Abstract
Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.
Highlights
Osteopontin (OPN), known as secreted phosphoprotein 1, is encoded by the gene SPP1 and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) familyPLOS ONE | DOI:10.1371/journal.pone.0148333 February 3, 2016Inhibition of Cellular Adhesion to Osteopontin Neoepitopes any additional role in the study design, data collection and analysis, or decision to publish
Integrin β1 containing integrins–depending on the combined α chain–are able to bind both binding motifs and β1 expression is high in HEK 293 (Fig 2A), adipose tissue (AT) endothelial cells (Fig 2B), AT preadipocytes (Fig 2C), and AT immune cells (Fig 2D)
HEK 293 cells that express RGD- and SVVYGLR-binding integrins appear suitable for investigation of cellular adhesion to both, full length OPN (flOPN), to which only RGD-dependent integrins can bind, and the OPN fragments mOPN and tOPN that feature the unmasked SVVYG or SVVYGLR integrin binding sites, respectively
Summary
Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We aimed to test whether the functionality of antibodies induced by peptide vaccination in vivo was comparable to the functionality of our purified monoclonal antibodies
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have