Journal of Neuroscience Research 33538-548 (1992)

The Neural Cell Adhesion Molecule (NCAM) Heparin Binding Domain Binds to Cell Surface Heparan Sulfate Proteoglycans S.G. Kallapur and R.A. Akeson Divisions of Basic Science Research (R.A.A.) and Neonatology (S.G.K.), Children’s Hospital Research Foundation, Cincinnati, Ohio

The neural cell adhesion molecule (NCAM) has been strongly implicated in several aspects of neural development. NCAM mediated adhesion has been proposed to involve a homophilic interaction between NCAMs on adjacent cells. The heparin binding domain (HBD) is an amino acid sequence within NCAM and has been shown to be involved in NCAM mediated adhesion but the relationship of this domain to NCAM segments mediating homophilic adhesion has not been defined. In the present study, a synthetic peptide corresponding to the HBD has been used as a substrate to determine its role in NCAM mediated adhesion. A neural cell line expressing NCAM (B35) and its derived clone which does not express NCAM (B35 clone 3) adhered similarly to plates coated with HBD peptide. A polyclonal antiserum to NCAM inhibited B35 cell-HBD peptide adhesion by only lo%, a value not statistically different from inhibition caused by preimmune serum. Both these experiments suggested no direct NCAM-HBD interactions. To test whether the HBD peptide bound to cell surface heparan sulfate proteoglycans (HSPG), HSPG synthesis was inhibited using P-D-xyloside. After treatment, B35 cell adhesion to the HBD peptide, but not to control substrates, was significantly decreased. B35 cell adhesion to the HBD peptide could be inhibited by lop7 M heparin but not chondroitin sulfate. Preincubation of the substrate (HBD peptide) with heparin caused dramatic reduction of B35 cell-HBD peptide adhesion whereas preincubation of B35 cells with heparin caused only modest reductions in cellHBD adhesion. Furthermore, inhibition of HSPG sulfation with sodium chlorate also decreased the adhesion of B35 cells to the HBD peptide. These results strongly suggest that, within the assay system, the NCAM HBD does not participate in homophilic interactions but binds to cell surface heparan sulfate proteoglycan. This interaction potentially represents an important mechanism of NCAM adhesion and fur0 1992 Wiley-Liss, Inc.

ther supports the view that NCAM has multiple structurally independent binding sites. 0 1992 Wiley-Liss, Inc.

Key words: adhesion, B35 cells, chlorate, xyloside INTRODUCTION The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily . NCAM participates in several developmental events including neuron-neuron , neuron-glial , and neuron-muscle interactions, neurite fasciculation, and formation of retinotectal cytoarchitecture (Fraser et al., 1984; Grumet et al., 1982; Keilhauer et al., 1985; Rutishauser and Edelman, 1980; Rutishauser et al., 1983; Thanos et al., 1984; Thiery et al., 1977; reviewed in Edelman, 1988; Edelman and Crossin, 1991; Jessell, 1988; Rutishauser and Jessell, 1988). Early studies suggested a homophilic mechanism of NCAM adhesion based on the observation that reconstituted lipid vesicles containing purified NCAM coaggregate (Hoffman and Edelman, 1983). However, other workers have reported that NCAM coated beads do not coaggregate unless micromolar amounts of heparin are added in the assay medium (Kadmon et al., 1990). A

Received April 14, 1992; revised August 6, 1992; accepted August 6, 1992. Address reprint requests to R.A. Akeson, Division of Basic Science Research, Children’s Hospital Research Foundation, Elland and Bethesda Ave., Cincinnati, OH 45229-2899. Abbreviations used: BCA, bicinchoninic acid; BME, basal medium Eagle’s; BSA, bovine serum albumin; CS, calf serum; DMEM, Dulbecco’s modified Eagle’s medium; DMSO. dimethyl sulfoxide; EDTA, ethylene diamine tetraacetic acid; FCS, fetal calf serum; GAG, glycosaminoglycan; HBD, heparin binding domain; HSPG, heparan sulfate proteoglycan; NCAM, neural cell adhesion molecule; OD, optical density; PBS, phosphate buffered saline.

