Secretion of a Novel Cell-Adhesive Protein Distinct from Fibronectin by Mouse L•P3 Cells Growing in Protein- and Lipid-Free Synthetic Medium

Abstract

Several cell lines growing in protein- and lipid-free synthetic medium secreted cell-adhesive protein(s) into the medium. The conditioned medium (CM) of one of these cell lines, mouse L•P3, showed the highest cell at-tachment-promoting activity (CPA) among them. Cell-adhesive protein(s) in the CM of L•P3 cells (L•P3-CM) were separated into two types by sequential affinity column chromatography employing gelatin-Sepharose 4B and heparin-Sepharose 4B. One was a gelatin- and 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 prepara-tion 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 cell-attachment 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•P3-CM is a novel cell-adhesive protein distinct from fibronectin or laminin. The secretion of the two types of cell-adhesive pro-teins by L•P3 cells is discussed.