Role of a 16S glycoprotein complex in cellular adhesion.

Abstract

Myogenic cells release into their culture medium a glycoprotein complex that mediates cellular adhesion.In the absence of calcium this complex has a sedimentation value of 16 S. it agegates in the presence of calcium. The 16S material both agglutinates and increases the rate of cell-substratum adhesion of a myoblast variant and inhibits the adhesion of a nerve-like cell line to culture dishes. It-is alsQ a hemagglutinin. The 16S particle is composed of glycbsamitoglycans and several proteins, including fibronectin and collagen. Macromolecules responsible for cellular adhesion are released from cells into their environment. For example, glycoproteins involved in the aggregation of retinal cells bave been isolated from growth-conditioned medium (1,2), and molecules secreted from fibroblast-like cells promote cell-substratum adhesion (3,4 (17), and identifying the GAGs in the individual peaks by enzymatic methods (18). Hydroxyproline was determined by using cells labeled with [14C]proline. The fractions of interest were isolated, hydrolyzed in 6 M HCI, and chromatographed on an amino acid analyzer; the radioactivity in the 4-hydroxyproline and proline peaks was determined.Preparation of Conditioned Medium and SAM. Conditioned medium was prepared from exponentially dividing cells by washing the cells twice in serum-free Hepes-buffered medium and incubating the cells in the same medium for 15 hr at 370C. To prepare SAM-coated dishes, growth-conditioned medium was placed in 35-mm. plastic petri dishes (Falcon) for 18 hr, and then the dishes were washed twice with 0.5 mM ethylene glycol bis(fl-aminoethyl ether)-NN,N',N'-tetraacetic acid (EGTA), three times with water, and once with Hepes medium. After the washing, 2 ml of Hepes medium containing 0.2% bovine serum albumin was added.Adhesion Assays. To assay cell-substratum adhesion, early stationary phase M3A or PC12 cells were labeled with [3H]-leucine (5 MCi/ml; 1 Ci = 3.7 X 1010 becquerels) for 15 hr. The cells were washed three times with Hepes medium containing 0.2% albumin and 0.2-ml aliquots were pipetted into 35-mm SAM-coated plastic petri dishes containing 2 ml of the same medium. At indicated times the dishes were swirled 10 times, the medium was aspirated, and the remaining attached cells were dissolved in Triton X-100 and their isotope content was determined.. The data were plotted as the fraction of input cells that adhered as a function of time. Variation between duplicates was less than 5% (19).Cell aggregation was measured by the disappearance of single cells from an agitated suspension (19, 20). Cells were washed twice in Hepes buffer containing 0.2% bovine serum albumin and added to 0.5-ml aliquots of the test medium made 0.2% in albumin. The cells were agitated on a rotary shaker at 100 rpm and the disappearance of single cells was monitored with a Coulter Counter.The rabbit erythrocytes used for the hemagglutination assays were prepared in two ways. Some were trypsin-treated and fixed with glutaraldehyde; others were trypsin-treated, fixed w...

Properties of extracellular adhesion-mediating particles in myoblast clone and its adhesion-deficient variant.

“…Evidence presented elsewhere has shown that L6 band A is fibronectin and that bands B and C (Fig. 8) are collagen-related (13,14). The identity of the remaining fractions is unknown.…”

“…Particles sedimenting between 5 S and 18 S in sucrose gradients have been isolated from a variety of cell types, including skeletal and smooth muscle (13,16), clonal nerve and glial cell lines, fibrobhsts, and chick neural retina (unpublished observations). These particles either stimulate or inhibit cellular adhesion, contain GAGs and a limited number of proteins, and in some cases have the adhesive specificity which is required if they play a role during normal development (16).…”

“…When growth-conditioned media of myoblasts were centrifuged at 100,000 g for 2 h, all of the adhesion-promoting activity was removed from suspension and recovered in the pellets (13). 0.4% of the total cell protein was in the paniculate material of both L6 and M3A conditioned medium.…”

“…All of the adhesion-promoting material from L6 growthconditioned medium sediments in calcium-free sucrose gradients as a 16 S peak (13). The 16 S particle was composed primarily of collagen, fibronectin, and several GAGs (13). To determine if the adheron from M3A cells is structurally different from that of L6, L6 and M3A cells were labeled with [3~S] sulfate in serum-free medium; unlabeled growth-conditioned media from 5 x 107 L6 and M3A cells were also prepared.…”

“…Myogenic cells release into their culture medium a glycoprotein complex which aggregates myoblasts and alters the adhesion of cells to the substratum of plastic petri dishes (13). When adsorbed onto culture dish surfaces, the material from clonal L6 skeletal muscle myoblasts stimulated the adhesion of myoblasts and inhibited the adhesion of a clone of sympathetic nervelike cells.…”