Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparan sulfate proteoglycan.
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 (K1) had 78% adherence to thrombospondin adsorbed to tissue culture plastic, CHO S745 cells, with less than 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 greater than or equal to 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.
Kaesberg
P R
PR
Department of Medicine, University of Wisconsin, Madison 53706.
Ershler
W B
WB
Esko
J D
JD
Mosher
D F
DF
D000327:Adsorption; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D011508:Chondroitin Sulfate Proteoglycans / Q000502:physiology*; D006224:Cricetinae; D005260:Female; D005353:Fibronectins / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000502:physiology*; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin / Q000494:pharmacology; D006497:Heparan Sulfate / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010053:Ovary; D010446:Peptide Fragments / Q000378:metabolism; D010969:Plastics; D011509:Proteoglycans / Q000502:physiology*; D019699:Thrombospondins; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002118:Calcium; D011508:Chondroitin Sulfate Proteoglycans; D005353:Fibronectins; D006023:Glycoproteins; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D010446:Peptide Fragments; D010969:Plastics; D011509:Proteoglycans; D019699:Thrombospondins; D014357:Trypsin