NCAM-Proteoglycan Interaction

recent report failed to demonstrate homophilic NCAMNCAM interaction in CHO cells transfected with NCAM (Murray and Jensen, 1992). Thus, there is no current On the mechanisms by which NCAM mediates adhesion. In an effort to identify domains On NCAM' and G1aser (1986) determined that a 25 kd proteolytic fragment bound heparin. A monoclonal antibody to this fragment inhibited cell adA monoclonal antibody to another region Of NCAM, however, did not decrease adhesion to the 25 kd proteolytic fragment but disrupted adto coated with intact NCAM ('Ole and G1aser, 1986; Cole et al., 1986). a polyclonal antibody to cell surface proteoglycan decreased retinal cell attachment to NCAM. Taken together, these experiments from different laboratories suggested the possible presence of distinct homophilic and heparin binding domains (HBD) in NCAM and also a potential third binding site involving as yet unidentified ligands. The existence of multiple binding sites On NCAM is further suggested by three independent studies: 1) the binding of NCAM to extracellular matrix constituents of a teratocarcinoma cell line (Werz and Schachner, 19881, 2) the binding of type I collagen fibers to 'CAM (Probstmeier et '1.9 19921, and 3 ) the ability Of loops Of NCAM t' sustain cell adhesion when expressed as recombinant fusion proteins (Frei et al., 1992). The NCAM HBD is in immunoglobulin like loop 2, a portion of the molecule which is identical in all NCAM isoforms (Reyes et al., 1991, Barthels et al., 1992). The importance of the HBD in the adhesion of NCAM was demonstrated by experiments in which mouse fibroblasts were transfected with either cDNA encoding wild type NCAM or NCAM polypeptides in which the heparin binding sequence was altered by substitution or deletion mutation. These mutants, when used as a substrate, had substantially reduced binding to heparin and could not support adhesion of retinal cells compared with wild type NCAM (Reyes et al., 1990). Furthermore, synthetic HBD peptide bound both, radiolabelled heparin, and supported adhesion to dissociated retinal cells (Cole and Akeson, 1989). In order to define the mechanisms of NCAM adhesion and to determine the relationship between homophilic adhesion and HBD mediated adhesion, the adhesion of an NCAM expressing neural cell line (B35) and its derived clone (B35 clone 3) not expressing NCAM was tested. Next, the effects of P-D-xylosides [which competitively inhibit the addition of glycosaminoglycan (GAG) chains to core proteins] on cell adhesion were tested. In this report we show that cells grown in P-D-xylosides have significantly decreased adhesion to HBD peptide, suggesting that the NCAM HBD interacts with cellular proteoglycans.

539

MATERIALS AND METHODS Cell Culture B35 cells (a generous gift of Dr. D. Schubert, Salk Institute, San Diego, CA) are a clonal cell line from a rat central nervous system tumor induced by nitroso&ylurea (Schubert et al., 1974). B35 cells express NCAM on the cell surface. B35 clone 3 cells were derived in our laboratory by treating B35 cells with the mutagen ethyl methyl sulfonate and then selecting for NCAM lacking variants by multiple rounds of treatment with polyclonal anti-NCAM and rabbit complement. The survivors were cloned. B35 clone 3 cells lack detectable cell surface NCAM polypeptide by immunofluorescence analyses with both polyclonal and monoclonal antibodies to NCAM. B35 clone 3 cells also had no detectable NCAM mRNA by northern analysis (Akeson and Haines, data not shown). All cells were maintained in culture in a medium consisting of Dulbecco's modified medium (DMEM) (Whittaker, Walkersville, MD) to which 5% calf serum (CS) and 5% fetal calf serum (FCS) (HyClone, Logan, UT) were added. Falcon tissue culture Lincoln park, NJ) were used. plates (Becton Cells were grown in an incubator with 10% CO, at 3 7 0 ~ . In some experiments involving growing cells in a medium with &lorate a lower concentration of Serum was used (below). The cells were detached using mM ethylene diamine tetraacetic acid (EDTA). Quantitation of Cell Adhesion Quantitation of cell adhesion was performed by measuring cell protein using the method described by Tuszynski and Murphy (1990). Ninety-six well microtiter plates (Corning, nontissue culture treated, Corning, NY) were used. Peptides were bound to individual wells using nitrocellulose (type BA85) dissolved in methanol as described by Lagenaur and Lemmon (1987). Since in some preliminary experiments, higher concentrations of nitrocellulose caused an increased background adhesion, wells were coated with 40 pL of nitrocellulose (0.45 pm, type BA85, Scheicher & Schuell, Keene, NH) in methanol solution (2.5 mm X 2.5 mm nitrocellulose dissolved in 12 mi methanol). Wells were allowed to dry in an air flow hood and then incubated with substrate (dissolved in sterile water) for 10 min at room temperature. Wells were then incubated with 1% bovine serum albumin (BSA) (Sigma, type V in PBS, St. Louis, MO) at room temperature for 1 hr to block unoccupied sites. After washing the wells with phosphate buffered saline (PBS), a cell suspension (20-40 pL) prepared in DMEM with no serum was plated in the wells at a concentration of 3 X 104/well. Excess adhesion medium (DMEM with 0.5% BSA, 200 pL) was then added to wells to reduce background adhesion. In some inhibition

