Role of Phosphate Groups in Inhibition of Calcium Oxalate Crystal Growth by Osteopontin

Osteopontin (OPN) inhibits the growth of calcium oxalate monohydrate (COM) and other crystal phases in a phosphorylation-dependent manner. In the present study, the role of OPN phosphate groups in adsorption to, incorporation into and inhibition of COM crystals was studied by comparing OPN isoforms differing in phosphorylation. OPN isoforms purified from rat bone (bOPN), which contains 10 phosphates, and cow milk (mOPN), which contains 25 phosphates, were compared with rat recombinant OPN (rOPN), which is not phosphorylated. Using fluorescence-labeled proteins and confocal microscopy, we show that mOPN and rOPN, like bOPN, adsorb preferentially to the edges between {100} and {121} faces of preformed COM crystals, and to a lesser extent to the {100} and {121} faces. Using scanning electron microscopy, we show that growth of COM in the presence of bOPN or mOPN results in a \u27dumbbell\u27 morphology, whereas crystals grown with rOPN are only slightly affected. COM crystals grown in the presence of low concentrations of fluorescence-labeled bOPN incorporate the protein into the crystal lattice. In crystals imaged in the {010} plane, incorporation of bOPN results in a cross-shaped pattern of fluorescence, consistent with preferential adsorption to {100}/{121} edges throughout the growth process