Homotypic interactions of soluble and immobilized osteopontin.

Abstract

Osteopontin (OPN) is an extracellular matrix protein found in bones and teeth, where it accumulates at matrix-matrix interfaces. We postulate that OPN interacts homotypically and heterotypically in the adhesion of apposing matrices. Using suspensions of OPN-coupled aldehyde/sulfate latex spheres, we measured the strength of homotypic OPN-OPN binding in vitro. Doublets formed through shear-induced collisions in a cone and plate rheoscope were subjected to shear stresses >0.6 Nm(-2) and the fraction broken up determined over 60 s. Rapid initial breakup of 35% of doublets was followed by very slow breakup of the remaining 65%. Monte Carlo simulation of the breakup kinetics pointed to the existence of low and high bond strength populations of doublets. Dynamic light scattering spectroscopy of soluble OPN showed that 27% by mass existed as dimers. We postulate that OPN dimers binding to monomers account for the low strength bonds since a strong bond has already formed between the molecules of the dimer. In contrast, OPN-OPN monomer bonds had higher tensile strength than bonds between the high-affinity interaction of IgG and protein G, previously studied. Antibody blocking studies showed that the self-binding region of OPN resides in the C-terminus. These data suggest that homotypic OPN-OPN bonds have physiologically significant strength, supporting the hypothesis that OPN-OPN binding and self-assembly participate in adhesion within mineralized tissues.