540

Kallapur and Akeson 0.6

T

El

ICOOH

r=0.996

I40 kD M a j o r NCAM Form

wlld t y p e

131 141 IleTrpLysHisLysGlyArgAspValIleLeuLysLysAsp~alArgPheIle

Fig. I . Schematic representation of 140 kd form of NCAM showing the amino acid sequence of HBD. The numbers refer to the loops of the extracellular portion of the molecule. The transmembrane region is designated by T and the leader (signal) sequence by L. experiments, soluble heparin or chondroitin sulfate was added at this stage to the adhesion medium. In preliminary experiments, the period of incubation was varied from 1-3 hr and no difference in adhesion was noted (data not shown). Therefore, in the experiments reported here, cells were incubated at 37°C with 10% CO, for 3 hr, after which the non-adherent cells were washed three times by immersing the plate in a beaker containing PBS at 37°C. This washing avoided the development of a false high optical density value due to traces of the adhesion medium, since the color development depends on the formation of a cuprous-amino acid complex. After draining all the fluid, 200 p L bicinchoninic acid (BCA) reagent (mixture of reagent A:B in the volume 50:l) (Pierce, Rockford, IL) was added to each well. The plate was then exposed to 60°C in a dry oven for 30 min (manufacturer’s enhanced protocol) (Pierce, 1989) and after allowing to cool, the resultant color developed was read in a microtiter plate reader (Biotek instruments, Winooski, VT) at 562 nm. Background values from wells with no cells added were subtracted from the actual reading to get the final reading. The OD values for various substrates shown in the figures were corrected for non-specific adhesion to nitrocellulose by subtracting the OD values obtained on wells coated with nitrocellulose only from OD values obtained on wells coated with substrates. When HBD peptide (25 pg/ml) was used as a substrate, about 80% of the input B35 cells adhered in this assay. To evaluate this method for assaying neural cells at cell concentrations less than those tested by Tuszynski and Murphy (1990), B35 cells in concentrations ranging from 5 x 10, to 5 x lo4 ceIls/weIl were tested and an excellent linear correlation ( r = .996) was observed (Fig. 2), indicating the reliability of the modified adhesion assay.

Antibody Rabbit anti-NCAM serum (antibody 161) was produced to NCAM immunoaffinity purified from adult rat

Ceiis/weii x l o m 3

Fig. 2. Correlation between the cellsiwell with the OD (BCA method). Individual wells in the 96 microtiter plate were coated with poly L-lysine and the B35 cells were incubated on these wells in an incubator for 3 hr at 37°C with 10% CO,. The cells were then washed gently with PBS before addition of BCA reagent to the wells. Each point is shown as mean ? SD and represents an average of ten readings.

brain using an anti-NCAM monoclonal antibody 3F4 column (Akeson et al., 1988). Antibody 161 binds to plates coated with intact NCAM but not to BHD peptide in immunoassays.

Xyloside and Chlorate Treatment of Cells Cells were grown in DMEM with 5% FCS and 5% CS and 4-methylumbelliferyl-~-D-xyloside (Sigma, St. Louis, MO) (hereafter referred to as xyloside) was added from a stock solution of 150 mg/ml xyloside in dimethyl sulfoxide (DMSO) (DMSO concentration 0.6%). The cells were inspected daily for morphology and they were counted before plating and after harvesting to determine growth kinetics. For chlorate treatment of cells, the cells were grown in basal medium Eagle’s (BME) medium (Sigma, St. Louis, MO), which has a low level of sulfur containing amino acids. Some cell types can metabolize sulfur from amino acids as an alternative source of sulfate for proteoglycan sulfation (Rapraeger et al., 1991). Cells were grown in 0.1 % FCS with 30 mM chlorate for 48 hr, harvested using 1 mM EDTA, and then replated in BME with 5 % FCS and 5% CS in 30 mM chlorate for another 24 hr. Finally they were switched back to 0.1% serum with 30 mM chlorate for another 2-3 days. They were then harvested with 1 mM EDTA for the experiments (Rapraeger et al., 1991). The cells were inspected

NCAM-Proteoglycan Interaction TABLE 1. Effects of Anti-NCAM on B35 cell-HBD Adhesion

