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.

FIGURE 1

Growth habit of COM. (a) Scanning electron micrograph of COM crystal grown under LSC (), viewed from a 〈010〉 direction. (b) Scanning electron micrograph of COM crystal grown under LSC, viewed from a 〈100〉 direction. Note the presence of a macrostep on the {100} face. (c) Perspective view of COM penetration twin, with crystal faces developed and major crystallographic directions indicated. The dotted lines indicate the twin plane.

FIGURE 2

Scanning electron micrographs of COM grown in the presence of OPN, poly-asp, or poly-glu. (a) COM crystal grown under LSC () in the presence of 5 μg/ml OPN. (b) COM crystal grown under LSC in the presence of 5 μg/ml OPN. (c) COM crystals grown under HSC () in the presence of 10 μg/ml OPN. (d) COM crystals grown under HSC in the presence of 11 μg/ml poly-asp. E. COM crystals grown under HSC in the presence of 11 μg/ml poly-glu. (Inset) High-magnification image (scale bar = 3 μm) of crystal with facets similar to faces of control crystals.

FIGURE 3

Confocal micrographs of Alexa-labeled polyelectrolytes adsorbing to COM crystals nucleated from a {010} face. (a, b) OPN. (c, d) Poly-asp. (e, f) Poly-glu. Panels a, c, and e are SCIM/fluorescence images of sections at the glass-crystal interface. The red (SCIM) and green (fluorescence) channels are superimposed. Panels b, d, and f are images using the green channel only.

FIGURE 4

Intensity plots of polyelectrolyte fluorescence in multiple optical sections through single COM crystals. Relative fluorescence intensity, after subtraction of background, is plotted against the normalized distance around the perimeter of the crystal. The average distances occupied by the different faces present are indicated.

FIGURE 5

Averaged intensity plots of polyelectrolyte fluorescence in multiple COM crystals. Each plot shown represents the average intensity of multiple optical sections through a single COM crystal. The gray bars represent the range of fractional perimeter distance values for the edges between {100} and {121} faces.

FIGURE 6

Averaged intensity plots of polyelectrolyte fluorescence in multiple sections of multiple COM crystals. These plots were generated by averaging the data shown in Fig. 5. The gray bars represent the range of fractional perimeter distance values for the edges between {100} and {121} faces.

FIGURE 7

Confocal micrographs of Alexa-labeled polyelectrolytes adsorbing to COM crystals nucleated from {100} and {010} faces. (a–d) Poly-asp. The crystals at upper right and lower left were nucleated from {010} and {100} faces, respectively. (a) SCIM/fluorescence image of poly-asp adsorption at the crystal-glass interface. (b) SCM/fluorescence image of poly-asp adsorption at the crystal-solution interface of the crystal shown in lower left. (c) Fluorescence image of section shown in a. (d) Fluorescence image of section shown in b. (e) SCM/fluorescence image of poly-glu adsorption to COM crystal nucleated from a {100} face. (f) SCM/fluorescence image of OPN adsorption to COM crystal nucleated from a {100} face.

FIGURE 8

Kinetics of OPN adsorption to COM crystals. Sigmoid binding curves were fitted to the data points using Origin.