Ganglioside inhibition of fibronectin-mediated cell

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1 Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp , July 1979 Cell Biology Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen (glycolipid/extracellular matrix/cell surface) HYNDA K. KLEINMAN*, GEORGE R. MARTIN*, AND PETER H. FISHMANt *Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health; and tdevelopmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland Communicated by Roscoe 0. Brady, April 9, 1979 ABSTRACT Fibronectin mediates the adhesion of cells to col1agen by first binding to the collagen substrate, followed by attachment of the cells to the fibronectin-collagen complex. Bovine brain gangliosides were found to block fibronectinmediated cell adhesion to collagen in a concentration-dependent manner. The gangliosides did not block the binding of fibronectin to collagen but did prevent the attachment of the cells to the fibronectin-collagen complex. Of the individual gangliosides tested, GT1 and GDia were the most effective inhibitors followed by GD1b >> GM1 >> GM2; GM3 was not an inhibitor. The inhibition of cell adhesion also was observed with the oligosaccharide portion of the gangliosides, but not with ceramides or with a variety of free sugars or glycosaminoglycans. Mild periodate oxidation of mixed gangliosides or of GDia modified their sialic acid residues and the oxidized gangliosides were no longer inhibitory; subsequent reduction with NaBH4 did not restore the inhibitory activity of the modified gangliosides. These results suggest that specific gangliosides or related sialic acid-containing glycoconjugates on the cell surface may act as the receptors for fibronectin. Most cells require an appropriate substratum for growth in culture. A variety of cells use fibronectin, a large extrinsic matrix protein to adhere either to plastic (1) or to collagen substrates (2). An identical or very similar protein, cold insoluble globulin, is present in serum, and the serum form of fibronectin promotes the attachment of freshly trypsinized cells. Fibronectin prepared from either cells or serum is equally active in promoting cell attachment (3). With collagen substrates, fibronectin binds to the collagen substratum first (2, 4). Then the cells, in the presence of Ca2+ or Mg2+ in an energy-dependent process (5), adhere to the fibronectin-collagen complex. Fibronectin binds to all mammalian collagens that have been tested so far and it also binds to fibrin (6). In the a l(i) chain of collagen, it binds to a specific amino acid sequence lacking carbohydrate (7-10). These interactions are not cation dependent and can be broken by 1 M KBr (11) or 6 M urea (12). Fibrinogen (13) and collagen affinity columns (11, 12) have proved useful in the purification of fibronectin from serum and from conditioned culture medium. Little is known about the interaction of fibronectin with the cell surface or the nature of the membrane component(s) to which fibronectin binds. One approach to their identification is to test various cell surface components for their ability to interact with the fibronectin-collagen complex and thereby prevent cell attachment. Gangliosides are well-defined membrane components, and a specific ganglioside, GM,, functions as the membrane receptor for cholera toxin (14). This role for GM, was elucidated from the original observation that gangliosides block the biological effects of the toxin (15). We now report that certain gangliosides block cell adhesion promoted by the fibronectin in serum. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. MATERIALS AND METHODS Materials. Mixed brain gangliosides were purchased from either Sigma or Supelco (Bellefonte, PA); GM1, GDia, GT1, and ceramides were obtained from Supelco. Highly purified GM3, GM2, GM1, GDia, GDlb, and GT1 were prepared as described (16). Precoated Silica Gel 60 thin-layer plates (250,um thick) were obtained from E. Merck (Darmstadt, West Germany) and resorcinol from Sigma. The glycosaminoglycans were obtained from the mucopolysaccharide reference standard kit of Mathews and Cifonelli. Fibronectin was prepared from serum by elution of the material that adsorbed to a collagen affinity column (11, 12). Cell Attachment Assay. Collagen-coated 35-mm bacteriological plates were prepared as described elsewhere (7). One milliliter of Eagle's minimal essential medium, containing 200,ug of bovine serum albumin and either 0.25% serum or 5 tg of purified fibronectin, was added to the coated dish, which was incubated at 37 C in 95% air/5% CO2. After 1 hr, freshly trypsinized Chinese hamster ovary (CHO) cells (4 X 105 in 0.1 ml) were added to the dish and allowed to attach for 1.5 hr. After this time, unattached cells were removed, and the attached cells were released from the substrate with trypsin and counted with a Coulter Counter. Treatment of Gangliosides. Oligosaccharides from mixed brain gangliosides were prepared by ozonolysis and purified by column chromatography as described (17). Gangliosides were oxidized