UC San Diego

The purpose of this dissertation was: 1) to biochemically characterize how purified matrix Gla protein (MGP) functions as a calcification inhibitor using two novel in vitro calcification assays; 2) to characterize MGP's binding to apatite mineral; and 3) to characterize MGP purified from bone, cartilage, and serum using mass spectrometry. Previous genetic and biochemical studies of MGP have convincingly demonstrated that impaired MGP function results in extensive calcification of the elastic lamellae in the artery media of mice, rats, and humans. Very few studies of purified MGP have investigated its role as an inhibitor of calcification. In this dissertation different amounts of purified MGP were added to a serum-initiated calcification assay containing devitalized arteries and demineralized tibias incubated in 5% serum. These experiments showed that calcification was abolished at 30 micrograms/mL MGP, and significantly reduced at 10 micrograms/mL MGP. Additional experiments demonstrated that MGP inhibited the initial formation of crystal nuclei in the elastin or collagen matrix as well as the subsequent growth of these nuclei. The inhibitory effect of MGP was also investigated on mineral formation in vitro in a solution of high ionic calcium and phosphate, higher concentrations than what are typically found physiologically, at neutral pH. Concentrations as low as 10 micrograms/mL of MGP in solutions of 4 mM ionic calcium and phosphate were able to completely inhibit the formation of a calcium phosphate mineral phase over a week long incubation. Binding studies showed that MGP had a stronger affinity for nascent calcium phosphate mineral than for hydroxyapatite. MGP's stronger affinity for nascent mineral supports the hypothesis that this mineral may more closely resemble the apatite found in vivo. Finally, mass spectrometry studies revealed that the degree of phosphorylation in MGP depended upon the tissue from which the protein is purified from. The data presented in this dissertation provide for the first time evidence of how purified MGP acts as an inhibitor of calcification and establishes that MGP accomplishes this function through direct interaction with calcium phosphate mineral