0.3

Pretreatment Pretreatment OD

None

0.27 2 0.009

T

Anti-NCAM

Preimmune serum

*

0.22 t 0.008

0.241 ~~

0.012

541

~

B35 cell suspension (1-2 X 106/ml) was incubated with either 25 pgiml polyclonal anti-NCAM serum (antibody 161) or 25 pgiml preimmune serum for 30 min at room temperature. The cells were then centrifuged at 800 rpm for 5 min followed by aspiration of the medium and gentle resuspension in serum free DMEM. The cells were then plated in individual wells which were coated with HBD peptide (25 pgiml) in a concentration of 3 X 104/well.The adhesion was allowed to proceed for 1 hr. B35 cells express about 2.5 X los NCAM moleculesicell (Akeson et al., 1988) and assuming anti-NCAM concentration in the immune serum to be 1% of the total IgG, this antibody concentration represents roughly a 100-fold molar excess of specific IgG over NCAM. The OD values are shown as mean t SEM and represent an average of eight observations per experiment repeated on three different occasions. P value is not significant ( P > .05) for OD between B35 cells incubated with preimmune serum vs. anti-NCAM antibody, whereas P value is significant ( P = .02) for OD between B35 cells with no pretreatment and anti-NCAM antibody by the paired Student’s t-test.

daily for morphology and were counted before plating and after harvesting to determine growth kinetics.

Peptides, Proteins, and Other Chemicals Heparin binding domain peptide was commercially synthesized (Immuno Dynamics, La Jolla, CA) (Table I). Bovine fibronectin (from EHS sarcoma, catalog no. 680-3010 IV) and laminin (isolated from EHS sarcoma, catalog no. 680-3017) were obtained from GIBCO BRL (Grand Island, NY) and Collagen type IV (isolated from EHS sarcoma) was from Biomedical Products (Bedford, MA, catalog no. 40233). Studies reported here used freshly reconstituted fibronectin, laminin, and collagen that had not been subjected to repeated freeze-thaw . GRGD peptide (gly-arg-gly-asp) was commercially purchased (Telios, La Jolla, CA) and the tetrapeptide RGDS (arg-gly-asp-ser) was obtained from Peninsula Laboratories (Belmont, CA). In the initial phases of experiments, RGDS tetrapeptide was used as a substrate, though later on GRGD was used. No difference in cell adhesion was found (data not shown) and therefore in this report the RGD peptide should be taken to mean either RGDS or GRGD tetrapeptide. Heparin (sodium salt from porcine intestinal mucosa, average molecular weight 16-17 X lo3) (H3125) and chondroitin sulfate (type A from whale cartilage, average molecular weight 50 X lo3) (C-4134) were purchased from Sigma Co. (St. Louis, MO). Sodium chlorate was purchased from Aldrich Chemical Co. (Milwaukee, WI).

E

N

-

0.2-

v) (D

m

9 0

0.1-

0.0

. 835

8 3 5 clone 3

Fig. 3. Comparison between B35 (NCAM expressing) and B35 clone 3 (NCAM non-expressing) cell adhesion to HBD peptide at a concentration of 25 pgiml in each well. Each bar is shown as mean ? SEM and represents an average of four observations on three different occasions. The representative blank values for these experiments were 0.074 for the B35 cells and 0.101 for B35 clone 3 cells. Input cells for each well in the microtiter plate was 3 X lo4 cells. The OD values are not statistically different (P = .14) by the unpaired Student’s ttest.

RESULTS Adhesion of Cells With and Without NCAM to HBD Peptide The HBD of NCAM could bind either to NCAM (homophilic adhesion) or another molecule (heterophilic adhesion). In order to test these alternative possibilities, the adhesion of NCAM expressing and non-expressing cells to wells coated with HBD peptide was tested. The amino acid sequence of the HBD peptide is identical to that of HBD of NCAM (Fig. 1). B35 cells and the derived NCAM lacking clone (B35 clone 3) were used in the experiments as prototypes of NCAM expressing and non-expressing cells respectively. Initial experiments varying the amounts of peptide used to coat the wells demonstrated a near maximal B35 cell adhesion peptide concentration of 25 pg/ml (data not shown). The adhesion of B35 and B35 clone 3 cells to HBD peptide was not statistically different at HBD peptide concentration of 25 pg/ml (Fig. 3). Adhesion of B35 and B35 clone 3 cells to HBD peptide was also similar at concentration of 5 and 20 pg/ml (data not shown). These experiments show that both NCAM expressing and non-expressing cells adhere strongly to HBD peptide. To further evaluate the mechanism of adhesion in these assays, the ability of a polyclonal anti-NCAM antibody (161) to perturb B35

542

Kallapur and Akeson

cell-HBD peptide adhesion was tested. B35 cells incubated with 100-fold molar excess of specific IgG to cell surface NCAM (see Table I legend) adhered with approximately 90% efficiency of controls and this adhesion was not statistically different from B35 cell-HBD adhesion when B35 cells were incubated with preimmune serum (Table I). These combined results indicate that, in this assay, the adhesion of cells to HBD peptide is not dependent on cell surface NCAM.

