Co-polymeric Glycosaminoglycans in Transformed Cells

THEJOURNAL OF BIOLOGICALCHEMISTRY Val. 256,.No.24, Issue of December 25, pp. 13044-13047, 1981 Printed m U S.A.

Co-polymeric Glycosaminoglycans in Transformed Cells TRANSFORMATION-DEPENDENT CHANGES IN THE SELF-ASSOCIATING PROPERTIES OF CELL-SURFACE HEPARAN SULFATE* (Received for publication, May 13, 1981)

Lars-Ake FranssonS and Ingrid Sjoberg From the Departmentof Physiological Chemistry 2, University of Lund, S-220 07 Lund, Sweden

Vincenzo P. Chiarugi From the Instituteof General Pathology, University of Florence, 50134 Florence, Italy

[3H,36S]Heparansulfate from extra-, peri-, or intracellular compartments ofa p r i m a r y cell culture (human lung fibroblasts), an established cell line (Balb-3T3) and SV40- or polyoma-transformed 3T3 cells have been examined with regard to charge density and self-associating properties. W h e n the various heparan sulfates were compared by ion exchange c h r o m a t o g r a p h y on DEAE-cellulose using bovine lung heparan sulfates HS2, HS3, and HS4 as reference compounds,lung fibroblast heparan sulfate eluted in a position intermediary to HS3 and HS4,3TS-heparan sulfatesshowed progressively m o r e retarded positions when going from intracellular (like HS2)over pericellular (HS2 and HS3 components) to extracellular material (HS3 and HS4 components), while heparan sulfatesfromtransformed cells were m o r e heterogeneous and of lower charge density than those f r o m normal cells. When assayed f o r aggregatability by affinity chromatography on immobilized and aggregating (A) forms of HS2,HS3, or HS4, lung fibroblast heparan sulfate had the highest affinity for HS4-A-agarose. The 3T3-pericellular HS2 fraction was partially bound to HS2-A-agarose, but not to HS3A-agarose. Conversely, the 3T3-pericellular HS3 fraction was partially and weakly bound to HS3-A-agarose but not to HS2-A-agarose. The 3T3-extracellular HS3 fraction was bound to HS3-A-agarose with a varying affinity, whereas the 3T3-extracellularHS4 fraction was not bound at all. The various heparan sulfate subfractions from transformed cells had no affinity for agarose gels substituted with the corresponding heparan sulfate species. This finding suggests that chains from transformed cells are nonassociating, which m a y have been caused by subtle changes in the co-polymeric structure.

teins), glycolipids, and glycosaminoglycans. The latter group includes hyaluronate and various forms of proteoglycans, ie. a protein core substituted withavarying number of side chains like chondroitin sulfate, dermatan sulfate, and heparan sulfate. Reports concerning transformation-dependent changes in these macromolecules prior to 1975 have been reviewed by Nicolson (2). In general, virus-transformed cells show enhanced rates of synthesis and increased cell-surface deposition of hyaluronate whereas the amounts of sulfated glycosaminoglycans are lower in transformed than in nontransformed cell lines. Later studies have c o n f i i e d and extended these results, i e . a close relationship exists between (a)an increase in hyaluronate, as well as alterations in the metabolism of heparan sulfate, and ( b )an ability of the cells to grow to high densities (3-9). Heparansulfate, inproteoglycanform, appearsto bea ubiquitous component of cell surfaces (10). Although there have been few comparative studies on the detailed co-polymeric structure of heparan sulfate from different cell lines, there is a growing notion that proteo-heparan sulfates may be cell/tissue-specific (11-14). The glycan side chains may play a role in cell-cell contact by virtue of their ability to selfassociate (15, 16). Comparative studies on the chemistryof heparan sulfates fromnormalandtransformed cells (ortumor cells) have revealed heterogeneity with regard to sulfate content(17, 18). There are also observations of decreased charge density (4) due to a lowering of the sulfate content (19), particularly0sulfate (no), inthe heparan sulfate chains derived from transformed cells. In the presentwork, attempts havebeen made to determine whether the transformation-dependent structural changesin heparan sulfate are accompanied by alterations in the selfassociating properties of these polymers. This property was assessed by affinity chromatography of heparan sulfatesfrom A variety of cell surface properties are modified in tumor transformed and nontransformed cells on gels substituted with cells or transformed cells compared with the normal counter- various forms of bovine lung heparan sulfate (21). part (1). Many experimental studies have been carried out EXPERIMENTALPROCEDURES with different cloned cell lines which can be transformed to a neoplastic state by oncogenic viruses. NormalandtransMaterials-Heparan sulfate was purified from heparin by-products formed cells in tissue culture havebeen compared with regard as described extensively elsewhere (15, 16, 22). The total pool was to structuralchanges involving cell surface proteins (glycopro- subfractionated according to charge density by stepwise precipitation

