TY - JOUR TI - Osteopontin and cell adhesion: role of post-translational modifications and the C-terminal region DO - https://doi.org/doi:10.7282/T3GF0TXZ PY - 2007 AB - Osteopontin (OPN) is an integrin-binding secreted phosphoglycoprotein found in many tissues and body fluids, where it acts as both a soluble cytokine and a component of mineralized matrices. It exhibits a broad range of functional activities in physiological and pathological processes, many of which are influenced by post-translational modifications. OPN is highly modified by phosphorylation and glycosylation that can vary depending on the source. This study identified two different forms of OPN produced by murine ras-transformed fibroblasts (FbOPN) and differentiating osteoblasts (ObOPN) based on the reactivity of a panel of anti-OPN monoclonal antibodies. By enzymatic dephosphorylation, mass, and sequence analyses, it was shown that FbOPN contained approximately four phosphate groups distributed over 17 potential phosphorylation sites, whereas ObOPN contained approximately 21 phosphate groups distributed over 27 sites. This is the first extensive characterization of OPN produced by transformed cells, and the FbOPN form is the least modified form of OPN that has been characterized to date. The forms of OPN also differed in their ability to support adhesion of human MDA-MB-435 tumor cells and mouse 275-3-2 ras-transformed fibroblasts. The MDA-MB-435 cells bound with approximately 60% greater efficiency to the FbOPN than ObOPN, whereas the 275-3-2 cells bound the ObOPN with 35% greater efficiency than FbOPN. Inhibition of adhesion with an Arg-Gly-Asp (RGD)-containing peptide inhibited the binding of both lines to the OPN forms, suggesting that different integrins preferentially bind to OPN based upon its phosphorylation state. This study has also identified a potential role for a novel region of OPN in cell adhesion. Two anti-osteopontin monoclonal antibodies, both of which recognize the C-terminal region of OPN, were shown to inhibit MDA-MB-435 cell adhesion to recombinant human OPN. Synthetic peptides corresponding to this region were bound by cells using a flow cytometry assay, suggesting a receptor interaction in this region of OPN. This interaction is hypothesized to be a signaling interaction because cells were not able to adhere to the synthetic C-terminal peptides and adhesion in the presence of these soluble peptides was not affected. KW - Microbiology and Molecular Genetics KW - Osteopontin KW - Cell adhesion molecules KW - Phosphoproteins KW - C-peptide LA - English ER -