Structural Biology of Calcium Phosphate Nanoclusters Sequestered by Phosphoproteins

Abstract

Biofluids that contain stable calcium phosphate nanoclusters sequestered by phosphopeptides make it possible for soft and hard tissues to co-exist in the same organism with relative ease. The stability diagram of a solution of nanocluster complexes shows how the minimum concentration of phosphopeptide needed for stability increases with pH. In the stable region, amorphous calcium phosphate cannot precipitate. Nevertheless, if the solution is brought into contact with hydroxyapatite, the crystalline phase will grow at the expense of the nanocluster complexes. The physico-chemical principles governing the formation, composition, size, structure, and stability of the complexes are described. Examples are given of complexes formed by casein, osteopontin, and recombinant phosphopeptides. Application of these principles and properties to blood serum, milk, urine, and resting saliva is described to show that under physiological conditions they are in the stable region of their stability diagram and so cannot cause soft tissue calcification. Stimulated saliva, however, is in the metastable region, consistent with its role in tooth remineralization. Destabilization of biofluids, with consequential ill-effects, can occur when there is a failure of homeostasis, such as an increase in pH without a balancing increase in the concentration of sequestering phosphopeptides.

Binding Mode Characterization of Osteopontin on Hydroxyapatite by Solution NMR Spectroscopy

“…These described binding modes of OPN(p) are in accordance with their reported differing roles in biomineralization. OPNp has been shown to be a natural inhibitor of calcification and mineral formation, growth and proliferation, [19,21,22,32,35] and to prevent crystal formation in soft tissues and biological fluids [25,28,32] . Whereas the non‐phosphorylated form always has shown less or no effect (e. g. recently, a weak binding of non‐phosphorylated quail OPN on HAP crystal surfaces has only mildly affected the crystallite properties [63] ).…”

“…OPNp has been shown to be a natural inhibitor of calcification and mineral formation, growth and proliferation,[ 19 , 21 , 22 , 32 , 35 ] and to prevent crystal formation in soft tissues and biological fluids. [ 25 , 28 , 32 ] Whereas the non‐phosphorylated form always has shown less or no effect (e. g. recently, a weak binding of non‐phosphorylated quail OPN on HAP crystal surfaces has only mildly affected the crystallite properties [63] ). This difference in function becomes clear by considering the elongated structure (in the apo and HAP‐bound state) of OPNp binding over the full length hence covering a larger surface area.…”

“…However, the different binding modes may explain the distinct biological functions of OPN and OPNp during osteogenesis and biomineralization: the rather “covering” binding mode of OPNp may explain its function as a mineralization inhibitor through physically blocking the mineral surface from further growth[ 19 , 21 , 23 , 32 ] and its property to stabilize transient pseudo‐coacervate phases of calcium phosphate. [ 23 , 25 , 28 , 29 ]…”

“…It was reported to be important in the differentiation and recruitment of osteogenic cells, especially in the interaction with osteoclasts,[ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ] and as an inhibitor of hydroxyapatite (HAP; Ca 10 (PO 4 ) 6 (OH) 2 ; principle component of bone: approx. 65 wt%) formation, growth and proliferation,[ 21 , 22 , 23 , 24 , 25 ] whereas an OPN deficiency can cause dystrophic calcifications in mice and humans and a decline in fracture/bone toughness. [ 19 , 22 , 26 , 27 ] In recent studies OPN was found to stabilize a transient pseudo‐coacervate phase of calcium phosphate in a saturated solution, enabling the entrance of these droplets into the collagen fibers and thus promoting intrafibrillar mineralization, and finally moderating extrafibrillar mineral coating.…”

Effect of pentasodium triphosphate concentration on physicochemical properties, microstructure, and formation of casein fibrils in model processed cheese

“…The amount of insoluble calcium from processed cheese samples-that is, calcium from residual CaP nanoclusters that were sequestered by the phosphoserine clusters on the caseins (Lenton et al, 2020)-was determined via the acid-base titration method described in Wolfschoon-Pombo and Andlinger (2013), which is an adaptation of the standard titrimetric method by Hassan et al (2004). Fifteen grams of processed sample and 120 mL of prewarmed (50°C) sodium hydroxide (3.3 mM) were mixed using the Ultra-Turrax dispersing instrument at 10,000 rpm for 2 min.…”