1. Anti-OPN Monoclonal Antibodies as Probes of OPN Structure and Function Christian C. Kazanecki, Josephine Cassella, Yao Li, Cassandra Louis, Tanya Gordonov, Esben S. Sørensen and David T. Denhardt Results ABSTRACT: OPN, a primarily secreted protein found in all body fluids, is post-translationally modified; there is also an intracellular form. It is functionally important in bone remodeling, the progression of autoimmune disease, cancer metastasis, inflammatory processes and stress responses. Our research has focused on how OPN interacts with cells and the consequences of these interactions. We have used MAbs to known epitopes in the protein and peptides representative of functional sequences in OPN to shed light on how OPN signals cells and the consequences of those signaling events. In this work we present the properties of some novel anti-OPN MAbs and we describe studies that suggest a possible interaction of the C- terminus of the protein with the central RGDSVVYGL sequence important in signal transduction pathways. Methods to characterize the MAbs ELISA: Performed to see how well an antibody binds to modified (native) and non-modified (recombinant) forms of OPN adherent to a plastic surface WESTERN BLOTTING: Used to test whether an MAb binds to native or recombinant OPN bound to a PVDF membrane. PEPTIDE ASSAY: Tests which specific 18-mer OPN peptide can bind an MAb Similar to an ELISA, only wells are coated with a peptide via an N- terminal biotin-GG linker Peptides cover the entire OPN protein in overlapping pieces. This reveals the approximate location of the epitope to which the MAb binds. OVERVIEW: Our characterization of the newer MAbs has used uncloned hybridomas because of our desire to identify MAbs that might not be effectively detected by Western Blotting or ELISAs (because the relevant epitope was bound to the plastic or PVDF membrane). Thus we use two additional screening methodologies to detect antigen-antibody associations free in solution (a peptide assay and a magnetic bead assay). We use both native (modified) and recombinant (unmodified) OPN to identify MAbs that specifically recognize either the native or the recombinant form. Antibodies specific only for the native form would be candidates for MAbs recognizing an epitope that is post-translationally modified; antibodies specific only for the recombinant form would presumably recognize peptide sequences that are post- translationally modified. The peptide assay makes use of peptides that collectively cover the entire OPN molecule with 2-4 amino acid overlaps; in some cases both a phosphorylated peptide and its non- phosphorylated counterpart were used. These peptides possess a biotin at the C terminus coupled to the peptide by two glycine residues; via the biotin, the peptides bind to the surface of neutravidin-coated wells in a 96-well format. The magnetic bead assay employs two different forms of OPN, the highly post-translationally modified native OPN purified from human milk and the unmodified form synthesized in E. coli. In this novel assay the two forms (differentially labeled) are mixed and allowed to react in solution with the a specific MAb. The antigen-antibody complexes are then captured on magnetic beads coupled to protein G. After washing the beads, the adsorbed IgG is eluted at low pH, and the fluorescence at the two wavelengths determined. MAbs that selectively bind the native protein are presumed to be antibodies that recognize post-translational modifications. We believe this novel method is important in that it detects the antibody-antigen reaction in solution. This is in contrast to the methods currently in use which entail the immobilization of the antigen on a plastic surface (ELISA) or a membrane (Western blotting). Acknowledgements This research was supported by funds from a Busch Biomedical Research Award, the Rutgers Technology Commercialization fund, and by a grant from the National Multiple Sclerosis Society. The support of Aresty Summer Fellowships to Tanya Gordonov and Cassandra Louis are gratefully acknowledged. During portions of this study Yao Li received support from a SURF Fellowship and Christian Kazanecki from an IGERT Fellowship. We thank Dr. Larry Steinman for many generous contributions including the peptides used in this research. MAGNETIC BEAD ASSAY: Recombinant and native OPN are differentially labeled and allowed to react with an MAb. ProteinG coupled to magnetic beads is then used to extract the MAb from the reaction. The ratio of the fluorescent signals indicates whether the MAb binds to rec or nat OPN (or both or neither). Monoclonal antibody recognition of OPN. Western blotting results showing antibody recognition of murine OPN. Conditioned media from various cell lines or 50 ng of recombinant murine OPN (GST-mOPN) were separated on 12% SDS-PAGE gels and transferred to PVDF membranes. The membranes were then cut into strips which were blotted with MAbs at 1  g/ml or polyclonal control at a 1:3000 dilution. 275-3- 2: ras-tranformed murine fibroblast cell line. 275: non-transformed murine fibroblast 3T3 cell line. MC3T3E1: pre-osteoblast cell line induced to differentiate for 12 days. Antibody inhibition of cell adhesion to recombinant human OPN. 96-well plates were coated with 150  M human recombinant his-tagged OPN, then blocked with 1% BSA. Antibodies were then added at 125  M and allowed to bind OPN for 2 hr. The wells were then washed and 5x10 4 275-3-2 cells were added and allowed to adhere for 3.5 h. Non- adherent cells were removed by washing and adherent cells were quantitated by staining with crystal violet. Data are representative of two independent experiments for the 275-3-2 cell line (n=4). *, p<0.001 Student’s t test. FROM: Characterization of Anti-Osteopontin Monoclonal Antibodies: Binding Sensitivity to Post-Translational Modifications. Christian C. Kazanecki, Aaron J. Kowalski, Tony Ding, Susan R. Rittling, and David T. Denhardt (JCB, in press) The post-translational modifications of OPN include phosphorylation, glycosylation, and cross-linking by transglutaminase. Here we describe the generation of unique monoclonal antibodies raised against recombinant OPN utilizing the OPN knock-out mouse. The antibodies exhibit differential binding to OPN produced by different cell lines. Most of the antibodies generated recognize OPN produced by ras-transformed mouse fibroblasts, however only one antibody recognizes the more phosphorylated protein produced by the differentiating pre-osteoblast murine cell line MC3T3E1. Using a novel biopanning procedure combining T7 phage gene fragment display and protein G precipitation, we have epitope-mapped these antibodies. Several of the antibodies bind to regions of the OPN molecule that are phosphorylated, and one binds the region of OPN that is glycosylated. Using phosphorylated and non-phosphorylated peptides, we show that the binding of two antibodies to the C-terminal end of OPN is inhibited by phosphorylation of this region. In addition, these two antibodies are able to inhibit cell adhesion to recombinant and weakly modified OPN. The sensitivity of these antibodies to PTMs suggests that caution must be taken when choosing anti-OPN monoclonal antibodies to detect this highly modified protein. SHOW EXAMPLES WITHOUT THE TEXT Representative data MODEL OF OPN FOLDING From Kazanecki, Uzwiak and Denhardt, in press Putative binding sites for some of the MAbs.