Urinary tract stone disease is a common chronic disorder in humans, with the majority of calculi being calcium oxalate (CaOx) stones1. The formation of urinary stones is a complex process involving multiple factors. Although the saturation product for CaOx is frequently exceeded in normal urine, most humans do not form stones. It has been presumed that inhibitors of CaOx crystallization have protective effects, although their precise role has not been defined. The majority of the inhibition of crystal growth observed in assays of normal urine is due to the presence of protein macromolecules2,3. We isolated a urinary protein inhibitor of CaOx crystal growth by immunoaffinity chromatography. The N-terminal amino acid sequence of this urinary protein is identical to that of human osteopontin (OPN), a phosphorylated matrix protein previously isolated from bone4,5. Its name connotes the potential bridging role in facilitating interactions between cells and mineralized matrix proposed on the basis of having a functionally active cell-binding Arg-Gly-Asp-Ser (RGDS) sequence and sequence domains rich in dicarboxylic acids6. To denote its urinary origin, we refer to urinary OPN by its contraction, uropontin (uOPN). This protein is a very potent inhibitor of CaOx crystallization in vitro. uOPN inhibits the growth of CaOx monohydrate (COM) crystals by 50% at 16 
2 nmol/L and inhibits the aggregation of COM crystals by 50% at 28 4 nmol/L7. Thus, levels of uOPN present in normal human urine are inhibitory for both of these aspects of COM crystallization7,8,9.
The primary structure of human OPN contains an abundance of acidic amino acids with 48 aspartic acid and 27 glutamic acid residues in its 298 amino acid peptide sequence. Many of the aspartic acid residues are clustered together in the N-terminal half of the protein. The human OPN sequence also contains 42 serines and 14 threonines. Many of these highly conserved residues are in suitable positions for phosphorylation. They have been shown to be phosphorylated in vivo by a combination of sequence analysis of S-ethylcysteine-derivitized peptides and mass spectroscopy10. The extent of phosphorylation of OPN is responsive to hormonal influences such as those of vitamin D11,12. Furthermore, decreased inhibition of hydroxyapatite crystal growth was seen after dephosphorylation of OPN13,14. These latter observations suggest that phosphorylation of full OPN molecules might also have a functional role in CaOx crystallization.
In addition to the obvious implications for clinical stone disease, studies of the process of crystallization leading to stone formation in the urinary tract and the elements contained in urine provide a model system for biologic control of mineralization in other body fluids. Although uOPN inhibits several aspects of COM crystallization7, specific structural features responsible for its effects on CaOx crystallization were not previously known. To define molecular features causing inhibition of crystallization through interactions with COM crystals, we synthesized peptides and phosphopeptides with sequences corresponding to those in the human OPN protein sequence.
Aspartic acid-rich proteins are closely associated with mineralization in a broad range of organisms and tissues4, and large polyaspartic acid polymers have inhibited CaOx crystal growth15,16. Accordingly, we selected from the full OPN sequence, a 54 amino acid segment with an abundance of highly conserved aspartic acid residues for the present investigations Figure 1. Within this region, the 10 consecutive aspartic acids in the mouse sequence are fully conserved in at least seven of the positions in each of five other mammalian species and the chicken; more than one nonconservative substitution is present in only the cow and chicken17. This region of OPN also contains highly conserved serines and a threonine in positions that have been shown to be phosphorylated in vivo10. Thus, we could evaluate the relative contributions of aspartic acid residues and phosphorylation to the functional activities of these OPN peptides. The effects of these peptides on CaOx crystal growth and crystal aggregation were determined. The secondary structure of these peptides was also analyzed to evaluate potential contributions of conformational changes induced by phosphorylation to functional activities. Our studies demonstrate a major contribution of phosphorylation to the inhibition of CaOx crystal growth by these small OPN peptides.
Figure 1.
