Specific Adsorption of Osteopontin and Synthetic Polypeptides to Calcium Oxalate Monohydrate Crystals

Abstract

Protein-crystal interactions are known to be important in biomineralization. To study the physicochemical basis of such interactions, we have developed a technique that combines confocal microscopy of crystals with fluorescence imaging of proteins. In this study, osteopontin (OPN), a protein abundant in urine, was labeled with the fluorescent dye AlexaFluor-488 and added to crystals of calcium oxalate monohydrate (COM), the major constituent of kidney stones. In five to seven optical sections along the z axis, scanning confocal microscopy was used to visualize COM crystals and fluorescence imaging to map OPN adsorbed to the crystals. To quantify the relative adsorption to different crystal faces, fluorescence intensity was measured around the perimeter of the crystal in several sections. Using this method, it was shown that OPN adsorbs with high specificity to the edges between {100} and {121} faces of COM and much less so to {100}, {121}, or {010} faces. By contrast, poly-L-aspartic acid adsorbs preferentially to {121} faces, whereas poly-L-glutamic acid adsorbs to all faces approximately equally. Growth of COM in the presence of rat bone OPN results in dumbbell-shaped crystals. We hypothesize that the edge-specific adsorption of OPN may be responsible for the dumbbell morphology of COM crystals found in human urine.

Modulation of Calcium Oxalate Dihydrate Growth by Selective Crystal-face Binding of Phosphorylated Osteopontin and Polyaspartate Peptide Showing Occlusion by Sectoral (Compositional) Zoning

“…Fluorescent molecules and fluorescently-tagged proteins have been previously and effectively used to study protein-mineral interactions (in COM studies (19,20,(77)(78)(79)). Extending this approach to COD and using a poly-Asp 86 -93 peptide not previously examined by others, we have visualized by light and confocal microscopy the incorporation behavior of this peptide into the crystal structure of COD and revealed distinctive and informative three-dimensional sectoral (compositional) zoning patterns (77)(78)(79)(80) that provide significant new insight into protein occlusion into biominerals.…”

Co‐ordinated functions ofMmsproteins define the surface structure of cubo‐octahedral magnetite crystals in magnetotactic bacteria

“…Because biominerals often show complicated architecture that cannot be achieved by chemical synthetic pathways in artificial systems, the process is attractive in the fields of materials development and science (Cha et al, 1999;Aizenberg et al, 2003;Garcia-Ruiz et al, 2009). Molecular studies on biomineralization have shown that the process involves a number of proteins that function as molecular templates (Du et al, 2005;Sugawara et al, 2006), a nucleation factor (Lakshminarayanan et al, 2002), a growth inhibitor (Marin et al, 2007;Taller et al, 2007), framework (Meldrum et al, 1991), and a catalyst (Cha et al, 1999). Recent transmission electron microscopic analyses of biominerals indicated that the micrometer-sized biomineral crystals consist of small grainy crystals, ranging in size from approximately 10 to 200 nm, depending on the biomineral types (Oaki et al, 2006;Nemoto et al, 2012).…”

“…The size and shape of the small crystals are uniform and well-regulated in individual biological systems, suggesting that these are the minimal unit of biomineral crystals, which assemble to form micrometer-scaled biomineral crystals (Cölfen and Antonietti, 2005;Oaki et al, 2006). Although many proteins have been isolated from a variety of biominerals, their functions have mainly been described in the synthesis of micrometer-sized biomineral crystals (Cha et al, 1999;Lakshminarayanan et al, 2002;Du et al, 2005;Sugawara et al, 2006;Marin et al, 2007;Taller et al, 2007). The control mechanisms of morphology and size of the minimum unit of biomineral crystals remain largely unknown.…”

MMS6 Protein Regulates Crystal Morphology during Nano-sized Magnetite Biomineralization in Vivo

“…Ansocalcin, a protein isolated from goose eggshell matrix, induces the construction of spherical calcite polycrystalline aggregates (6). Osteopontin, a protein that is abundant in urine, interacts with the specific crystal edges of calcium oxalate and influences the morphology of the crystals (7). In recent years, it has been determined that hierarchical organized structures with highly uniform pseudo-hexagonal structures are involved in the formation of the sturdy nacreous layer of mollusk shells and that rhombohedral nano-sized crystals are involved in the development of egg shells (3, 8 -9).…”