Crystallization of Calcium Oxalates Is Controlled by Molecular

Sep 3, 2009 - Hydrophilicity and Specific Polyanion-Crystal Interactions. Bernd Grohe,*,† Adam Taller,† Peter L. Vincent,. ^. Long D. Tieu,† Kem...
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Crystallization of Calcium Oxalates Is Controlled by Molecular Hydrophilicity and Specific Polyanion-Crystal Interactions )

Bernd Grohe,*,† Adam Taller,† Peter L. Vincent,^ Long D. Tieu,† Kem A. Rogers,§ Alexander Heiss,z Esben S. Soerensen, Silvia Mittler,^ Harvey A. Goldberg,†,‡ and Graeme K. Hunter*,†,‡ †

CIHR Group in Skeletal Development and Remodeling and School of Dentistry, ‡Department of Biochemistry, Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Canada, ^Department of Physics and Astronomy, University of Western Ontario, London, Canada, zInstitute for Biomedical Engineering, Biointerface Group, and Central Facility for Electron Microscopy, RWTH Aachen University, Germany, and Department of Molecular Biology, University of Aarhus, Denmark )

§

Received April 1, 2009. Revised Manuscript Received August 6, 2009 To gain more insight into protein structure-function relationships that govern ectopic biomineralization processes in kidney stone formation, we have studied the ability of urinary proteins (Tamm-Horsfall protein, osteopontin (OPN), prothrombin fragment 1 (PTF1), bikunin, lysozyme, albumin, fetuin-A), and model compounds (a bikunin fragment, recombinant-, milk-, bone osteopontin, poly-L-aspartic acid (poly asp), poly-L-glutamic acid (poly glu)) in modulating precipitation reactions of kidney stone-related calcium oxalate mono- and dihydrates (COM, COD). Combining scanning confocal microscopy and fluorescence imaging, we determined the crystal faces of COM with which these polypeptides interact; using scanning electron microscopy, we characterized their effects on crystal habits and precipitated volumes. Our findings demonstrate that polypeptide adsorption to COM crystals is dictated first by the polypeptide’s affinity for the crystal followed by its preference for a crystal face: basic and relatively hydrophobic macromolecules show no adsorption, while acidic and more hydrophilic polypeptides adsorb either nonspecifically to all faces of COM or preferentially to {100}/{121} edges and {100} faces. However, investigating calcium oxalates grown in the presence of these polypeptides showed that some acidic proteins that adsorb to crystals do not affect crystallization, even if present in excess of physiological concentrations. These proteins (albumin, bikunin, PTF1, recombinant OPN) have estimated total hydrophilicities from 200 to 850 kJ/mol and net negative charges from -9 to -35, perhaps representing a “window” in which proteins adsorb and coat urinary crystals (support of excretion) without affecting crystallization. Strongest effects on crystallization were observed for polypeptides that are either highly hydrophilic (>950 kJ/mol) and highly carboxylated (poly asp, poly glu), or else highly hydrophilic and highly phosphorylated (native OPN isoforms), suggesting that highly hydrophilic proteins strongly affect precipitation processes in the urinary tract. Therefore, the level of hydrophilicity and net charge is a critical factor in the ability of polypeptides to affect crystallization and to regulate biomineralization processes.

1. Introduction Biomineralization is the process by which organisms deposit mineral phases (usually crystalline, but sometimes amorphous) within tissues. The hallmark of biomineralization is a high degree of specificity in terms of mineral type, location, orientation and growth habit (size and shape). In contrast, ectopic calcification, the deposition of crystals within soft tissues, is characterized by great variability in the nature of the mineral phase. In both cases, however, extracellular-matrix and secreted proteins play critical roles in mineral formation. Therefore, both biomineralization and ectopic calcification can be seen as interfacial phenomena in which the precipitation process is modulated by proteins that can interact with growing crystals and thereby, for example, promote/ inhibit nucleation, affect growth and determine which polymorph forms.1 Kidney stone formation is an ectopic calcification process that has been intensively studied, not only because of its severe effects on human health but also because the calcium oxalate monohydrate (COM) crystals that represent the most common mineral *To whom correspondence should be addressed. Phone: 519-661-2185. Fax: 519-850-2459. E-mail: [email protected]. (1) Lowenstam, H. A.; Weiner, S., On Biomineralization. Oxford University Press: New York, 1989; p ix, 324 p.