with cetylpyridinium chloride in the presence of decreasing concentrations of NaCl (23). The following fractions were obtained: HS1 (0.2 Research Council (567), “Greta och Johan Kocks Stiftelser,” and the to 0.4 M NaCI), HS2 (0.4 to 0.6), HS3 (0.6 to 0.8), HS4 (0.8 to LO), Medical Faculty, University of Lund. The costs of publication of this HS5 (1.0 to 1.2),and HS6 (complexes not soluble in 1.2 M NaCI). The article were defrayed in part by the payment of page charges. This subfractions HS2-4 were separated intomore or less association-prone article must therefore be hereby marked “aduertisement” in accord- variants (15) bygel chromatography as describedinapreceding ance with 18 U.S.C. Section 1734 solely to indicate this fact. report (21), where the analytical data for these materials were also $ T o whom requests for reprints should be addressed. given. The fractions containing aggregatable species were designated

* This work was supported by grants from the Swedish Medical

13044

Co-polymeric Glycans HS2-A, HS3-A, and HS4-A, respectively. The procedure for immobilizing heparan sulfates on agarose gels were outlined in a preceding report (21). In brief, partially periodateoxidized chains were coupled to adipic acid dihydrazide-substituted Sepharose 4B and the resulting aldimines were stabilized by reduction. Other sources of materials were as follows:glycosaminoglycans from Dr. M. B. Mathews, University of Chicago; Insta-gel, Packard; microgranular DEAE-cellulose (DE-521, Whatman; other chemicals were of analytical grade. Preparation of Radiolabeled Heparan Sulfates--jH- and %-labeled heparan sulfate was prepared from human lung fibroblasts according to methods that have been described in detail elsewhere (14, 23, 24). The cells,grown in monolayer, were maintained on sulfate-poor medium before the addition of radioactivity (5 pCi of Na25S04 and 1 pCiof ~-[l-~H]glucosamine/ml of medium). After incorporation of radioactivity for 12 h, medium and cells were collected separately. 3H- and 35S-labeledheparan sulfate was also obtained from a Balb3T3 cell line and SV40 or polyoma-transformed ones as described previously (25). After incorporation of radioactivity for 48 h, medium and cells were collected separately. In this case, the cells were digested briefly with trypsin and material released by this treatment (pericellular material) as well as a cell residue (intracellular material) were recovered. All the material obtained from the above experiments was processed in the same way (14,24).Thus, thefractions were digested with papain, and labeled glycosaminoglycanswere isolated by ion exchange chromatography. After depolymerization of the galactosaminoglycan components by digestion with chondroitinase ABC, the heparan sulfates were separated from split products by gel chromatography on Sephadex G-50(void volume fraction). Chromatographic Methods-Radiolabeled heparan sulfates were subjected to ion exchange chromatography on columns (6 X 140 mm) of DE-52 DEAE-cellulose that were equilibrated with 0.1 M sodium acetate, pH 5.0. Elution was performed with a linear gradient of 0.1 to 2.5 M sodium acetate, pH 5.0 (total volume, 100 m l ) at a rate of 3 ml/h. The shape of the gradient was checked by conductivity measurements. The effluent was analyzed for radioactivity in a Packard 2650 liquid scintillation counter with automatic quench-correction using Insta-gel (0.5 ml of sample and 5 mi of liquid) as scintillator. Binding studies (affiity chromatography) were performed at room temperature on columns (6 X 50 mm) containing agarose gels substituted with various self-associating heparan sulfate species (HS2-A, HS3-A, or HS4-A). The columns were equilibrated with 0.15 M NaC1, and samples were applied in 100 pl of 0.15M NaC1. Elution was carried out with a linear gradient of0.15 M NaCl to 1.5 M guanidinium chloride (total volume, 100 ml) at a rate of 3 ml/h. For analysis, see above. In a few cases, the samples were applied in 100pl of 4 M guanidinium chloride. After this solution had drained into the column (pre-equilibrated with 0.15 M NaCl) a head of -1 ml of 0.15 M NaCl was added, and the outlet of the column was kept closed for 12 to 15 h with three changes of the liquid head above tbe gel. Then, elution was performed as described above.

