The objective of this research is to characterize the process of membrane-mediated biologic calcification. This proposal tests the hypothesis that specific proteolipids structure phosphatidylserine and phosphoinositides so that interaction with Ca and Pi results in Ca-proteolipid-Pi (CaPrPX) and subsequent Ca-phospholipid-Pi (CPLX) formation and calcium hydroxyapatite (HA) deposition. Using proteolipid-dependent calcification of Bacterionema matruchotii as a model, this research will identify and characterize Ca-phosphate-phospholipid-protein interactions which facilitate mineral formation and their metabolic regulation. While calcifiable proteolipid contains 4 major protein species, only the MR 10,000 species is incorporated into CPLX, suggesting it has a specific and important role in HA formation. This research will biochemically characterize the MR 10,000 SDS-PAGE proteolipid of B. matruchotii by determining its relationship to other proteolipids in the parent extract via Western blots using anti-10K antibody, and by comparison of peptide maps. Amino acid composition, N- and C-terminal amino acids susceptibility to proteases, and bound and associated lipid will be analyzed. Proteoliposomes will be used to examine interaction of proteolipid and phospholipid to form a calcifiable domain. Calcifiability of individual proteolipid classes (10K proteolipid, HPLC purified proteolipids, parent extracts and proteolipids present in CaPrPX) will be assessed in vitro. Those which support CaPrPX, CPLX and/or HA formation will be further characterized with respect to structure. Proteoliposomes will also be used to assess whether calcifiable proteolipids have inophoretic activity. B. matruchotii cultures will be perturbed and the incorporation of radiolabeled precursors of proteolipid and associated lipid will be quantitated to assess metabolic regulation of CPLX formation and HA deposition. The validity of the model for eukaryotic membrane-mediated calcification will be assessed by analyzing concentration and composition of proteolipid, CaPrPX and CPLX In ectopic calculi and pathologic soft tissue calcifications. These studies have significance for understanding the basic mechanisms of membrane-mediated HA formation, whether it be normal regulated matrix vesicle calcification or unregulated pathologic mineral formation.
Showing the most recent 10 out of 13 publications