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phase of kidney stones are highly amenable to experimental analysis.2 The formation of oxalate stones is a complex process involving nucleation, growth, aggregation, and retention of crystals in a dynamic urinary environment.2,3 It is affected by the level of Ca2þ and C2O42- supersaturation, stone-forming promoters (membranes, defective cells, and proteins) and inhibitors (citrate, urinary proteins, and other compounds).4 Most of the inhibitory activity resides in proteins such as Tamm-Horsfall protein (THP), osteopontin (OPN), urinary prothrombin fragment 1 (PTF1), and bikunin.2,4 Some of these are capable of catalyzing the precipitation of calcium oxalate dihydrate (COD), which is less adherent to epithelial cell surfaces and, therefore, more easily discharged with the urine.5 Other proteins are capable of being incorporated into calcium oxalate (CaOx) crystals6,7 or inhibiting the attachment of crystals to epithelial surfaces.3,8 (2) Khan, S. R.; Kok, D. J. Front. Biosci. 2004, 9, 1450-1482. (3) Kok, D. J.= World J. Urol. 1997, 15, (4), 219-228. (4) Lieske, J. C.; Toback, F. G. Curr. Opin. Nephrol. Hypertens. 2000, 9, (4), 349-355. (5) Wesson, J. A.; Worcester, E. M.; Wiessner, J. H.; Mandel, N. S.; Kleinman, J. G. Kidney Int. 1998, 53, (4), 952-957. (6) Cook, A. F.; Grover, P. K.; Ryall, R. L. BJU Int. 2009, 103, (6), 826-835. (7) Hunter, G. K. G., B.; Jeffrey, S.; O’Young, J.; Soerensen, E. S.; Goldberg, H. A. Cells Tissues Organs 2009, 189, (1-4), 44-50 (online: Aug.15/2008). (8) Atmani, F.; Khan, S. R. J. Am. Soc. Nephrol. 1999, 10 Suppl 14, S385-S388.