in the presence of sodium periodate (18, 19), and in some cases the oxidized gangliosides were further reduced with NaBH4. Both treated and untreated gangliosides were assayed for sialic acid content (20), and the oxidized gangliosides were further characterized by thin-layer chromatography and detected with resorcinol reagent (21). RESULTS We first tested the effect of various gangliosides on cell adhesion by adding them with the fibronectin or serum during the preincubation period. As shown in Fig. 1A and Table 1, the mixed brain gangliosides and GT1 and GDia inhibited cell adhesion in a concentration-dependent manner. GDia, GDlb, and GT1 alone were more active than the mixture, whereas GM1 and GM2 were slightly inhibitory. GM3, at the concen- Abbreviations: GM1, galactosyl-n-acetylgalactosaminyl-[n-acetylneuraminyl]-galactosylglucosylceramide; GM2, N-acetylgalactosaminyl-[N-acetylneuraminyl]galactosylglucosylceramide; GM3, N- acetylneuraminylgalactosylglucosylceramide; GD3, N-acetylneuraminyl-lN-acetylneuraminyl]-galactosylglucosylceramide; GDIa, N- acetylneuraminylgalactosyl-n-acetylgalactosaminyl-[n-acetylneuraminyll-galactosylglucosylceramide; GDlb, galactosyl-n-acetylgalactosaminyl -[N - acetylneuraminyl-n-acetylneuraminyll - galactosylglucosylceramide; GT1, N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl - [N - acetylneuraminyl-n-acetylneuraminyl] -galactosylglucosylceramide; CHO cells, Chinese hamster ovary cells. 3367

2 3368 Cell Biology: Kleinman et al. Proc. Natl. Acad. Sci. USA 76 (1979).. 8a \ 12 A ~~~~~~~~~~20 x 16- ~~~~0 X~ ~ ~ ~ ~ ~~~/ 16 / 12-..!/* ~ - ~- 4-4 A 0,, I I I I I % serum Amol/ml FIG. 1. (A) Effect of gangliosides and ceramides on cell adhesion. Gangliosides and ceramides were dissolved at 10 mg/ml in Eagle's medium and different amounts were added to the collagen-coated culture dishes containing serum. After 1 hr, the cells were added and cell adhesion was assayed. Values represent means of duplicate measurements, which did not differ by more than 10%. 0, Ceramides. Gangliosides: 0, GM1; o, GT1; A, GDia; A, mixture. (B) Effect of increasing serum concentrations on ganglioside-mediated inhibition of cell attachment. Increasing amounts of serum in the presence of 0.29,gmol (0) or 0.58,gmol (-) of mixed brain gangliosides per ml were first incubated for 1 hr on the collagen-coated dishes. The cells were then added and cell adhesion was assayed. Values represent means of duplicate determinations, which did not differ by more than 10%. *, No additions. tration tested, appeared to contain no activity. Different preparations of gangliosides gave qualitatively similar results. However, some differences were noted in the potency of the gangliosides between different preparations. The degree of inhibition of cell attachment depended upon the concentration of serum (Fig. 1B) or fibronectin (not shown) used. At all serum concentrations tested, gangliosides inhibited cell attachment, but at higher serum levels more ganglioside was required to achieve a given degree of inhibition of cell attachment. Several observations suggest that the inhibition of cell at- Table 1. Effect of various gangliosides on cell attachment Cells attached Additions (50 nmol) X 10-3 SD Inhibition, % None GM GM2* 38.1 ± GM GDlb 18.1 t GDia 11.7 i GTjt 2.1 I Mixed brain gangliosidest Glass vials (12 mm in diameter), coated with 1 Asg of collagen, were incubated for 1 hr in 0.1 ml of Eagle's medium containing 0.2% serum and highly purified gangliosides where indicated. Then CHO cells (6 X 104 cells in ml of Eagle's medium) were added and incubated an additional 1.5 hr. The number of cells attached was then determined. * Contained 97% GM2 and 3% GM1. t Contained 92% GT, and 8% GD1at Obtained from Sigma. B tachment by gangliosides is not due to toxicity. First, cell viability, as estimated by the ability of cell to exclude trypan blue, was not increased by incubation with the mixture of gangliosides. Second, cellspreincubated with themixture of gangliosides and then washed with medium to remove gangliosides attached normally. Third, after attachment, the addition of the mixture of gangliosides to the cells had no effect on cell growth. These results indicate that the effects observed with certain gangliosides on cell attachment are not the result of toxicity of the gangliosides to the cells. The inhibition of cell adhesion by gangliosides was further characterized by testing the ability of the oligosaccharide portions of the gangliosides to interfere with cell attachment. Oligosaccharides prepared from commercially available mixed gangliosides inhibited adhesion of cells to collagen (Fig. 2), whereas the ceramides had no effect on cell attachment (Fig. 1A). However, inhibitory activity of the oligosaccharides was less than that observed with the intact gangliosides. To determine whether free sugars were active, we tested various free sugars, including those present in gangliosides. Sialic acid, N-acetylglucosamine, N-acetylgalactosamine, glucose, and galactose at concentrations as high as 100 ttmol/ml were without effect on cell adhesion (data not shown). Glucosamine and glucuronic acid did interfere with cell adhesion at high concentrations (1 mmol/ml), but neither sugar is present in gangliosides. In addition, various polysaccharides, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, and chondroitin sulfate, were tested at 2 mg/ml and found to be inactive. Hyaluronic acid at 2 mg/ml inhibited cell adhesion by 50%. The importance of the sialic acid residue as part of the functional component of the ganglioside was determined by

