In continuation of our past research interests, we plan to carry out an integrated series of experiments designed to determine the relationship between the structures of vitamin K-dependent bone proteins and their functions in bone metabolism. Our efforts will be primarily focused on bone Gla protein (BGP; osteocalcin), an abundant bone protein typically 50 residues in size, and matrix Gla protein (MGP), a larger protein we discovered just 2 years ago. We plan to determine the complete primary structure of MGP from species, such as rat and man, which have the greatest relevance for bone research. Our sequencing strategy is based on our recently completed sequence of bovine MGP, a 79-residue protein which has 5 residues of Gamma-carboxyglutamic acid (Gla). We also intend to obtain crystals of BGP suitable for X-ray diffraction analysis and to isolate and characterize other novel, Gla-containing bone proteins. We will carry out a series of in vitro experiments to establish the relationship between the structures of BGP and MGP and their ability to bind to hydroxyapatite and to inhibit mineralization. These studies will include tests of the possible role in hydroxyapatite binding of homologous structures within the Gla-containing region of these closely related proteins, such as the pentapeptids Gla domain (Gla-X-X-Arg-Gla) and the recurring tripeptide Gla-Leu-Asn. We will also determine the nature of the binding interaction which apparently exists between MGP and the organic matrix of bone. Our primary system for the analysis of vitamin K-dependent bone protein function will be the Warfarin treated rat, a system which we have shown reduces BGP levels in bone to 3% of normal. We plan to further characterize the accelerated mineralization disorders which we have identified in those animals, to develop a bone organ culture system in which to further study this effect, and to evaluate the ability of BGP and MGP to restore normal mineralization rates. We will also examine other tissues, such as fracture callus, for evidence of accelerated mineralization due to Warfarin treatment and will test bone from such animals for possible evidence that accelerated mineralization reduces collagen cross-linking. Finally, we will explore the hypothesis that MGP functions in the bone morphogenetic response. This hypothesis is based in part on the observation that the bone morphogenetic protein is isolated from bone by virtue of its co-precipitation with MGP upon arrival of the denaturants which are needed to dissolve MGP.
