Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage

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.

Secreted-Osteopontin Contributes to Brown Adipogenesis In Vitro via a CD44-Dependent Pathway

“…PRDM16 is identified as a switch of the browning program, which cooperates with PGC-1α, peroxisome proliferator activated receptor γ (PPARγ), and PPARα to induce the expression of brown adipocyte-specific genes [16]. Osteopontin (OPN), a secreted multifunctional glycoprotein, is originally derived from skeleton and similar to bone morphogenetic proteins (BMPs ) in bone development and differentiation [17]. OPN exists as 2 forms in mammals, namely secreted-OPN (sOPN) and intracellular-OPN (iOPN).…”

Peptide-based vaccination against OPN integrin binding sites does not improve cardio-metabolic disease in mice

“…by attacking different vital proteins. Interestingly, another study exhibited OPN-specific antibodies after active immunization with human OPN-derived peptides in the DIO model (WT mice) [44]. Concerning these results, the question is raised whether naïve lymphocytes specific for the original (murine) central OPN region-exist or whether T- and/or B-cells will be eliminated by negative depletion.…”

Antibody-mediated targeting of cleavage-specific OPN-T cell interactions

“…In 3 independent experiments, 10 6 primary human T cells from 3 donors were activated with 2 μg/ml PHA (Sigma, Darmstadt, Germany) in serum free RPMI medium and stimulated with 37.5 nM, 75 nM, 150 nM and 300 nM recombinant full length (FL)-OPN, Thr-cOPN, MMP-cOPN [20]. Additionally, GST-OPN-CTF (C-terminal fragment) was tested in one experiment using T cells from 3 donors.…”

“…Monoclonal antibodies recognizing human OPN were produced as previously described [20]. Antibody 9–3 was raised against peptide C-GDSVVYG while antibody 21–5 was raised against peptide C-TYDGRGDSVVYG-CO-NH 2 .…”

“…Peptide 1, C-TYDGRGDSVVYG-CO-NH2 represents the C-terminus of MMP-cOPN, peptide 2, C-VVYGLR-COO, represents the C-terminus of Thr-cOPN, peptide 3, C-RGDSVVYG-COO, represents the C-terminus of the MMP-cOPN and a scrambled control peptide from the whole of the region, C-SGRVYGDLVGRD-CO-NH2, were synthesized (EMC microcollections, Tübingen, Germany). Vaccination experiments were performed[20] by coupling the peptides to KLH and injecting the antigens into C57BL/6J mice (30 μg peptide, subcutaneously) at biweekly intervals with alum (Brenntag, Mülheim, Germany) as an adjuvant. 2 weeks after the fourth vaccination, mice were sacrificed and the postimmune serum was analyzed.…”