Sequences of amino acids from 62 to 116 of the OPN sequence in the four pairs of peptides used for the crystallization studies shown in Figures 2 and 3. The molecular weights of the four unmodified peptides ranged from 1591 to 3031, and the molecular weights of the four phosphorylated peptides ranged from 1828 to 3268. Three pairs of the peptides contain 3 serine (S) or threonine (T) phosphorylation sites, while peptide 99 to 115 has 5 serines. Peptide 62 to 85 has a single casein kinase phosphorylation site. Peptide 93 to 106 has three and peptide 99 to 115 has five of these sites. The fraction of aspartic (D) or glutamic (E) acids in the four peptides were comparable with 54% of the amino acids in the first two peptides and 47 or 57% of the amino acids in the two later peptides being either D or E.
Full figure and legend (11K)METHODS
Peptides were synthesized on Milligen 9050 and Rainin PS3 automatic synthesizers using 9-fluorenylmethoxycarbonyl amino acids according to standard procedures18. Individual phosphorylated serine residues were incorporated as Fmoc-Ser (PO3HBzl)-OH19, purchased from Novabiochem, Ltd. (Newton, MA, USA). Global phosphorylation of all potential serine and threonine residues in the peptides was achieved with polyphosphoric acid after cleavage20. Peptides and phosphopeptides were detached from the solid support with trifluoroacetic acid (TFA) and were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using an aqueous acetonitrile gradient elution system containing 0.1% TFA as an ion-pairing reagent. In the case of global phosphorylation, the earliest eluting HPLC fractions were collected (representing the highest level of phosphate incorporation). The integrity of the peptides and phosphopeptides was verified by matrix-assisted laser ionization/desorption mass spectroscopy in the Protein Microchemistry Laboratory of the Wistar Institute (Philadelphia, PA, USA).
Inhibition of CaOx crystal growth was measured using a seeded, solution-depletion assay. Loss of absorbance at 214 nm was monitored continuously after addition of COM seed crystals and reflects consumption of oxalate into the COM seed crystals21. After calculation of the initial rate, inhibition expressed as a ratio of rate with protein to the control rate. Inhibition of CaOx crystal aggregation was determined using a particle size assay22. Spontaneous sedimentation of particles from a well-equilibrated COM crystal slurry was measured by monitoring absorbance at 620 nm for 300 seconds after aggregation was initiated. The slope of absorbance reflects free-fall velocity, which is proportional to particle size and was calculated as previously described22.
Circular dichroism (CD) spectra were taken on a Jasco J720 instrument at room temperature in a 0.2 mm path-length cell. Double-distilled water and spectroscopy-grade trifluoroethanol (TFE) were used as solvents. The peptide concentration was about 0.5 mg/mL, determined each time by quantitative RP-HPLC23. Since secondary structures of peptides (especially phosphopeptides) provided by the current computer-assisted curve-analyzing algorithms show a high error rate, the CD spectra evaluations were based on comparisons with known peptide conformations24,25.
RESULTS
We studied the effects on growth and aggregation of COM crystals of the series of peptides with the sequences shown in Figure 1 and their phosphorylated counterparts. This region contains an abundance of aspartic acid residues as well as serines and a threonine in positions suitable for phosphorylation. Thus, we were able to evaluate the contribution of aspartic acids and of phosphorylation to the function of these peptides.
As shown in Figure 2a, growth of COM crystals was inhibited by 
50% at 1000 nmol/L of the earlier two unmodified peptides, but was not inhibited by the other two unmodified peptides Figure 2b, with aspartic residues more evenly distributed within their sequences. Phosphorylation markedly increased inhibition of COM crystal growth so that each of the four phosphorylated peptides inhibited growth by at least 50% at concentrations of 
200 nmol/L.
Figure 2.
(A and B) Inhibition of CaOx crystal growth by synthetic peptides (open symbols) and phosphorylated synthetic peptides (closed symbols) corresponding to amino acids in the portion of the osteopontin sequence shown in Figure 1 as follows: 62 to 85 (triangles) and 66 to 91 (diamonds), 93 to 106 (circles) and 99 to 115 (squares).
