Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins.

Abstract

Proteases were used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-protens by pronase (1 mg/ml for 20 min at 37.degree. C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to clevage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37.degree. C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 .mu.M cycloheximide. Under these conditions only 3 glycoproteins (gp) of MW 130,000, 120,000 and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells, gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37.degree. C), under conditions not affecting adhesion. These glycoproteins may be involved in fibronectin-mediated cell adhesion in some as yet unknown way.