Analysis and Molecular Modeling of the Formation, Structure, and Activity of the Phosphatidylserine-Calcium-Phosphate Complex Associated with Biomineralization

Abstract

The nucleational core of matrix vesicles contains a complex (CPLX) of phosphatidylserine (PS), Ca 2؉ , and inorganic phosphate (P i ) that is important to both normal and pathological calcification. Factors required for PS-CPLX formation and nucleational activity were studied using in vitro model systems and molecular dynamic simulations. Ca 2؉ levels required for and rates of PS-CPLX formation were monitored by light scattering at 340 nm, assessing changes in amount and particle size. Fourier transform infrared spectroscopy was used to explore changes in chemical structure and composition. Washing with pH 5 buffer was used to examine the role of amorphous calcium phosphate in CPLX nucleational activity, which was assessed by incubation in synthetic cartilage lymph with varied pH values. Addition of 4 Ca 2؉ /PS was minimally required to form viable complexes. During the critical first 10-min reaction period, rapid reduction in particle size signaled changes in PS-CPLX structure. Fourier transform infrared spectroscopy revealed increasing mineral phosphate that became progressively deprotonated to PO 4 3؊ . This Ca 2؉ -mediated effect was mimicked in part by increasing the Ca 2؉ /PS reaction ratio. Molecular dynamic simulations provided key insight into initial interactions between Ca 2؉ and P i and the carboxyl, amino, and phosphodiester groups of PS. Deduced interatomic distances agreed closely with previous radial distribution function x-ray-absorption fine structure measurements, except for an elongated Ca 2؉ -N distance, suggesting additional changes in atomic structure during the critical 10-min ripening period. These findings clarify the process of PS-CPLX formation, reveal details of its structure, and provide insight into its role as a nucleator of crystalline calcium phosphate mineral formation. One of the essential features of mineral-inducing matrix vesicles (MV)2 is the presence of a nucleation core (1, 2) that contains lipid-calcium-phosphate complexes (CPLX) capable of inducing mineral formation when incubated in a synthetic cartilage lymph (SCL) (3-6). A critical component of CPLX is the acidic phospholipid, phosphatidylserine (PS) (3,7,8), which is known to have a high affinity for Ca 2ϩ (9,10). PS is largely confined to the inner leaflet of the MV membrane (11), and electron micrographs of calcifying MV show the electrondense calcium phosphate precipitate juxtaposed along the inner leaflet of the MV membrane (12).Discovery of the nucleation core stems from the early finding that acidic phospholipids, especially PS, are complexed with Ca 2ϩ at sites of early mineralization (13,14). PS-Ca 2ϩ -P i complexes (PS-CPLX) are present at the early stages of almost all calcifying tissues as follows: growth plate cartilage (14), tumors (15), bone (16), and especially MV (8). Synthetic PS-CPLX formed in the absence of Mg 2ϩ has been found to be a powerful nucleator that rapidly induces hydroxyapatite (HA) formation when incubated in SCL (3, 4); however, when formed from Mg 2ϩ -and HCO 3 Ϫ -containing P i -r...

Mineralization of Annexin-5-containing Lipid-Calcium-Phosphate Complexes

“…Additionally, oxygen atoms from P i groups of ACP help fill the coordination sphere around Ca 2ϩ , which results in the typical 1:1:1 Ca 2ϩ :P i :PS molar ratio of PS-CPLX. This suggested arrangement of atoms is consistent with spacefilling molecular models and data from RDF-EXAFS analysis of PS-CPLX (71) and has now been confirmed by molecular dynamic simulation (70).…”

“…Thus, with probable alternate packing of PS and PE head groups, the PS:PE-CPLX formed on the membrane surface may be arranged by Anx-A5 to facilitate epitaxial growth of crystalline mineral formation. This hypothesis can now be directly tested using molecular dynamic simulation, similar to that used for visualizing the formation of PS-CPLX (70).…”

Gain-of-function mutations in the phosphatidylserine synthase 1 (PTDSS1) gene cause Lenz-Majewski syndrome

“…Nevertheless, phosphatidylserine and its calcium-binding properties have been recognized as being important in bone mineralization, physiological and pathological calcification and dentine formation 39,40 . These processes could well be affected by changes in phosphatidylserine metabolism in LMS, as they involve the rearrangement of apoptotic cell membranes and the release of phosphatidylserineenriched vesicles 40,41 , and are tightly regulated by the osteoblast, the chondrocyte and the odontoblast lineages, cells similar to the fibroblasts studied here.…”

Lipids in biocalcification: contrasts and similarities between intimal and medial vascular calcification and bone by NMR

“…70 ppm from the ␥ -carbon of hydroxyproline, the sample was resuspended in concentrated sodium hydroxide and sodium hypochlorite for another few weeks. This procedure was repeated enriched in acidic phospholipids, such as phosphatidylserine, that could coordinate calcium ions in the initial mineral nucleation stages ( 8,9 ), and they have been found to be loaded with mineralization-supportive proteins and other macromolecules ( 9 ).…”