Morphology of B35 Cells on H B D Peptide Substratum The morphology of B35 cells incubated for 1-3 hr on a non-tissue culture treated plastic surface coated with 25 pg/ml HBD peptide and quenched with BSA was dramatically different compared to B35 cells on the same plastic surface without any added peptide and quenched with BSA. Without any added peptide the B35 cells had a spheroid morphology and clumped together whereas on HBD substratum B35 cells were flattened and had neurite outgrowth within 3 hr (Fig. 6, compare E and F). This morphology was similar to B35 cells grown on tissue culture plates in normal culture medium (Fig. 6, compare E and A; note that in Fig. 6 E and F, the cells have been photographed after a 3 hr incubation and prior to washing). Thus a HBD peptide coated plastic surface is permissive to cells for both attachment and initial neurite outgrowth. Specificity of B35 Cell Adhesion to H B D Peptide An alternative mechanism by which the HBD peptide could mediate cell adhesion is by binding to cell surface proteoglycans. As an initial test of this mechanism, the ability of exogenously added micromolar concentrations of heparin to inhibit cell-HBD interaction was examined. B35 cell adhesion to HBD peptide was inhibited by and l o p 7 M heparin but not by equimolar or excess concentrations of chondroitin (Fig. 4). The inhibitory effect of heparin was not detected at concentrations of lo-* M or less. The effect of independent preincubation of the cells and substrate with glycosaminoglycans was then tested. Preincubation of B35 cells alone with heparin ( lop6 M concentration for 30 min at room temperature) caused only a modest decrease in B 35-HBD adhesion (Fig. 5 ) whereas preincubation of the substrate alone with heparin but not chondroitin under similar conditions produced a dramatic decrease in B35HBD adhesion (Fig. 5 ) . These experiments demonstrate a specific B35 cell adhesion to HBD peptide and suggest that this adhesion does not involve NCAM but is mediated through interactions with cell surface heparan-like proteoglycans.

Chondroitin sulfate

0.2

E N VI c

9 0

0.1

0.0

1 0 - 6 M

1 0 - 7M

1 O-'M

GAG concentration

Fig. 4. Specificity of B35 cell adhesion to HBD peptide: effect of soluble heparin. Each bar on the graph is shown as mean I SEM and represents an average of four sets of observations repeated at least two times. The GAG was added to the adhesion medium immediately after the addition of cells at a concentration of lop6 M to lop8M. Input cells for each well in the microtiter plate was 3 x lo4 cells. The OD values between no GAG and addition of soluble heparin are significantly different (*P= ,0001)by the unpaired Student's t-test for heparin doses lop6 and lod7 M but not for lop8 M (P = .12).

Effects of P-D-Xyloside Treatment on B35 Cells P-D-xylosides inhibit carbohydrate addition to proteoglycan core proteins. Thus cell adhesion mediated by proteoglycan carbohydrates can potentially be inhibited by P-D-xyloside. Several previous biochemical studies have shown inhibition of HSPG synthesis using xyloside concentrations of 0.5 -5 .0 mM and varying exposure times. In our preliminary experiments, xyloside concentrations higher than 5 mM caused extensive detachment of B35 cells from the culture plate after 3-6 days of growth (data not shown). Therefore, xylosides at a concentration of 3.5 mM were used to study the biologic effects of altered HSPG synthesis by B35 cells. At this xyloside concentration, the B35 cells showed decreased adhesion to the tissue culture plate compared with nontreated controls and the cells had a more spheroid form (Fig. 6, compare A and B). To test if the decreased cell-tissue culture plate adhesion was due to decreased cell viability, the cells were washed and tested for ability to replate. Treated and washed B35 cells readhered to a tissue culture plate in 10% serum containing medium lacking xyloside. The morphology of cells was no different from the untreated cells 18 hr after replating (Fig. 6, compare C and A) and all the cells in the culture medium replated. These experiments demonstrate that xyloside in the concentration defined above is not toxic