in Transformed Cells

13045

on HS2-A, HS3-A, or HS4-A-agarose gels (Fig. 2). The samples were applied either in 0.15 M NaCl or in 4 M guanidinium chloride. Both modes weretested because, ina previous study (21), it was observed that theoptimum conditions for binding

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FIG. 1. Ion exchange chromatography of [SH/36S]heparan sulfates from(a)human lung fibroblasts, (6) 3T3-intracellular pool, (c) 3T3-pericellular pool, and (d)3T3-medium pool. The various radiolabeled heparan sulfates were obtained as described under “Experimental Procedures” and chromatographed on DEAEcellulose. The points of elution (peak positions) of bovine lung heparansulfate subfractions HS2,HS3, and HS4, as well as that of chondroitin sulphate (CS) are indicated in the top graphs. Fractions were pooled as indicated by vertical lines.

,

RESULTS

Charge Density and Binding Properties of Heparan Sulfates from Normal Cells-’H- and 35S-labeledheparan suifates from human lung fibroblasts (representing both intraand pericellular pools) and from three different pools of 3T3 cell cultures gave the ion exchange profiles shown in Fig. 1 . When compared with the bovine lung standards HS2, HS3, and HS4, lung fibroblast heparan sulfate eluted in a position intermediary to HS3 and HS4 (Fig. l a ) , whereas heparan sulfate from the 3T3 intracellular pool (Fig. Ib) appeared at the same elution volume as HS2. The 3T3-heparan sulfates showed a progressively more retarded position in the chromatogram, when going from intracellular (Fig. Ib) over pericellular (Fig. IC)to extracellular (Fig. Id) material. In addition, the heparan sulfates of the two latter pools were heterogeneous and two subfractions were pooled from each; HS3 was common to both. The heparan sulfate derived from lung fibroblasts (Fig. l a ) was assayed for aggregatability by affinity chromatography

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FIG. 2 (left). Affinity chromatography of [3H]heparan sulfate from human lung fibroblastson agarose gels substitutedwith (a)HS2-A, (b) HS3-A, and (c) HSCA. The samples were applied as described under “Experimental Procedures” either in 0.15 M NaCl (-) or in 4 M guanidinium chloride (- - -). FIG. 3 (right). M n i t y chromatography of C3H]heparan sulfates from 3T3 cells on variously substituted agarose gels. Heparan sulfates were obtained from the medium, the pericellular and intracellular compartments and chromatographed as described under “Experimental Procedures.” The following experiments were performed a and b, HS2 and HS3, respectively, from the pericellular pool on HS2-A-agarose;c and d, the same fractions on HS3-A-agarose; e and f,HS3 and HS4, respectively, from the medium pool on HS3-Aagarose. The samples were all applied in 0.15 M NaCl.

