Conformational analyses on soluble and surface bound osteopontin.

Abstract

Immunohistology of calvarial sections revealed that staining with monoclonal anti-osteopontin antibodies (clone MPIIIB10) is minimal unless sections are first treated with EDTA. In contrast, following treatment of sections with EDTA, strong staining of mineralizing osteoid areas and osteoblast-like cells was noted (Fig. 1B). Immunostaining for osteopontin appeared to be specific in that controls which substituted rabbit IgG or normal mouse ascites fluid for monoclonal antibody, or which omitted monoclonal antibody uniformly gave background results (Fig. 1C). In an effort to circumvent problems of antibody accessibility we examined the immunoreactivity of OP when adsorbed to plastic and hydroxyapatite surfaces. Although OP bound to plastic surfaces is reactive with MPIIIB10 antibodies, OP adsorbed to hydroxyapatite crystal surfaces is not recognized by these antibodies as assessed by two detection methods. These results demonstrate that most or all of OP bound to hydroxyapatite exhibits a different conformation than when bound to plastic surfaces. On the basis of immunohistologic results with calvarial sections, we suggest that the conformation of native OP in bone and of isolated OP adsorbed to hydroxyapatite may be similar. Finally, solution circular dichroism and Fourier-transformed infrared spectroscopic studies indicate that the conformation of bone OP is dependent upon its concentration, and, secondarily to the presence or absence of calcium ion. With both spectroscopic methods, addition of calcium appeared to increase the extent of disordered structure. We suggest that these findings support our hypothesis that bone matrix proteins exhibit a different conformation when adsorbed on hydroxyapatite crystal surfaces. Assumption of a more organized secondary structure in concentrated OP solutions (i.e., 15 mg/ml) is consistent with these results in that local concentrations of OP within a semisolid matrix may approach or exceed levels used here.