Synthesis of human osteocalcins: gamma-carboxyglutamic acid at position 17 is essential for a calcium-dependent conformational transition.

Human osteocalcins having two (positions 21 and 24) or three (positions 17, 21 and 24) gamma-carboxyglutamic acids (Gla) were synthesized in solution employing both the Boc strategy and the HF procedure for the final deprotection. During synthesis, the gamma,gamma-dicarboxyl functional groups of the Gla residues were protected by the cyclohexyl group. The identity of the synthetic peptides was confirmed by amino acid analysis, mass spectrometric measurement and peptide mapping. Circular dichroism measurement showed that the conformation of osteocalcin containing three Gla residues dramatically changed on addition of calcium ions, whereas the peptide containing glutamic acid at position 17 did not. These findings clearly show that the Gla residue at position 17 is essential for a calcium-dependent conformational transition of osteocalcin.