title: 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.
authors.affiliation: Department of Medicine, Montreal General Hospital, Montreal, QC, Canada.
authors.forename: H L
authors.initials: HL
authors.lastname: Goldsmith
authors.forename: J M
authors.initials: JM
authors.lastname: Labrosse
authors.forename: F A
authors.initials: FA
authors.lastname: McIntosh
authors.forename: P H
authors.initials: PH
authors.lastname: Mäenpää
authors.forename: M T
authors.initials: MT
authors.lastname: Kaartinen
authors.forename: M D
authors.initials: MD
authors.lastname: McKee
mesh_terms: D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D002449:Cell Aggregation; D003198:Computer Simulation; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D046911:Macromolecular Substances; D051379:Mice; D008863:Microspheres; D008892:Milk / Q000737:chemistry; D008956:Models, Chemical; D008970:Molecular Weight; D009010:Monte Carlo Method; D053495:Osteopontin; D011485:Protein Binding; D012212:Rheology / Q000295:instrumentation / Q000379:methods*; D012795:Sialoglycoproteins / Q000737:chemistry* / Q000378:metabolism; D013314:Stress, Mechanical
chemical_list: D046911:Macromolecular Substances; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin