Modulation of calcium oxalate dihydrate growth by selective crystal-face binding of phosphorylated osteopontin and polyaspartate peptide showing occlusion by sectoral (compositional) zoning

Abstract

Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin/uropontin (OPN) are commonly found in kidney stones. To investigate the effects of OPN on COD growth, COD crystals were grown with phosphorylated OPN or a polyaspartic acid-rich peptide of OPN (DDLDDDDD, poly-Asp(86-93)). Crystals grown with OPN showed increased dimensions of the {110} prismatic faces attributable to selective inhibition at this crystallographic face. At high concentrations of OPN, elongated crystals with dominant {110} faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp(86-93) dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp(86-93) followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into {110} crystal sectors. Computational modeling of poly-Asp(86-93) adsorption to COD {110} and {101} surfaces also suggests increased stabilization of the COD {110} surface and negligible change to the natively stable {101} surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN. In summary, OPN and its poly-Asp(86-93) sequence similarly affect COD mineral growth; the {110} crystallographic faces become enhanced and dominant attributable to {110} face inhibition by the protein/peptide, and peptides can incorporate into the mineral phase. We, thus, conclude that the poly-Asp(86-93) domain is central to the OPN ability to interact with the {110} faces of COD, where it binds to inhibit crystal growth with subsequent intracrystalline incorporation (occlusion).

FIGURE 1.

a, immunohistochemical localization of OPN in human kidney stone by light microscopy. Concentric layers/lamellae of organic matrix, rich in OPN, radiate from apparent nidi of calcification. b and c, at the ultrastructural level, colloidal-gold immunolabeling for OPN viewed by transmission electron microscopy shows OPN variably concentrated either at crystallite surfaces (b), here seen as voids after sample decalcification, or within the crystallites (within the void) and associated with a flocculent organic material evident after decalcification (c). d, scanning electron micrograph of COD crystals precipitated from normal male human urine by oxalate addition. Typical di-pyramidal COD crystals show prominent {101} crystallographic faces. e, Western blots for OPN of normal human urine (lane 1) and the protein extract of COD crystals precipitated from normal human urine (lane 2).

FIGURE 2.

a–d, scanning electron micrographs of COD grown in solution in the presence of full-length, phosphorylated OPN at the indicated concentrations. With increasing amounts of OPN, the {110} prismatic crystallographic faces of COD grow in dimension and become predominant at the expense of the {101} faces, the latter being the predominant crystal face formed in the absence of added protein (lower inset in panel a). At the highest concentration of OPN used (d), most COD crystals are elongated and often intergrown as penetration twins. Schematic insets show SHAPE software-derived renditions of the crystals used for face identification and c axis determination (extension bars). Scale bars equal 5 μm.

FIGURE 3.

Scanning electron micrographs of COD grown in solution in the presence of synthetic, low (a) and high (b) molecular weight aspartic acid-rich peptides at the indicated concentrations. a, increases in linear poly-Asp^86–93 concentration (sequence from OPN bovine sequence), although not as effective as full-length OPN, induced a similar elongating effect on COD crystal morphology, increasing the {110} faces at the expense of the {101} faces. b, high molecular weight and poly-Asp (Sigma) was more potent in similarly generating rod-shaped crystal morphologies, with some additional “rounding” effect at the poles of the elongated crystals. Schematic insets show SHAPE software-derived renditions of the crystals used for face identification and c-axis determination (extension bars). Scale bars equal 5 μm.

FIGURE 4.

Conventional fluorescence micrographs and laser confocal micrographs of COD crystals grown in the presence of fluorescently-tagged poly-Asp^86–93 at the indicated concentrations. a and b, pyramidal, compositional (sectoral) zoning is observed in COD crystals reflecting selective incorporation during growth of fluorescently tagged poly-Asp^86–93 specifically into {110} crystal sectors. Because of differences in orientation, the zoning pattern changes in appearance despite the crystals having similar morphology. Concentration of peptide affects the relative dimensions of the sectors as well as the area and shape of {110} prismatic faces (as in Fig. 3a). In this non-confocal image, fluorescence may originate from the interior of the crystal as well as from exterior faces. c, laser scanning confocal microscopy with focal plane imaging performed within crystal interiors (here shown for a crystal at a scanning depth of 5, 10, and 15 μm from the top of the crystal) confirms sectoral compositional zoning within COD crystals, revealing the boundaries of {110} crystal sectors. The overall morphology of the imaged crystal is shown by transmitted light in the left-most panel. Schematic insets show SHAPE software-derived renditions of the crystals used for face identification and c axis determination (blue extension bars).

FIGURE 5.

Summary of data using SHAPE software renditions of effects of full-length OPN and poly-Asp^86–93 peptide on COD crystal growth morphology (a and b) and of occlusion of poly-Asp^86–93 into COD in a sectoral compositional zoning pattern arising from {110} face binding of this peptide (c). Increases in OPN or poly-Asp^86–93 concentrations in growth solution kinetically result in similar effects on COD crystal morphology; the same faces {110} are significantly inhibited and become predominant, and COD crystals elongate along their c-axis. {110} sectoral zones become dominant with increases in poly-Asp^86–93 concentration, and the three schematics in the bottom row represent 2-dimensional planes taken from the center of COD crystals where poly-Asp^86–93 incorporation (in increasing solution concentration from left to right) is shown by green triangles in two (of four) apposing pyramidal sectors. The {110} sectors (green) gain in proportion relative to other crystal-face sectors (black) as poly-Asp^86–93 concentration increases.

FIGURE 6.

Molecular modeling of poly-Asp^86–93 peptide binding to {110} and {101} crystallographic faces of COD. Atomic configurations used for modeling at the surfaces of the {110} face (a) and {101} face (b) of COD. c–f, representative, top-scoring (low energy) models of poly-Asp^86–93 adsorbed onto COD. c and e, two lateral views (∼90° to each other) of poly-Asp^86–93 bound to a high calcium density plane in the {110} face of COD (with the N terminus of the peptide extending beyond the image plane in panel c, and with the N terminus of the peptide to the right in panel e. Some degree of lattice matching for five carboxylate (COO⁻) side chains aligned with calcium atoms occurs on the {110} face in this profile. d and f, two lateral views (∼90° to each other) of poly-Asp^86–93 bound to the {101} face of COD (with the N terminus of the peptide extending beyond the image plane in panel d and with the N terminus of the peptide to the right in panel f. Hydrogen bonding is schematically illustrated by dashed yellow lines connecting donor-acceptor pairs. Green, calcium; gray, carbon; red, oxygen; white, hydrogen).