Co-polymeric Glycans in Transformed Cells

13046

may vary from onespecies to another. As there is a competition between binding of free chains to the immobilized ones and a self-associationamong the free chains, the latter event should be especially disfavored if the concentration of free chains in an associative solvent was very low. This seems to be the case in the present situation (Fig. 2) since binding was most pronounced when the samples were applied in 0.15 M NaC1. Therefore, this mode of application was used throughout this study. Moreover, it was noted that lung fibroblast heparan sulfate had the highest affinity for HS4-A-agarose (Fig. 2c). The heparan sulfates from 3T3 cells comprised five major fractions: an intracellular (Fig. l b ) , the pericellular HS2 and HS3 fractions (Fig. IC), and the extracellular HS3 and HS4 fractions (Fig. Id). When the latter four were subjected to affinity chromatography on heparan sulfate-agarose, the results shown in Fig. 3 were obtained. The pericellular HS2 fraction was partially bound to HS2-A-agarose (Fig. 3a) but e not to HS3-A-agarose (Fig. 3c). Conversely, the pericellular 12t HS3 fraction was partially and weakly bound to HS3-A-agarose (Fig. 3d) but not to HS2-A-agarose (Fig. 3b). The extracellular HS3 fraction was partially bound to HS3-A-agarose (Fig. 3e) witha varying affinity, whereas the extracellular HS4 fraction was not bound at all (Fig. 3f). Charge Density and Binding Properties of Heparan Sulfates from Transformed Cells-The ion exchange chromatography profiles of the 3H- and 35S-labeledheparan sulfates 10 20 10 20 derived from the three pools of normal, SV40- and polyomaEffluentvolume (ml) transformed 3T3 cells are shown in Fig. 4. In general, the heparan sulfates from transformed cells were moreheterogeFIG. 5. Affinity chromatography of (a)HS2 and (b)HS3 from neous and of lower charge density than those from the normal the pericellular pool of normal 3T3 cells on HS2-A- and HS3A-agarose,respectively, of (e) HS2and (d)HS3 from the

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corresponding poolof SV40-3T3 cells on the same gels, and(e) HSla and ( f ) HS2 from the corresponding pool of Polyoma3T3 cells on the same gels. The heparan sulfates were prepared and chromatographed (applied in 0.15 M NaCl) as described under “Experimental Procedures.”

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counterpart (see e.g.Fig.4, g and h). In both normal and transformed cells, the heparan sulfates from the medium (Fig. 4, c, z) were more retarded on the column than the corresponding cell-associated materials. Heparan sulfates that eluted at different ionic strengths were isolated from the Pericellular as well as extracellular pools of SV40-transformed (HS2, HS3, and HS4 in Fig. 4, e and f ) and polyoma-transformed cells (HSla, HSlb, HS2, HS3, and HS4 in Fig. 4, h and i). When the various heparan sulfate fractions were subjected to affinity chromatography on heparan sulfate-agarose the results seen in Fig. 5 were obtained. Whereas the HS2 and HS3 fractions from normal cells showed affinity for HS2-Aand HS3-A-agarose,respectively (Fig. 5, a and b), thecorresponding heparan sulfate fractions from SV40- and polyomatransformed cells were not bound (Fig. 5, c to f).

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DISCUSSION

The results reported herein indicate that theaggregatability of radiolabeled heparan sulfates fromcell cultures can be studied using affinity chromatography on heparan sulfateEffluent volume (ml) agarose. It was concluded in a preceding report (21) that FIG.4. Ion exchange chromatography of [SH/36S]heparan association-prone chains chiefly bind to matrices substituted (a,4 g),pericellular (b, e, h) and with heparan sulfates of the same kind. This concept is sulfates from the intracellular medium pools (c, f , i ) of normal (a to e), SV40 (d to f ) , and strengthened by the present findings as HS2 fractions generpolyoma-transformed (g to i ) cell lines. The heparan sulfates ally bind to HS2-A-agarose, HS3 fractions to HS3-A-agarose, were prepared and chromatographedas describedunder “Experimental Procedures.”HS2 3,4 indicates the elution position of the heparan and HS4 fractions to HS4-A-agarose. This result may give the as indicated impression that all heparan sulfate chains that elute in a sulfate subfractionsof bovine lung. Fractions were pooled particular position upon ion exchange chromatography have by vertical lines. 30 40 50 60

