Immunochemical studies of conformational alterations in bone .gamma.-carboxyglutamic acid containing protein

Abstract

The Ca2+-dependent transition of the vitamin K dependent bone protein bone Gla-containing protein (BGP) was investigated by use of anti-BGP antibody that reacts with the Ca2+-dependent conformation of BGP. Antibody binding occurred in the presence of Ca2+ or Mg2+ with a Kd(app) of 1.75 mM for Ca2+. Upon removal of Ca2+ with ethylenediaminetetraacetic acid, antibody binding was eliminated. Upon thermal acid decarboxylation of BGP, Ca2+-independent binding of the antibody was restored. Thus, the epitope not expressed by fully carboxylated BGP in the absence of calcium ion was restored either by addition of Ca2+ or by decarboxylation of the protein. Circular dichroic studies of fully carboxylated and fully decarboxylated BGP indicated that addition of Ca2+ to the fully carboxylated protein or decarboxylation to produce the glutamic acid containing equivalent of BGP resulted in increased order structure (apparent alpha-helix) in the protein, and this alteration was coincident with antibody binding. These data suggest that carboxylation of this vitamin K dependent protein may lead to increased disorder in the protein as compared to the glutamic acid containing equivalent. Upon Ca2+ binding a structure more equivalent to the Glu-containing protein is obtained.

The Three-Dimensional Structure of Bovine Calcium Ion-Bound Osteocalcin Using 1H NMR Spectroscopy

“…This suggests that the same hydrophobic interactions occur in the monomer and are intramolecular. Finally, CD spectra collected at protein concentrations where the protein is predominantly monomeric (50 µM) indicated that approximately 20% percent of the protein is in an α-helical conformation (23). This finding is in agreement with that determined from the structure in the present study (24% α-helical) at concentrations where the dimer predominates.…”

“…Our study is in general agreement with the pieces of structural information on osteocalcin reported previously. Data obtained from CD and 1D NMR studies showed that Ca 2+ -induced an α-helical conformation in 15-34% of the protein and shifted aromatic and aliphatic side chain resonances (21)(22)(23)54). Fluorescence, ultraviolet CD, and UV absorption studies showed an alteration in the environment of tyrosine and phenylalanine residues, suggesting decreased exposure to polar solvent (19).…”

“…Most likely, after it is secreted from the osteoblast, it is mineral bound and in the monomeric form. [21][22][23]54). Fluorescence, ultraviolet CD, and UV absorption studies showed an alteration in the environment of tyrosine and phenylalanine residues, suggesting decreased exposure to polar solvent (19).…”

Structure, Activity, and Distribution of Fish Osteocalcin

“…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.…”

“…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).…”