RECOMBINANT GST-HUMAN OSTEOPONTIN FUSION PROTEIN IS FUNCTIONAL IN RGD-DEPENDENT CELL-ADHESION

Osteopontin (OPN) is a secreted phosphoprotein expressed by many tumor cells, as well as a limited set of normal cells. Native OPN has been shown to support cell adhesion in an RGD-peptide-inhibitable fashion. Here we expressed human OPN in E. coli as a recombinant fusion protein with glutathione-S-transferase (GST). We report that the GST-OPN fusi on protein has functional activity. PAP2 (ras-transformed, metastatic murine NIH 3T3) and MDA-MB-435 human mammary carcinoma cells bound to GST-OPN in an in vitro cell adhesion assay nearly as well as to native bovine OPN. Adhesion to the recombinant fusion protein was blocked by addition of GRGDS peptide, suggesting that the cells adhere to the re combinant and native OPN proteins by similar, integrin-mediated mechan isms. Adhesion to both sources of OPN also was inhibited by thrombin t reatment of the protein. Thrombin cleaves GST from OPN in the fusion p rotein, and also cleaves internally in OPN, adjacent to the RGD sequen ce of the protein. Our results suggest that (a) thrombin cleavage of n ative OPN may be a natural regulator of OPN function, and (b) the majo rity of OPN cell binding activity is mediated by the RGD sequence in t he protein backbone, with little or no requirement for post-translatio nal modifications that occur in native OPN for adhesive function as me asured here. (C) 1994 Wiley-Liss, Inc.