Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3343-3347, July 1979 Cell Biology Role of carbohydrate in biological function of the adhesive glycoprotein fibronectin KENNETH OLDEN*, ROBERT M. PRATTt, AND KENNETH M. YAMADA* *Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health; and tLaboratory of Developmental Biology and Anomalies, National Institute of Dental Research, Bethesda, Maryland 20205 Communicated by DeWitt Stetten, Jr., March 28, 1979 ABSTRACT We have investigated the role of the carbohy- MATERIALS AND METHODS drate moiety in the biological activity of fibronectin in vitro by Cell Culture. Secondary chicken embryo fibroblasts (CEF) using tunicamycin to inhibit the glycosylation of this glyco- protein. Tunicamycin is a glucosamine-containing antibiotic were cultured in plastic tissue culture dishes in Ham's FlO that specifically inhibits glycosylation of protein asparaginyl medium supplemented with 10% (vol/vol) tryptose phosphate residues mediated by dolichol pyrophosphate. Fibronectin broth, 5% (vol/vol) heat-inactivated calf serum, 0.056% sodium synthesized in the presence of 0.5 ptg of tunicamycin per ml was bicarbonate, 50 units of penicillin per ml, 50 Ag of streptomycin not glycosylated, as determined by amino sugar analysis, lack per ml, and 2 mM glutamine as described (1, 12). of incorporation of [14Ciglucosamine and [3H]mannose, and Isolation of Fibronectin. Confluent primary cultures were concanavalin A binding studies. Nonglycosylated fibronectin that was isolated from chicken embryo fibroblasts and added preincubated for 3.5 hr in culture medium with or without 0.50 to transformed cells in vitro was as effective as the glycosylated ,ug of tunicamycin per ml. Cells were passaged with 0.25% protein in promoting a more normal fibroblastic phenotype, trypsin, replated at the same cell density in the preincubation including cell flattening, elongation of cell processes, and par- medium, and incubated for 24 hr. Control and tunicamycin- allel alignment of cells. The nonglycosylated protein was also treated cultures were homogenized separately in 50 mM sodium as effective as the glycosylated species in mediating cell at- phosphate, pH 11.0/1% Triton X-100, and the pH was re- tachment to collagen and spreading on plastic, as well as in to 11.0 with NaOH. The were centri- agglutination of formalin-fixed sheep erythrocytes. The non- adjusted homogenates glycosylated protein was twice as sensitive as the glycosylated fuged at 100,000 X g at 4°C for 1 hr to sediment insoluble protein to proteolytic hydrolysis in vitro as had been suggested material. The supernatant was diluted 1:10 with 100 mM Na by previous studies with intact cells [Olden, K., Pratt, R. M. & phosphate, pH 7.0/1% Triton X-100/50 mM NaCl. Fibronectin Yamada, K. M. (1978) Cell 13,461-4731. We conclude that the was isolated from the homogenates by incubation with antifi- carbohydrate moiety of fibronectin is not required for the bronectin covalently coupled to Sepharose 4B. The column was mediation of a number of biological activities characteristic of washed with 5 vol of 0.1 M borate buffer, pH 8.5/0.5 M NaCl. this glycoprotein. The supernatant containing fibronectin was slowly applied to the column (10 ml/hr), and unbound material was eluted with The role of the carbohydrate moiety covalently attached to the 0.1 M borate buffer. Nonspecifically adsorbed protein was proteins is not known. In previous studies, inhibition of glyco- eluted from the resin with 0.1 M borate buffer, pH 8.5/0.5 M sylation of fibronectin, a major cell surface glycoprotein (also NaCl. Fibronectin was then eluted from the resin with 0.2 M known as CSP or LETS), did not affect its synthesis or secretion acetic acid, pH 2.9/0.5 M NaCl. The eluates were immediately (1-4). Similar results have been obtained for other glycoproteins adjusted to pH 11.0 with NaOH, dialyzed overnight against (5-7). These results left the possibility that the biological ac- buffer A (0.15 M NaCl/1.0 mM CaCl2/10 mM cyclohexyl- tivities (e.g., the cell surface adhesive interactions affected by propane sulfonic acid, at pH 11.0) and stored in 200-yl aliquots fibronectin) might require the carbohydrate moiety. at -60°C. Because tunicamycin inhibits the glycosylation of fibronectin Protein Synthesis and Glycosylation. Protein synthesis was (1), the major cell surface glycoprotein of chicken embryo fi- measured by the incorporation of L-[U-'4C]leucine (2 ,Ci/ml, broblasts, we used this antibiotic to evaluate the influence of 1 Ci = 3.7 X 1010 becquerels). Glycosylation of fibronectin and the carbohydrate moiety on many of the known biological ac- total macromolecular trichloroacetic acid-insoluble material tivities of fibronectin. This protein is an adhesive glycoprotein were measured by the incorporation of D-[U-'4C]glucosamine that helps maintain normal cell morphology, cell surface ar- (2 tiCi/ml) and D-[2-3H]mannose (0.5 ,Ci/ml) and also by chitecture, and cell interactions in cultured cells (2, 3, 8-11). amino sugar analysis (13). Radioactively labeled fibronectin We present evidence that the carbohydrate moiety is not was immunoprecipitated from cell homogenates and homog- required for these biological activities mediated by fibronectin enized in 2% sodium dodecyl sulfate as described (1, 4, 12). in cell culture and in assays for cell adhesiveness. In addition, Sodium dodecyl sulfate gel electrophoresis was performed we directly confirm a previous suggestion, based on in vivo with 1-mm-thick polyacrylamide slab gels according to the studies, that nonglycosylated fibronectin is more susceptible method of Laemmli (14) with stacking gels of 4% acrylamide to proteolytic degradation than the glycosylated protein (1). and resolving gels of 7.5% acrylamide as described (1, 4, 12, 15). Gels were stained with Coomassie blue and destained in 7% The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- acetic acid. The polypeptide band corresponding to fibronectin vertisement" in accordance with 18 U. S. C. §1734 solely to indicate was cut from the gel and dissolved in 1.5 ml of 30% hydrogen this fact. peroxide. Radioactivity was measured by liquid scintillation 3343 Downloaded by guest on October 2, 2021 3344 Cell Biology: Olden et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Incorporation of leucine and sugars into cellular fibronectin and trichloroacetic acid-insoluble fraction of cells Incorporation into fibronectin/ltg Incorporation, cpm/,ug total cell protein total cell protein Precursor Control Tunicamycin % inhibition Control Tunicamycin % inhibition L-Leucine 1432 ± 77 1045 ± 91 27 i 3 43 ± 6 23 ± 3 47 D-[U-14C]Glucosamine 1125 ± 93 259 ± 37 77 ± 2 32 ± 7 0.63 ± 1 98 D-[2-3H]Mannose 4519 + 203 181 ± 21 96 ± 3 124 ± 12 1.24 ± 1 99 Cells were incubated for 24 hr with the radioactive precursors in medium with and without tunicamycin. Then fibronectin was immunoprecipitated and electrophoresed, and radioactivity was quantitated. The data presented represent the average of three experiments (plus and minus represent the range). In a typical experiment with 0.5,ug tunicamycin per ml, the amount of [14C]leucine incorporated into total acid-precipitable material was 1365 cpm/,gg of protein for untreated cultures and 954 cpm/,ug of protein for tunicamycin-treated cultures. To correct for this difference in inhibition (27%) of protein synthesis, we applied 52 ,l of tunicamycin-treated cell homogenate to the gel compared to 40 Al of the untreated cell homogenate. Similar aliquots were used for determination of the percentage inhibition of glucosamine or mannose incorporation into total acid-precipitable material. The percentage inhibition of leucine incorporation into fibronectin was determined by dividing the cpm in fibronectin from tunicamycin-treated cultures by that found in fibronectin from untreated cultures. The percentage inhibition of glucosamine or mannose incorporation into fibronectin was determined by dividing the glu- cosamine/leucine ratio of tunicamycin-treated culture by the glucosamine/leucine ratio of untreated cultures. We emphasize that the inhibition of glycosylation is therefore expressed as the inhibition for the fibronectin actually synthesized and not merely as inhibition of overall carbohydrate incorporation into fibronectin. spectrometry after dilution to 20 ml with Aquasol (New En- The radiochemicals L-[U-14C]leucine (specific activity 325 gland Nuclear). Ci/mole), D-[U-14C]glucosamine (specific activity 238 Ci/ Other Procedures. Affinity-purified goat antibodies against mole), and D-[2-3H]mannose (specific activity 2 Ci/mmol) were fibronectin were covalently coupled to CNBr-activated Seph- obtained from New England Nuclear. Phenylmethylsulfonyl arose (Pharmacia) at a ratio of 10 mg of antibody protein per fluoride, Triton X-100, and Pronase (Streptomyces griseus g of hydrated gel following the procedures recommended by protease, grade B, no. 53702) were purchased from Calbiochem, the company (Pharmacia). Hemagglutination of formalinized electrophoresis reagents from Bio-Rad, and CNBr-activated sheep erythrocytes and effects of fibronectin on cell spreading Sepharose 4B from Pharmacia. Plastic tissue culture dishes were and morphology were assayed as described (15, 16). Protein was from Costar, and nonwettable bacteriological petri dishes for determined by the method of Lowry et al. (16), with bovine the collagen attachment assay were obtained from Falcon. serum albumin as protein standard. Materials. Tunicamycin was a gift from Gakuza Tamura via RESULTS the Drug Evaluation Branch of the National Cancer Institute. Isolation of glycosylated and nonglycosylated fibronectin Table 2. Effect of tunicamycin on amino sugar composition of cellular fibronectin To quantitate the inhibition of fibronectin glycosylation by Amino sugar/ tunicamycin, we isolated the protein by immunoprecipitation cellular fibronectin from treated and untreated cultures labeled for 24 hr with ei- monomer, ther [3H]mannose or [14C]glucosamine. The results are shown Addition mol/mol % inhibition in Table 1.