3 Cell Biology: Kleinman et al. Proc. Natl. Acad. Sci. USA 76 (1979) x wa) -C 11 ('w(1 ii -A were not acting by binding to the collagen substratum. The effect of the gangliosides on the binding of the cells to the fibronectin-collagen complex was next tested. After the fibronectin or serum (not shown) had been preincubated with the collagen substratum for 1 hr, gangliosides were added and allowed to incubate for another hour. Half of the plates were rinsed to remove unbound gangliosides, and then cells were added. As shown in Fig. 3B, gangliosides inhibited cell attachment when added to the collagen-fibronectin complex. Further evidence that gangliosides interact directly with fibronectin is seen in preliminary studies (S. Rennard and H. K. Kleinman, unpublished observations), which show that considerably more GDia than GM1 binds to fibronectin. In addition, this binding does not require and is not affected by the presence of collagen. In previous studies (22, 23), gangliosides more complex than GM3 and GD3 were not detected in CHO cells by conventional techniques. When we treated these cells with neuraminidase, we observed a 7-fold increase in cholera toxin binding (Table 3). In agreement with Critchley and Vicker (23), the untreated CHO cells bind very little 125I-labeled toxin and thus contain only trace amounts of GM1. These results indicate that neu- C pmol/ml FIG. 2. Effect on cell adhesion of oligosaccharides derived from mixed gangliosides. The assay was carried out as described in the legend for Fig. 1. Oligosaccharides were prepared from bovine mixed brain gangliosides. Values represent means of duplicate determinations, which did not differ by more than 10%1. A, Oligosaccharides; 0, gangliosides. preparing mixed gangliosides or GDia with one or two of the carbon groups in the terminal polyhydroxyl chain of the sialic acids removed by mild periodate treatment. Alteration of the sialic acids in the mixed gangliosides or in GDIa greatly reduced their ability to interfere with cell adhesion (Table 2). Subsequent reduction of the aldehyde groups on the sialic acid residues did not restore the inhibitory activity of the modified gangliosides. The mechanism by which the gangliosides inhibited cell adhesion was examined. The gangliosides did not appear to act by binding to the cell. Pretreatment of the cells for 1 hr, followed by washing, had no effect upon cell adhesion. We tested whether the gangliosides were binding to the collagen by incubating the collagen-coated plates with the gangliosides for 1 hr. Then half of the plates were washed twice to remove any unbound gangliosides. Serum or fibronectin was then added to the plates, and cell attachment was assayed as usual. As shown in Fig. 3A, the gangliosides did not inhibit cell attachment when added during the initial incubation period, indicating that they Table 2. Effect of oxidation and reduction of gangliosides on their ability to inhibit cell attachment Ganglioside Cells attached X 0-4 Inhibition,% None added Mixed brain, untreated Mixed brain, oxidized Mixed brain, oxidized and reduced GDia, untreated GDia, oxidized and reduced Collagen-coated dishes were incubated in the presence of 0.25% serum and commercially obtained (treated and untreated) gangliosides (0.9,umol/dish). lw x m (LU (U I 2 () 0T A B -+ + Ganglliosides Wash Fibronectin Fibronectin Gangliosides Wash FIG. 3. Effect of gangliosides on binding of fibronectin to collagen and binding of cells to fibronectin-collagen complexes. (A) Collagen-coated plates were preincubated for 1 hr with either Eagle's medium or Eagle's medium containing 0.29 Amol of mixed brain gangliosides per ml. Then the preincubation solutions were removed and the plates were washed gently twice with 1 ml each of Eagle's medium. A final concentration of 0.2% serum was added to all the plates, which were further incubated for 1 hr before addition of the cells. Cell adhesion was determined after 1 hr. (B) After a 1-hr preincubation with 0.2% serum, the collagen-coated plates were further incubated for 1 hr with either Eagle's medium or Eagle's medium containing 0.29,gmol of mixed brain gangliosides per ml. The incubation solutions were then removed and the plates were gently washed twice with 1 ml each of Eagle's medium. The cells were then added and allowed to attach. All values were corrected for background (attachment of cells to plates containing no serum). Each value represents the mean of triplicate determinations, which did not differ by more than 10%.