NCAM-Proteoglycan Interaction 0.3

~~~~~

Chondroitin sulfate Heparin sulfate

T

E

0.2

N

In (D

c

0.1

0.c

None

A

Incubation medium

B

835 cells

substrate

only

C

D

GAG addition

Fig. 5 . Specificity of B35 cell adhesion to HBD peptide. A: Native B35 cell adhesion to HBD peptide. B: GAG in a dose M was added to the adhesion medium immediately of after the addition of cells and incubated for 3 hr at 37°C with 10% C02. C: B35 cells were preincubated with M GAG for 30 min at room temperature followed by washing once with serum free DMEM and subsequent plating in the individual wells. D: Substrate (HBD peptide) was preincubated with M GAG and washed with 200 FL PBS followed by plating of B35 cells. Input cells for each well in the microtiter plate was 3 x lo4 cells. The representative blank value for B35 cells was 0.046. In all the experiments, the substrate was HBD peptide 25 pgiml. Each bar on the graph is shown as mean 5 SEM and

represents an average of eight sets of observations repeated three times. to B35 cells and can be used to study the effects of altered proteoglycan synthesis by the cells.

Tests of Adhesion of Cells with Decreased HSPG to HBD Peptide In preliminary experiments, B35 cells exposed to 3.5 mM xylosides for less than 3 days adhered with almost the same efficiency to HBD peptide as control cells presumably due to incomplete turnover of existing fully glycosated HSPG. Cells exposed to 3.5 mM xylosides for longer than 6 days showed extensive detachment from the tissue culture plate and disruption of cell morphology. Therefore adhesion experiments were carried out with B35 cells exposed to 3.5 mM xyloside for 3-6 days. B35 cells grown in xylosides for this period have a significantly decreased adhesion to the HBD peptide (Fig. 7). These results are consistent with the hy-

543

pothesis that the HBD of NCAM binds to HSPG. However, it is possible that xyloside treatment of cells nonspecifically decreases their ability to adhere to any substrate. To test this possibility, adhesion assays were performed using the substrates fibronectin, collagen type IV, and laminin. For most cells adhesion to these substrates is mediated by integrin related receptor mechanisms independent of NCAM. The adhesion of untreated and xyloside treated B35 cells to fibronectin, laminin, and collagen type IV were similar (Fig. 7). Thus the xyloside treatment of B35 cells specifically decreased the cell-HBD peptide adhesion, supporting the possibility that the HBD of NCAM specifically bound to HSPG. B35 cells grown in xyloside did not completely reduce the adhesion to HBD peptide. Heparin in a dose M, but not chondroitin, was able to of lop6 or completely inhibit this residual cell adhesion to the HBD peptide (Fig. 8) (note that in Fig. 8, the OD values are much lower than those for untreated B35 cells in Figs. 6 and 7). This inhibitory effect of heparin was lost at lo-* M concentration, which is similar to the dose dependent effect of heparin on non-xyloside treated cells. These experiments demonstrated both that xyloside pretreatment of cells largely inhibited B35 cell adhesion to HBD peptide and that the remaining adhesion was heparin sensitive. As B35 cells express NCAM polypeptide there is again no evidence for direct HBD-NCAM interaction. Chlorates in the culture medium decrease the sulfation of proteoglycans (Rapraeger et al., 1991). Thus the role of this sulfation in cell adhesion can be examined by testing the adhesion of cells grown in a medium with excess chlorates. The morphology of B35 cells grown in a culture medium containing 30 mM chlorate was similar to that grown in a normal medium (Fig. 6, compare D and A). B35 cells grown in chlorates had significantly decreased adhesion to HBD peptide (Fig. 9). As a control for non-specific effects of chlorates, no significant difference in the adhesion of control and chlorate treated B35 cells to fibronectin and RGD peptide was found in one experiment (data not shown). Decreased sulfation of proteoglycans thus specifically decreased B35 cell adhesion to HBD peptide. These experiments further support the possibility that the HBD of NCAM binds to the HSPG and further indicate that HBD binds to the sulfate containing component(s) of the HSPG.

DISCUSSION The importance of the HBD in the adhesion of NCAM has been demonstrated by earlier experiments (Cole and Glaser, 1986; Reyes et al., 1990). The results from the present study suggest that within the limits of the assay the HBD participates in heterophilic interactions with cell surface HSPG. Three independent lines of

544

Kallapur and Akeson

Fig. 6. Photograph of B35 cells on different substrata and culture conditions (Zeiss, phase contrast, magnification 325 x). A: Normal B35 cells cultured in DMEM with 5% CS and 5% FCS showing good neurite outgrowth. B: B35 cells cultured in DMEM with 5% CS and 5% FCS with 3.5 mM xyloside for 5 days showing lack of neurite outgrowth and spheroid morphology while still remaining attached to the tissue culture plate. C: B35 cells cultured as in B for 4.5 days and then replated in DMEM with 5% CS and 5% FCS and photographed 18 hr after replating. The morphology is indistinguish-

