No abstract

Osteocalcin (bone Gla protein) is an extracellular matrix protein synthesized by osteoblasts that is a marker of bone. Osteocalcin probably originated in the ancestors of Teleostei or bony fish and of the Tetrapoda or amphibians, reptiles, birds, and mammals. We have characterized the Cyprinus carpio (carp) osteocalcin for mineral binding to hydroxyapatite, amino acid sequence, and extent of secondary structure. Hydroxyapatite binding is enhanced in the presence of calcium. The ␣-helical content of teleost osteocalcin increases and ␤-sheet structure decreases upon calcium binding, similar to findings in calf osteocalcin. The gene structure and primary sequence of prepro-osteocalcin from 2 pufferfish compared with carp shows that there are many conserved features in teleost osteocalcin genes. Using an immunoassay for carp osteocalcin, we determined that the relative content of osteocalcin is highest in dorsal fin spines and other bones and lowest in scales. The carp osteocalcin antibodies, cross-reactive to other species of fish, were used to study the role of osteocalcin in teleost model systems.Bone contains a vitamin K-dependent protein containing ␥-carboxyglutamic acid (Gla) 1 called osteocalcin (Oc) or bone Gla protein (1, 2). Bony fishes (Teleostei) and land vertebrates (Tetrapoda) contain the protein Oc (2). Protein sequence comparisons reveal the highest sequence conservation in the Glacontaining domain (3, 4). Gla and the conformation conferred by the Gla domain appear necessary for Oc to bind to hydroxyapatite (HA) (5, 6). Calcium binding causes a conformational change that coincides with increased affinity for hydroxyapatite (7,8).Bone mineral binding, tissue distribution, and structural studies of osteocalcin have been performed almost exclusively on proteins from mammals and birds (Tetrapoda). Bony fish (Teleostei) are the most successful organisms in aquatic environments. The common ancestor of tetrapods and teleosts evolved bone over 200 million years ago and a comparison of Oc in the two orders can provide insights into the evolution of Oc structure and function. For example, the amino acid sequences of most tetrapods contain a conserved C-terminal RRFYGPV sequence that is missing in teleosts, while a truncated N terminus and extended C terminus are missing in tetrapods (3, 4).The present studies of Cyprinus carpio osteocalcin and sequences derived from other fish genome sequences (Fugu rubripes and Tetraodon nigroviridis) confirm the primary structure differences observed in osteocalcins of tetrapod and teleost. A higher proportion of carp Oc is ␣-helical when compared with the calcium-free bovine Oc. The studies also characterize mineral binding behavior and tissue distribution of teleost Oc using a C. carpio Oc radioimmunoassay. EXPERIMENTAL PROCEDURESPurification of Osteocalcin-Carp (C. carpio) bones were removed and cleaned of adherent tissue after placing in boiling water for 1-1.5 min as described previously (3). Bones were broken into 8 -125-mm 3 pieces, water washed for 20 min, then lyoph...

Background. Matrix Gla protein (MGP) is a vitamin K-dependent, extracellular matrix protein. MGP is a calcification inhibitor of arteries and cartilage. However MGP is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells. The presence of MGP in those instances does not correlate well with the calcification inhibitory role. This study explores a potential mechanism for MGP to bind to matrix proteins and alter cell matrix interactions. Methods. To determine whether MGP influences cell behavior through interaction with fibronectin, we studied MGP binding to fibronectin, the effect of MGP on fibronectin mediated cell attachment and spreading and immunolocalized MGP and fibronectin. Results. First, MGP binds to fibronectin. The binding site for MGP is in a specific fibronectin fragment, called III1-C or anastellin. The binding site for fibronectin is in a MGP C-terminal peptide comprising amino acids 61–77. Second, MGP enhances cell attachment and cell spreading on fibronectin. MGP alone does not promote cell adhesion. Third, MGP is present in fibronectin-rich regions of tissue sections. Conclusions. MGP binds to fibronectin. The presence of MGP increased cell-fibronectin interactions.