INHIBITION OF ARG-GLY-ASP (RGD)-MEDIATED CELL-ADHESION TO OSTEOPONTINBY A MONOCLONAL-ANTIBODY AGAINST OSTEOPONTIN

Osteopontin (OPN), a secreted phosphoprotein, has been implicated in v arious biological phenomena (e.g. bone development, sepsis, tumor prog ression, and metastasis). Its role in any context is poorly understood . OPN contains a conserved Gly-Arg-Gly-Asp-Ser (GRGDS) sequence, and b inds to cells via integrin-mediated mechanisms. Using recombinant huma n osteopontin-glutathione S-transferase fusion protein and our improve d hybridoma fusion partner (Sp2/mIL6), we raised murine monoclonal ant ibodies against osteopontin. We characterized two antibodies that reco gnize not only recombinant but also native human osteopontin. These an tibodies do not cross-react with mouse osteopontin (recombinant protei n or that secreted by ras-transformed NM 3T3 cells), or bovine bone os teopontin, suggesting that they recognize epitopes unique to human OPN . One antibody specifically inhibited adhesion of MDA-MB-435 human bre ast cancer cells and ras-transformed NIH 3T3 cells to human osteoponti n. This antibody failed to recognize osteopontin cleaved by thrombin, which cleaves adjacent to the cell binding domain. We previously showe d that thrombin cleavage reduces osteopontin cell binding activity. Th us we postulate that this monoclonal antibody recognizes and interfere s with the function of the RGD/thrombin cleavage region of human OPN.