able from control B35 culture. D: B35 cells grown in 30 mM sodium chlorate BME medium for 5 days. The morphology is indistinguishable from control B35 culture. E: B35 cells cultured as in A photographed 3 hr after plating on a plastic surface with HBD peptide immobilized by nitrocellulose at 25 p&ml concentration. Neurite outgrowth is seen. F: B35 cells cultured as in A 3 hr after plating on a plastic surface with nitrocellulose only and no substrate showing clumping of cells and lack of neurite outgrowth.

NCAM-Proteoglycan Interaction

545

0.1r 0 30

835

T

B35 3.5mM xylosids

Ghondroitin sulfate

0.1;

0.1c

E

9

0.20

N

N

u) (D

v, W

L

i

9

i?

0 OE

O.OE

0

0 0.10

0.04

0 02

0.oc

0.00

HRD

Fibronectin

Laminin

Collagen type IV

peptide

Substrates

lO7M

1 0 6 M

GAG

1

OEM

concentration

Fig. 7. Adhesion of B35 cells grown in a culture medium containing xyloside at 3.5 mM concentration for 3-6 days. Each bar on the graph is shown as mean SEM and represents an average of four sets of observations repeated three times. The representative blank values for these experiments were 0.101 for the B35 cells and 0.144 for xyloside treated B3.5 cells. Input cells for each well in the microtiter plate were 3 X lo4 cells. All the substrates were plated at a concentration of 25 pgiml. The OD values for the xyloside treated vs. untreated cells are statistically significantly different by the unpaired Student’s t-test for HBD peptide (*P = .001) but not for fibronectin, laminin, or collagen type IV (P > .05).

Fig. 8. Specificity of residual adhesion of xyloside treated 8 3 5 cells to HBD peptide: Effect of addition of soluble heparin. Each bar on the graph is shown as mean & SEM and represents an average of three observations repeated at least two times. The representative blank value for these experiments was 0.140. Input cells for each well in the microtiter plate was 3 X lo4 cells. The OD values between no GAG and addition of soluble heparin are significantly different ( t P = ,0001) by the unpaired Student’s t-test for heparin doses lop6 and lo-’ M but not for M ( P = .69) (asterisks represent OD values less than blank values for B35 cells with soluble heparin: *OD value = -0.089; **OD value = -0.076).

evidence support this conclusion: 1) comparison of the adhesion of NCAM expressing and non-expressing B35 cells to HBD peptide; 2) inability of a polyclonal antiNCAM antibody to decrease B35-HBD adhesion; and 3 ) examination of the effects of inhibitors of the HSPG synthesis and assembly. The strength of the latter line of evidence is dependent on the specificity of the inhibitors used. Recently, the effects of xylosides on cultured neural PC12 cells have been characterized (Margolis et al., 1991). After treatment with a dose of 1 mM xyloside for 13 days (compared to 3.5 mM for 3-6 days in this study), PC12 cells had an increased tendency to form clumps, a decreased rate of growth, decreased adhesion to the tissue culture plate, and decreased adhesion to poly L-lysine (Margolis et al., 1991). Both PC12 and B35 cells are of neural origin and the xyloside effects are similar. The decreased adhesion of cells to tissue culture plates is most likely due to decreased HSPG on the cell surface leading to a net reduction of negative charge. The decreased rate of growth of the cells may be due either to decreased adhesion of the cells to the tissue culture plate (Folkman and Moscona, 1978) or to abolition of the

binding sites for growth factors like heparin binding growth factors (Rapraeger et al., 1991). Some studies have noted toxic effects of high doses of xylosides on cells (Kolset et al., 1990), while others failed to find such an effect (Gressner, 1991). Toxicity to cells is unlikely in this study, since the xyloside treated cells replated efficiently to new culture dishes in normal medium (Fig. 6C) and the adhesion of xyloside treated cells to other substrates was unaffected. In the present study, xyloside treated B35 cells had decreased adhesion to HBD peptide, but not to control substrates. Thus, xyloside induced inhibition of proteoglycan synthesis causes changes in the morphology and adhesiveness of B35 cells similar to the effects noted on other cell types. Furthermore the specificity of xyloside effects on the cells was independently tested in this study by growing B35 cells in chlorate containing medium. Cells grown in a chlorate rich medium had decreased cellHBD but not cell-fibronectin or cell-RGD peptide adhesion. This result provides additional evidence for HBDHSPG interaction and demonstrates that this interaction depends on HSPG sulfation. It has been proposed that NCAM has both a ho-

*

546

Kallapur and Akeson 0.3

5

635 835 rn 30mM chiorate

0.2

N

Y) LD

c

9 0

0.1

0.0 5.0pgiml

25.opgiml

HBD Peptide concentration

Fig. 9. Effect of growing B 35 cells in 30 mM chlorate. Each bar on the graph is shown as mean Z? SEM and represents an average of four sets of observations repeated at least four times. The representative blank values for these experiments were 0.073 for the B35 cells and 0.151 for the chlorate cells. Input cells for each well in the microtiter plate was 3 x lo4 cells. The OD values for the chlorate treated vs. untreated cells are significantlydifferent by the unpaired Student’st-test ( P = ,0001).