4 3370 Cell Biology: Kleinman et al. Table 3. Effect of neuraminidase treatment on binding of cholera toxin to CHO cells Cell 125I-Labeled toxin specifically treatment bound, cpm/mg protein None 56,500 ± 3,117 Neuraminidase 401,400 ± 16,400 CHO cells grown in monolayer culture were washed with phosphate-buffered saline and incubated for 1 hr at 370C in the presence and absence of Vibrio cholerae neuraminidase (100 units, Calbiochem) in 4 ml of Dulbecco's phosphate-buffered saline containing Ca2+ and Mg2+. The medium was removed; the cells were detached with trypsin, washed, and suspended in Tris-buffered saline, ph 7.4 (t5 X 106 cells per ml). Portions of 0.1 ml were incubated in 0.2 ml of the same buffer containing 0.1% bovine serum albumin and 0.5 nm 125I-labeled cholera toxin (24) for 45 min at 370C. The cells then were collected on 1-,gm Millipore filters (24). Values are the mean ± SD of triplicate determinations and have been corrected for nonspecific binding (cpm bound in the presence of 0.2 AtM unlabeled toxin). Binding was proportional to the amount of cell suspension added ( ml), and the same difference between control and neuraminidase-treated cells was obtained under these latter conditions. raminidase-sensitive gangliosides (i.e., GDia and GTI) on the cell surface are being converted to GM1, which is neuraminidase resistant, and new toxin-binding sites are being generated. Similar increases in cholera toxin binding after the treatment of other cultured cell lines with neuraminidase have been observed (23, 25). DISCUSSION Evidence is presented here that gangliosides inhibit cell adhesion by binding to fibronectin. Based on experiments in which gangliosides were washed off prior to the addition of fibronectin and cells to the collagen substrates, it appears that they do not act by complexing to the collagen. Because gangliosides effectively inhibit cell adhesion after the fibronectin has bound to the collagen, we suggest that the gangliosides may occupy the site on fibronectin that binds to the cell. However, we cannot exclude the possibility that gangliosides may bind to another region of the fibronectin molecule and prevent its interaction with the cell membrane. The oligosaccharide of the ganglioside is the active portion because (i) ceramides did not alter cell adhesion, (ii) the isolated oligosaccharides inhibited cell adhesion, and (iii) removal of one or two carbon groups from ganglioside sialic acid resulted in reduction of activity. In addition, there appeared in general to be an increase in potency with increasing size of the sugar portion of the gangliosides as well as the number and location of sialic acid residues. GT1 and GDia, complex gangliosides whose specific functions are not known, inhibited cell adhesion to a much greater extent than any of the other gangliosides tested. These included GM1, which is the choleragen receptor (14). Also, if the gangliosides are the cell surface receptors for fibronectin, as these data suggest, their lipid portions would be buried in the membrane bilayer and only the oligosaccharide portions would be available to bind fibronectin. It is unclear why significant inhibition is observed only at relatively high ganglioside concentrations (above the critical micellar concentration), suggesting a low affinity between gangliosides and fibronectin. However, because the ganglioside applied to the fibronectin-collagen complex can be extensively washed and still inhibit cell adhesion, there might be a high affinity between gangliosides and fibronectin. Preliminary results of studies directly examining the interaction of gangliosides with fibronectin indicate that GDIa can bind to fibronectin because this complex can be precipitated by antifibronectin antibodies. Proc. Natl. Acad. Sci. USA 76 (1979) One of two possibilities may explain the necessity for relatively high concentrations of gangliosides. First, the concentration of ganglioside required for inhibition depends upon the concentration of serum or fibronectin used in this study. Because a significant amount of serum is required to support cell attachment, a large amount of ganglioside may be required to neutralize this amount of fibronectin. Second, gangliosides can spontaneously insert themselves through their ceramide portions into the cell membranes (26). At low ganglioside concentrations (below the critical micellar concentration), gangliosides attached to fibronectin may be incorporated into the cell membrane and allow cell attachment. Higher levels may be required to show inhibition. Similar considerations are believed to underly the biphasic effect of GM1 on the binding of cholera toxin (17, 24). Alternatively, binding of gangliosides by fibronectin may be a cooperative process and occur better with gangliosides in a membrane or a micellar form. Gangliosides influence the growth (27) and morphology (28) of cells, and there are several examples of a correlation between the levels of complex gangliosides and fibronectin. Studies with established mouse cell lines indicate a reduction in the complex gangliosides, especially GDia, with transformation by viruses, chemicals, and x-irradiation (14, 21, 29). The presence of fibronectin on the cell surface is also reduced with transformation (30-32). On the other hand, GDia synthesis increases in certain cultured cells where their cell-to-cell contacts are increased (33, 34), and fibronectin accumulation also increases at high cell density (35). 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