated beads (less than 2%) showed aggregate sizes greater than 4.5 pm, this particle size was taken as the cut-off value for non-specific aggregation (Kadmon et al. , 1990a). The experimental values for aggregation were taken as the percentage of partial volumes greater than 4.5 pm. Values are from at least three experiments performed at least in triplicate. The statistical significance of differences between values was determined by one way ANOVA and Newman-Keuls multiple comparison among means on non-normalized data. Differences were reported as significant when P was at least < 0.05.
RESULTS Determination of the Contribution of the Protein Backbone to Homophilic Binding of PO To determine whether the hornophilic binding of PO was mediated by its protein backbone, the non-glycosylated, extracellular domain of PO from the rat expressed in bacteria (POED) was used. Binding was determined by a solid phase binding assay, in which POED was substrate-coated into microtiter plates and biotinylated POED was used as probe for homophilic binding. POED bound well to POED in this assay (Fig. 1). Polyclonal PO antibodies preincubated with substrate-coated POED before addition of biotinylated POED resulted in a complete inhibition of the binding of POED to POED (Fig. 1). Identical results were obtained, when the biotinylated POED was preincubated with the antibodies before the antigen-antibody mixture was added to substrate-coated POED (not shown). Fab fragments of monoclonal L2 antibody 412 did not interfere with binding (Fig. 1). With the recombinant proteins expressed in bacteria not being glycosylated, these experiments indicate that PO is able to interact with itself via the protein backbone.
Coulter Counter Analysis Preparation of protein-coated beads. Human PO and NCAM in PBS containing 0.1% deoxycholate were coated onto latex beads (3 p m in diameter; Sigma) according to Kadmon et al. (1990a). Briefly, 75 pl of a 10% suspension of beads were added to 900 p1 of the protein solutions (all at 200 pg/ml) and dialyzed against two changes of PBS overnight at 4°C. The beads were pelleted by centrifugation and the supernatants collected for determination of remaining protein by the Bradford assay (Bradford, 1976). The coupling efficiency was determined by measurement of protein concentrations in the solutions before and after incubation with the beads. For PO, the coupling efficiency was 95% and for NCAM Determination of the Involvement of Carbohydrates 25% of the protein offered in solution. After coating, the in Homophilic Binding of PO To determine whether in addition to protein-protein beads were blocked with 1% fat free BSA in PBS for 1 hour at room temperature, washed three times with PBS, binding, also carbohydrates may mediate binding besuspended in 1 ml 1% BSA in PBS and stored at 4°C tween PO molecules, PO from human sciatic nerve caruntil use. All experiments were performed within 2 rying the L2/HNK- 1, L3 and other oligosaccharides (Field et al., 1992) was substrate-coated into microtiter weeks after coating the beads. Analysis of bead aggregation. Beads were incu- plates and binding was probed with biotinylated POED.
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+ anti L2
Fig. 1. Determination of binding of POED to POED in the absence (control) or presence of polyclonal PO antibodies ( + anti PO) and Fab fragments of monoclonal L2 antibody 412 ( + anti L2). Antibodies were preincubated with substrate-coated POED before addition of biotinylated POED. Binding of POED to POED in the absence of antibodies was set to 100%. Bars represent mean values 2 SEM from three independent experiments carried out in triplicate. Hatched bars are control and value not different from the control, while the black bar is significantly different from the control at P
