CELL STRUCTURE AND FUNCTION 13, 481-493 (1988) C by Japan Society for Cell Biology Secretion of a Novel Cell-Adhesive Protein Distinct from Fibronectin by Mouse L•EP3 Cells Growing in Protein- and Lipid-Free Synthetic Medium

Several cell lines growing in proteinand lipid-free synthetic medium secreted cell-adhesive protein(s) into the medium. The conditioned medium (CM) of one of these cell lines, mouse L•EP3, showed the highest cell attachment-promoting activity (CPA) among them. Cell-adhesive protein(s) in the CM of L•EP3 cells (L•EP3-CM) were separated into two types by sequential affinity column chromatography employing gelatin-Sepharose 4B and heparinSepharose 4B. One was a gelatinand heparin-binding cell-adhesive protein (GCP), and was identified as a cellular form of mouse fibronectin. The other was a gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). The CPA of GNCP preparation was effective for the cell-attachment and spreading of both epithelial and fibroblastic cells. The CPA of GNCP preparation was not blocked by the antiserum and scarcely inhibited in the presence of the synthetic cell attachment-promoting peptide Gly-Arg-Gly-Asp-Ser-Pro, a competitive inhibitor of fibronectin. This suggests that the structure of the cellattachment site of GNCP is different from that of fibronectin. The GNCP preparation showed little cross-reactivity with anti-mouse laminin antiserum in enzyme-linked immunosorbent assay (ELISA). These results demonstrate the possibility that GNCP in L•EP3-CM is a novel cell-adhesive protein distinct from fibronectin or laminin. The secretion of the two types of cell-adhesive proteins by L•EP3 cells is discussed. Fibronectin is a well-characterized, high molecular weight glycoprotein in blood plasma, in connective tissues, and on the cell surface of a large number of cell types. This protein has many biological and biochemical activities, including cell adhesion, spreading, growth, motility, and differentiation, as well as interaction with gelatin, heparin, and fibrin (12, 21, 33). There are known to be at least two distinct types of fibronectin: plasma and cellular fibronectins. These fibronectins, even when derived from different species, are very similar in physical, biochemical, imAbbreviations used: CPA, cell-attachment promoting activity; GCP, gelatinand heparin-binding cell-adhesive protein; GNCP, gelatin-non-binding and heparin-binding cell-adhesive protein; L•EP3-CM, conditioned medium of L•EP3 cells.