mophilic binding site and a separate heparin binding site. The initial basis for this proposal was experiments in which a monoclonal antibody C,H, decreased retinal cell-NCAM adhesion but did not inhibit NCAM-heparin interactions (Cole et al., 1985). There is recent evidence suggesting that NCAM binds to collagen (Probstmeier et al., 1992) and may possess ligands that mediate heterophilic adhesion (Murray and Jensen, 1992). The theoretical possibilities are that within the NCAM sequence the HBD and the other binding sites are 1) completely independent, 2) identical or closely related, or 3) independent but interrelated. In a recent study, it was shown that NCAM coated latex beads failed to aggregate unless micromolar amounts of heparin were included in the assay medium (Kadmon et al., 1990). In independent experiments from our laboratory, HBD lacking NCAM was expressed on transfected L cells. When the HBD lacking NCAM was as a substratum, the retinal cell adhesion to NCAM decreased to almost baseline levels (Reyes et al., 1990). One interpretation of both these results is that direct NCAM-NCAM interactions not involving HBD are not important for cell adhesion. However, the possibility also exists that an initial HSPG-NCAM interaction is necessary for subsequent NCAM-NCAM interactions. This latter interpretation is in keeping with a previously suggested model in which NCAM-heparin binding was proposed to promote a conformational change in NCAM,

facilitating NCAM-NCAM interaction (Cole and Glaser, 1986). While consistent with this model, the experiments reported here do not, however, directly provide evidence for either the possibility of NCAM conformational changes or even for the presence of an independent homophilic binding site. During development, NCAM is present in high abundance in neural tissue and its expression is spatially and temporally regulated, suggesting an important role for the molecule. A recent study has reported as many as 16 membrane associated and nine soluble fraction associated core proteins of the proteoglycans during various stages of development in the rat central nervous system (Herndon and Lander, 1990). NCAM has been reported to copurify from brain with a heparan sulfate containing proteoglycan, further suggesting the relevance of these interactions in vivo (Cole and Burg, 1989). Potential NCAM-HSPG interactions should be quantitatively numerous and could therefore play an important role in development. The possible physiological importance of NCAM-HSPG interactions is also illustrated by comparisons with other HSPG-extracellular matrix protein interactions. A laminin-HSPG complex has been shown to enhance neurite outgrowth when used to coat culture dishes (Lander et al., 1985; Davis et al.. 1987; Sandrock and Matthew, 1987). The role of myelin associated glycoprotein in neuron-glial interactions may also depend on HSPG (Poltorak et al., 1987). In addition, the HBD of fibronectin promotes neurite outgrowth when used to coat culture dishes (Rogers et al., 1985; Mugnai et al., 1988). Extracellular matrix heparan sulfate binds various growth factors (Schubert et a]., 1987). Thus, NCAMHSPG interaction could indirectly increase the concentration of growth factors in the vicinity of cells, given the high abundance of NCAM during development. Thus experimental data from several laboratories now clearly demonstrate three NCAM mediated interactions: collagen binding, homophilic interaction, and heparin binding. Data presented here suggest the latter two are mediated by independent segments of the molecule. However, the structural and the mechanistic relationships between NCAM segments mediating these interactions remain to be established. In this context, a study demonstrating differential cell binding potential of different IgG like loops of NCAM when expressed as fusion proteins is especially relevant (Frei et al., 1992). It is interesting to note that the immune system immunoglobulin supergene family member I-CAM has three well documented binding affinities. Each of these is mediated by a distinct region of the molecule (Staunton et a]., 1990; Ockenhouse et al., 1992; Berendt et al., 1992). Further work is clearly necessary to define the relationships of the binding regions of NCAM. The results here indicate that analyses of NCAM’s biological actions should take

NCAM-Proteoglycan Interaction

into account its ability to interact with heparan sulfate containing proteoglycans.

ACKNOWLEDGMENTS We would like to thank Ms. Sue Haines for B35 and B35 clone 3 cells, Ms. Shirley Arnold for assistance, Dr. Jeffrey Whitsett for manuscript review, and Ms. Nada Gorman for assistance with manuscript. This work was supported by grant NICHD 21065.

NOTE ADDED IN PROOF After the submission of this paper, a proposed homophilic binding site was identified within a decapeptide contained in IgG like loop 3 of the chicken NCAM molecule (Rao et al., 1992). As the NCAM HBD is found in Ig- like loop 2 in all species, the findings of Rao et a1 are consistent with the conclusions presented above that the HBD does not participate in homophilic interactions.

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