Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparan sulfate proteoglycan.

Abstract

Thrombospondin is a 420-kD platelet alpha-granule glycoprotein that binds specifically to heparin. We examined adhesion to thrombospondin of CHO K1 cells and three mutant CHO lines with varying deficiencies in glycosaminoglycan (GAG) synthesis. In an experiment in which the parent line (Kl) had 78% adherence to thrombospondin adsorbed to tissue culture plastic, CHO S745 cells, with < 6% normal GAG synthesis had 11% adherence. CHO S677 cells, with decreased heparan sulfate proteoglycan but increased chondroitin sulfate proteoglycan, had 42% adherence. CHO S803 cells, with decreased heparan sulfate proteoglycan and normal chondroitin sulfate proteoglycan, had 31% adherence. Heparin inhibited K1 cell adhesion to thrombospondin, but not fibronectin, in a concentration-dependent manner. Dermatan sulfate but not chondroitin sulfate was also inhibitory. There was markedly decreased K1 cell adhesion to a thrombospondin core fragment that lacked the heparin binding NH2-terminal domain. Purified heparin binding domain, although poorly adhesive when adsorbed to substratum, inhibited cell adhesion to intact thrombospondin. Adhesion was better for all cell lines tested, including three human tumor cell lines, when thrombospondin was adsorbed at pH 4.0 compared with pH 7.4. When adsorption of thrombospondin was done at pH 7.4, cell adhesion was better when thrombospondin was adsorbed in the presence of 2 0.6 mM calcium, compared to 0.1 mM calcium or EDTA. These findings suggest that thrombospondin can adsorb to plastic with varying degrees of exposure of a cell adhesion domain. We conclude that the thrombospondin cell adhesion receptor on CHO cells is a heparan sulfate proteoglycan, and that cell adhesion to thrombospondin depends on conformation of adsorbed thrombospondin.

Calcium indirectly regulates immunochemical reactivity and functional activities of the N-domain of thrombospondin-1

“…Calcium influenced TSP1 binding to perlecan (Vischer et al, 1997), and divalent cations enhanced TSP1 binding to sulfatide (Roberts et al, 1985). Immobilization in the presence of Ca 2+ also enhanced proteoglycan-dependent Chinese hamster ovary cell adhesion on TSP1 (Kaesberg et al, 1989). All of these results are consistent with the present data and can now be explained as indirect effects on the N-module of calcium binding to the TSP1 signature domain.…”

Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

“…We used the CHO-K1 cell line (ATCC CCL-61), one mutant CHO cell line defective in glycosaminoglycan biosynthesis (CHO-745 cells ATCC: CRL-2242 (18), and the CHO B2 cell line lacking the ␣5 subunit (19), a generous gift from Dr. R. Juliano (Chapel Hill, NC). The cells were harvested by treatment with 1% EDTA at ϳ80 -90% confluence and were suspended (3 ϫ 10 5 cells/ml) in 25 mM Tris, pH 7.4, containing 150 mM NaCl, 1 mM MgCl 2 , 0.1 mM CaCl 2 , and P20 0.005%.…”

Differential expression of thrombospondin 1, 2, and 3 during murine development

“…Further investigation established that TSP was also secreted by a variety of cultured cells that include endothelial cells (McPherson et al, 1981;Mosher et al, 1982;Canfield et al, 1990), smooth muscle cells and fibroblasts (Raugi et al, 1982;Jaffe et al, 1983), keratinocytes (Wikner et al, 1987), macrophages (Jaffe et al, 1985), and glial cells (Asch et al, 1986). These findings, coupled with reports describing several cell-surface receptors for the protein Taraboletti et al, 1987;Lawler et al, 1988;Kaesberg et al, 1989;Frazier, 1991;Prater et al, 1991;Guo et al, 1992;Leung et al, 1992), indicated a broader spectrum of functions than that initially recognized in coagulation.…”