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Co-polymeric Glycans the same structure. This may be partly true as the elution position is mainly governed by the degree of sulfation which, in turn, is correlated with the uronic acid composition (for a more extensive discussion, see Ref. 22). However, the structural variability among heparan sulfate chains is sufficiently large to accommodate a host of sequential arrangements within a certain population with the same average charge density. As shown in the present study, heparan sulfates from transformed cellsdid not bind to agarose gels that were substituted with chains that had the corresponding elution position in ion exchangechromatography. Even if the elution position is also affected by size,previous work (20) has indeed shown that heparan sulfates from SV40-transformed3T3 cells have a lower 0-sulfate content. Although no differencesin the L-iduronate/D-glucuronateratio were observed (20), it is conceivable that changes in the sequences can take place without changes in gross uronate composition. Therefore, the present result prompts us to examine the co-polymeric features of heparan sulfates from normal and transformed cells. Subtle changes in the sequential arrangements of L-iduronate- and D-glucuronate-bearing repeats may well affect the aggregatability of the chains. A complete loss of this property or an alteration in the specificity of association might ensue. Acknowledgment-The expert technical assistance of Birgitta Havsmark is greatfully acknowledged. REFERENCES 1. Robbins, J. C., and Nicolson, G . L. (1975) in Cancer: A Comprehensiue Treatise (Becker, F. F., ed) Vol. 4, pp. 3-54, Plenum Press, New York 2. Nicolson, G. L. (1976) Bwchim. Bwphys. Acta 458,l-72 3. Winterbourne, D. J., and Mora, P. (1977) J. Supramol. Struct. 7, 91-100 ~.~ 4. Winterbourne, D. J., and Mora, P. T . (1978) J. Biol. Chem. 253, 5109-5120 ~~

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5. Underhill, C. B., and Keller, J. M. (1975) Biochem. Biophys. Res. Commun. 63,448-454 6. Underhill, C. B., and Keller, J. M. (1977) J.Cell Physiol. 90,5359 7. Hopwood, J. J., and Dorfman, A. (1977) J.Biol. Chem. 252,47774785 8. Vannucchi, S., and Chiarugi, V. P. (1977) J.Cell Physiol. 90,503510 9. Dunham, J. S., and Hynes, R. 0.(1978) Biochim. Biophys. Acta 506,242-255 10. Dietrich, C. P., and Montes de Oca, H. (1978) Biochem. Biophys. Res. Commun. 80,805-812 11. Atherly, A. B., Barnhart, B. J., and Kramer, P. (1977) J. Cell Physiol. 90,375-386 12. Linker, A. (1979) Biochem. J. 183, 711-720 13. Oldberg, A., Kjellen, L., and Hook, M. (1979) J.Biol. Chem. 254, 8505-8510 14. Sjoberg, I., and Fransson, L.-A. (1980) Biochem. J. 191, 103-110 15. Fransson, L.-A., Nieduszynski, I. A., and Sheehan, J. K. (1980) Biochim. Biophys. Acta 630,287-300 16. Fransson, L.-A., Havsmark, B., Nieduszynski, 1.A., and Huckerby, T.N. (1980) Biochim. Biophys. Acta 633,95-I04 17. Nakamura, N., Hurst, R.E., and West, S. S . (1978) Biochim. Biophys. Acta 538,445-457 18. Keller, K. L., Underhill, C. B., and Keller, J. M. (1978) Biochim. Biophys. Acta 540,431-442 19. Johnston, L. S., Keller, K. L., and Keller, J. M. (1979) Biochim. Biophys. Acta 583,81-94 20. Keller, K. L., Keller, J. M., and Moy, J . M. (1980) Biochemistry 19,2529-2536 21. Fransson, L.-A., Havsmark, B., and Sheehan, J. K. (1981) J.Biol. Chem. 256,13039-13043 22. Fransson, L.-A., Sjoberg, I., and Havsmark, B. (1980) Eur. J. Biochem. 106,59-69 23. Malmstrom, A., Carlstedt, I., Aberg, L., and Fransson, L. A. (1975) Biochem. J.151,477-489 24. Sjoberg, I., and Fransson, L.-A. (1977) Biochem. J. 167, 383-392 25. Chiar~gi,V. P., and Dietrich, C. P. (1979) J. Cell. Physiol. 99. 201-206