Full figure and legend (38K)However, in contrast to the potent inhibition of COM crystal growth by these phosphorylated peptides, they did not inhibit aggregation of COM crystals Figure 3. None of the concentrations as high as 10,000 nmol/L of any of the four unmodified peptides or their phosphorylated counterparts reduced the aggregation of COM crystals by 50%.
Figure 3.
Inhibition of CaOx crystal aggregation by the four synthetic peptides (
) and the four phosphorylated peptides (
) shown in Figure 2 (mean SD).
The secondary structure of these OPN peptides was analyzed by CD spectroscopy to determine if the effects on functional activities of OPN peptides were the consequence of changes in peptide conformation induced by phosphorylation. We had previously studied synthetic peptides corresponding to sequences of tau, a neural protein that accumulates in Alzheimer's disease. Phosphorylation of tau peptides resulted in major conformational effects that included reversible 
-pleated sheet formation or stabilization of turn structures26,27. Effects induced by divalent cations on the conformation of these neural peptides were profoundly influenced by phosphorylation26,28,29. Our studies of the CD spectra of the full OPN molecule showed that exposure to calcium ions increased the fraction of -pleated sheet structure30. However, the present studies of effects of phosphorylation on the structure of OPN peptides did not show comparable changes when analyzed using identical conditions. Notably, phosphorylation generally resulted in a decrease or loss of ordered structure of the unmodified OPN peptides. Peptides of this length generally assume the conformation of a single conformational element; this primarily consisted of turns in the spectra of these OPN peptides in TFE with less ordered structure in water. Peptide 93-106 was the only one having spectra suggestive of -pleated sheets in TFE. However, the structure of phosphorylated peptide 93-106 in water was completely unordered. In contrast to major conformational changes induced in phosphorylated tau peptides26,28,29, the addition of Ca2+, Mg2+, or Al3+ to the OPN phosphopeptides did not generate -pleated sheet formation or other new secondary structure.
Since the four phosphorylated peptides from the OPN sequence inhibited the growth, but not the aggregation of COM crystals, effects of a model polypeptide, polyaspartic acid (molecular weight of 11,000 D), on the growth and the aggregation of COM crystals were also compared. This analysis Figure 4 showed inhibition of crystal growth by molar concentrations of polyaspartic acid that were slightly higher than inhibitory levels of full uropontin molecules. By contrast, inhibition of COM crystal aggregation by 50% required 
mol/L (that is, 1000 times higher) levels of polyaspartic acid. The effect of polyaspartic acid on COM crystal aggregation is quite weak when compared with 50% inhibition by 28 4 nmol/L of uropontin using identical experimental conditions7.
Figure 4.
Inhibition of CaOx crystal growth (
) and aggregation (
) by polyaspartic acid (molecular weight 11,000).
DISCUSSION
Aspartic acid-rich proteins (AARPs) are closely associated with mineralization in a broad range of organisms and tissues4. Furthermore, a series of elegant in vitro studies have demonstrated tissue-specific patterns of modulation of crystal growth by AARPs from other mineralizing tissues31,32,33. However, contributions of individual domains within an AARP to the modulation of CaOx crystallization were not previously known. The present investigation showed that phosphorylation markedly increased the inhibition of COM crystal growth by each of the four OPN peptides analyzed. These findings show that the full protein is not required for functional activity, since these small domains of the OPN primary sequence were able to independently inhibit COM crystal growth. Our studies also showed that molecular features responsible for functional activities of uropontin are at least partially separable; the phosphorylated OPN peptides were potent inhibitors of COM crystal growth, but not of COM crystal aggregation.
