Abstract
Osteocalcin, the most abundant noncollagenous protein of bone matrix, has been demonstrated to inhibit bone growth by gene knockout experiments (Ducy, P., Desbois, C., Boyce, B., Pinero, G., Story, B., Dunstan, C., Smith, E., Bonadio, J., Goldstein, S., Gundberg, C., Bradley, A., and Karsenty, G. (1996) Nature 382, 448-452). Its specific functional mechanism in bone metabolism is, however, largely unknown. In this study, we provide evidence that osteocalcin has an inhibitory effect on tissue transglutaminase activity, as measured by cross-linking of osteopontin, another bone matrix protein. Using a set of synthetic peptides, we found that the inhibitory activity resided within the first 13 N-terminal amino acid residues of osteocalcin. An N-terminal peptide also inhibited cross-linking of another tissue transglutaminase substrate, beta-casein. The inhibitory peptide was shown to have affinity for the substrates of transglutaminase rather than for the enzyme. Since the N terminus of osteocalcin exhibits homology to the substrate recognition site sequences of two transglutaminases, we conclude that the inhibitory effect is most likely due to competition with the enzyme for the transglutaminase-binding region of the substrates, osteopontin and beta-casein, which prevents access of the enzyme to them to perform its function. The interference of osteocalcin with osteopontin cross-linking gives osteocalcin a new potential function as the first protein inhibitor of tissue transglutaminase. This suggests a specific role and a plausible mechanism for it as a modulator of maturation, stabilization, and calcification of bone matrix.
Highlights
One of the most abundant noncollagenous proteins of adult bone is osteocalcin (OCN),1 which is a small osteoblast-specific calcium-binding protein of 46 –50 amino acid residues, containing three vitamin K-dependent Gla residues [1, 2]
OCN is known to interact with osteopontin (OPN) in vitro [7]
OPN is a substrate of tissue transglutaminase (TG) [17,18,19]
Summary
Bone is osteocalcin (OCN), which is a small osteoblast-specific calcium-binding protein of 46 –50 amino acid residues, containing three vitamin K-dependent Gla residues [1, 2]. The gene knockout experiments by Ducy et al [6] have demonstrated that mice lacking OCN develop bones with increased mass and strength This implies the importance of OCN in promoting bone resorption and inhibiting mineralization. TG is produced in mineralizing cartilage and bone [22, 23], and it is thought to participate in matrix cross-linking before the tissue undergoes calcification [21] It might be involved in the initiation and regulation of the mineralization processes [22]. We provide evidence that OCN reduces the formation of TG-catalyzed high molecular mass complexes of OPN most likely by competing Osteocalcin, an Inhibitor of Transglutaminase Activity with the enzyme for the binding site of the protein substrate by a sequence homologous to tissue transglutaminase
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