Hauschka Laboratory - Bone Cell Biology
In 1974-5, we discovered and named osteocalcin, a unique and abundant protein of mineralized bone. This small vitamin K-dependent Ca2+-binding protein, 49-aa residues long in humans, employs 3 gamma-carboxyglutamic acid ("Gla") residues for tight adsorption to hydroxyapatite mineral surfaces in bone matrix. Osteocalcin has highly conserved structural motifs (Gla-helix, b-turns, Ca2+-binding sites) that were inferred from physicochemical studies in solution, amino acid sequence comparison among vertebrate species, and predictive algorithms. The recent x-ray diffraction structure [Hoang QQ et al Nature 425: 977 (2003)] confirms and extends the understanding of osteocalcin and its interaction with bone mineral.
Gla is invariably synthesized by vitamin K-dependent, enzyme catalyzed posttranslational carboxylation of specific Glu residues. Proof of Gla in osteocalcin expanded the known role of vitamin K beyond the hepatic synthesis of Gla blood coagulation factors. Unexpected tissue sources of Gla-containing proteins now include: bone, cartilage, dentin, cementum, kidney, pancreas, lung, blood vessel walls, tumor cells, and even venom ducts of invertebrate marine cone snails, where a subset of conotoxins share the Gla a-helix first described in osteocalcin.
Osteocalcin protein sequence studies may provide a unique window into recent vertebrate evolution. Sensitive radioimmunoassay detects undegraded osteocalcin in bone samples of modern and extinct species approaching 500,000 years of age, where intact DNA is absent. With paleobiologists at Michigan State Univ. and in the U.K. we recently sequenced the first intact protein (osteocalcin) recovered from bone of an extinct species, the steppe bison, Bison priscus (Nielsen-Marsh CM, et al 2002).
Osteocalcin comprises about 10% of the total non-collagen proteins in bone. Osteocalcin is approximately stoichiometric to the number of collagen molecules and hydroxyapatite-like nanocrystals with which it associates in the microarchitecture of bone.
Osteocalcin studies in developing embryos and bone cell models have validated its high specificity as a marker for the mature osteoblastic phenotype. The osteocalcin gene is the most thoroughly studied of all bone-specific genes, serving as a model for regulation by 1,25 (OH)2-vitamin D3, glucocorticoids, growth factors, and the transcription factors Runx2/Cbfa1 and Osterix. Our studies of osteocalcin are currently focused on analysis of functional domains for protein-protein interactions and a search for osteocalcin-dependent signaling pathways and cellular processes.