Potent inhibition of crystal growth appears to require a protein structure that binds to crystals on the basis of specific patterns of charge density. Although an abundance of carboxylic acid residues in AARPs suggests the potential for interaction with crystal surfaces, conformations adopted by these proteins may also underlie specific interactions that lead to modulation of crystal growth. For example, the aspartic acid-rich proteins extracted with ethylenediaminetetraacetic acid (EDTA) from mollusk shells adopted a -sheet conformation upon exposure to calcium34. Exposure of immunopurified rat OPN to calcium ions also significantly increased the -sheet structure in CD spectra30. However, in contrast to findings in our studies of full OPN molecules and neural peptides26,27,28,29, conformational changes of OPN peptides favoring interactions with crystals did not result from phosphorylation and/or exposure to divalent cations. Thus, by default, the contribution of phosphorylation to functional activity of these peptides appears to result from changes in localized patterns of charge density.
Inhibition of CaOx crystal growth by homopolymers of aspartic acid15,16 suggested that the close proximity of a series of aspartic acid residues within OPN might also cause growth inhibition. It should be noted that chains of aspartic acid residues in these model polymers were at least five- to tenfold longer than the longest series of aspartic acids in native OPN molecules. In the present studies, a minor role for close proximity of aspartic acids within the OPN sequence was suggested by inhibition of crystal growth by unmodified peptides 62-85 and 66-91 Figure 2a, but not by the later unmodified peptides Figure 2b. However, micromolar levels of unmodified peptides 62-85 and 66-91 were required to achieve inhibition comparable to <20 nanomolar levels of full uOPN molecules in identical experimental conditions7 or <40 nanomolar levels of polyaspartic acid Figure 4.
Molar concentrations of the four phosphorylated OPN peptides required for 50% inhibition of COM crystal growth were orders of magnitude lower than those for the unmodified OPN peptides. Inhibitory molar levels of these phosphopeptides were 1 to approximately 15 times higher than molar levels of uOPN required for comparable inhibition of growth. On a weight basis, inhibition of crystal growth by phosphorylated peptides 62-85 and 66-91 was roughly equal (75 to 95%) to inhibition by uOPN. It is noteworthy that, by weight, inhibition of crystal growth by phosphorylated peptides 99-115 and 93-106 was more potent (2-fold and 18-fold, respectively) than inhibition by uOPN. Since inhibition of crystal growth by these two phosphorylated peptides exceeds that of uOPN by weight, phosphorylation of the threonine and serines in positions 98-113 may substantially contribute to the inhibition of crystal growth exerted by full native molecules.
A capacity to inhibit the growth and also the aggregation of CaOx crystals, as well as other aspects of crystallization, is shared by several urinary proteins, including uOPN, prothrombin fragment 1, and bikunin7,35,36. Although the structural basis has not been defined, studies of Tamm-Horsfall protein, a potent inhibitor of CaOx crystal aggregation22, suggest that structural requirements for this functional activity differ from those for inhibition of crystal growth. Larger molecular size may be a factor contributing to inhibition of CaOx aggregation, since the 616 amino acid peptide sequence of Tamm-Horsfall protein37 is roughly twice as long as that of the full OPN sequence. However, it seems certain that inhibition of aggregation requires more than multiple crystal growth inhibiting domains, since Tamm-Horsfall protein does not inhibit the growth of CaOx crystals22,38. The present studies of phosphorylated and unmodified OPN peptides demonstrate that these two functional activities are segregated within OPN, since the phosphorylated OPN peptides were potent inhibitors of crystal growth, but not of crystal aggregation. Defining the structural basis for the inhibition of CaOx crystal aggregation by uOPN remains a goal for future studies.
Studies of patients with nephrolithiasis have shown that the inhibition of CaOx crystal growth by their unfractionated urinary proteins is frequently decreased39. Investigations of functional capacities of uOPN and other individual urinary proteins of such patients will better define the nature of this functional defect. The present studies strongly indicate that phosphorylation of OPN is an important determinant of inhibitory activity. Thus, analysis of the extent of phosphorylation of uOPN will contribute to the molecular definition of the functional abnormality in such patients with decreased inhibition of crystallization by their uOPN.
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Acknowledgments
Research presented in this study was supported by grant R01 DK33501 from the National Institutes of Health (J.R.H.).
