| Osteopontin (OPN) is an arginine-glycine-aspartate (RGD)-containing, secreted, phosphorylated glycoprotein that is ubiquitously expressed and is fairly highly conserved among vertebrates. Numerous studies have been reported showing the protein function to be multifaceted. It is found in every body fluid examined and high levels of OPN can be found in bone, kidney, and the mammary gland. It is produced in abundance by epithelial cells and macrophages. Of particular interest, OPN is highly expressed in a number of pathological settings and during tumorigenesis. There is evidence that different isoforms of OPN may perform different functions in the context of homeostasis and pathology. It would be of great benefit to gain a better understanding of the structure/function relationship of OPN, particularly, if posttranslational modifications modulate OPN function. In my research, I have created mouse anti-OPN monoclonal antibodies employing OPN−/− mice. These mice mount a strong immune response to immunization with OPN. Monoclonal antibodies created from spleen cells derived from these mice recognize OPN from multiple species with high specificity and affinity. Characterization of these monoclonal antibodies shows that they recognize amino acid sequences that are well conserved among multiple mammalian species. A modified immunoprecipitation-based phage technique was developed to rapidly identify epitopes utilizing the display of OPN gene fragments. Uses for these monoclonal antibodies include immunoassays such as western blotting, ELISA and immunohistochemistry. Examination of OPN produced by mouse osteoblasts and mouse fibroblasts with two of these monoclonal antibodies, AKm2A1 and AKm3D9, showed differential posttranslational modification. Two monoclonal antibodies, AK1H3 and AKm2C5, both significantly inhibited OPN-dependent cell adhesion. Collaborative studies have demonstrated the ability of antibody AKm2C5 to inhibit OPN-dependent osteocalcin gene up-regulation and enhance collagenase 3 expression and that antibody AKm1G4 recognizes an intracellular form of the protein.;Examination of the posttranslational modification state of these different forms of OPN, utilizing mass spectroscopy of tryptic peptides, continues in a collaborative effort to further delineate the precise modification differences. Future applications of these antibodies, including possible therapeutic applications are discussed. |