Published on Web 09/03/2009

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Because of the complexity of stone formation and the effects of urinary proteins at many stages of the process, the exact physiological roles of these proteins are hard to predict. However, accumulating evidence suggests that highly anionic proteins rich in aspartic and glutamic acid (such as OPN) strongly adsorb to urinary crystals and inhibit their growth, whereas weakly acidic proteins (PTF1, THP, bikunin, albumin) appear to predominantly prevent aggregation and/or adhesion of crystals to epithelial cells.2,4,8,9 These interactions appear to be linked to the positive surface charge of COM under physiological conditions.10 Detailed molecular-scale investigations of the interactions between urinary proteins and COM crystals show a high degree of specificity in terms of the charged groups present in the protein and on the crystal face.11 In addition to the amino-acid sequence of the protein, post-translational modifications contribute to inhibitory activities. For example, phosphorylation of OPN greatly increases its potency in inhibiting crystal formation.12,13 Fluorescence imaging and molecular dynamics simulations have shown that phosphopeptides based on sequences found in rat OPN interact with the {100} face, the most calcium-rich face of COM, resulting in strong effects on growth perpendicular to this face.14 However, some other polyanions affect mineralization processes quite differently. Full-length OPN, for instance, was found to preferentially interact with the edges between {100} and {121} faces of COM,7,15 polyaspartic acid with {121} faces,15,16 and polyglutamic acid nonspecifically with all faces of COM.15 All these polypeptides adsorbed to COM crystals and affected their growth. However, the chemistry underlying facespecific adsorption, growth-habit modification, and polymorph selection by crystal-binding proteins is not well understood. In the present study, we have investigated the relationship between protein structure and the regulation of calcium oxalate crystal formation by examining the effects of a number of urinary proteins and model compounds (PTF1, THP, bikunin, albumin, lysozyme, fetuin-A, four forms of OPN, poly-L-aspartic acid, and poly-L-glutamic acid) on different aspects of the crystallization process. Combining scanning confocal microscopy with fluorescence imaging, we visualize the adsorption of these polypeptides to the various faces of the COM crystal. Scanning electron microscopy is used to characterize the effects of these macromolecules on nucleation, growth habit, and the volume of precipitate. In a crucial deviation from previous paradigms, we have interpreted these findings in terms of isoelectric point, molecular charge, polarity, and hydrophilicity, rather than aminoacid sequence. By this analysis, we demonstrate that net molecular charge and hydrophilicity of proteins are the critical factors in determining their effects on crystallization of calcium oxalates. (9) Mueller, P. W.; Macneil, M. L.; Steinberg, K. K. Fresenius J. Anal. Chem. 1990, 338, (4), 543-546. (10) Fernandez, J. C.; Delasnieves, F. J.; Salcedo, J. S.; Hidalgoalvarez, R. J. Colloid Interface Sci. 1990, 135, (1), 154-164. (11) De Yoreo, J. J.; Qiu, S. R.; Hoyer, J. R. Am. J. Physiol.-Renal Physiol. 2006, 291, (6), F1123-F1131. (12) Wang, L. J.; Guan, X. Y.; Tang, R. K.; Hoyer, J. R.; Wierzbicki, A.; De Yoreo, J. J.; Nancollas, G. H. J. Phys. Chem. B 2008, 112, (30), 9151-9157. (13) Langdon, A. W., G. R.; Rogers, K. A.; Soerensen, E. S.; Denstedt, J.; Grohe, B.; Goldberg, H. A.; Hunter, G. K. Calcif. Tissue Int. 2009, 84, 240-248. (14) Grohe, B.; O’Young, J.; Ionescu, D. A.; Lajoie, G.; Rogers, K. A.; Karttunen, M.; Goldberg, H. A.; Hunter, G. K. J. Am. Chem. Soc. 2007, 129, 14946-14951. (15) Taller, A.; Grohe, B.; Rogers, K. A.; Goldberg, H. A.; Hunter, G. K. Biophys. J. 2007, 93, (5), 1768-1777. (16) Jung, T.; Sheng, X. X.; Choi, C. K.; Kim, W. S.; Wesson, J. A.; Ward, M. D. Langmuir 2004, 20, (20), 8587-8596.

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2. Materials and Experimental Methods 2.1. Chemicals. For precipitation of calcium oxalates, the reagents used and the solution preparation were as previously described.17 Poly-L-aspartic acid sodium salt (poly asp10, ∼9.15 kDa and poly asp35, ∼35.7 kDa), poly-L-glutamic acid sodium salt (poly glu10, ∼10.25 kDa), lysozyme (from chicken egg white, ∼14.3 kDa), human serum albumin (globulin free, 66.5 kDa), and the urinary trypsin inhibitor fragment Arg-Gly-Pro-Cys-ArgAla-Phe-Ile (bikunin fragment, BIF, ∼1.0 kDa) were obtained from Sigma-Aldrich. Human urinary trypsin inhibitor (bikunin, 41 kDa) was purchased from ProSpec (Rehovot, Israel) and Tamm-Horsfall protein (THP, ∼85 kDa) from Chemicon International. Prothrombin fragment 1 (PTF1, ∼31 kDa), isolated from prothrombin via established methods,18 was a generous gift of Jeffrey I. Weitz (McMaster University, Hamilton, Canada). Bovine fetuin-A (fetuin, ∼50 kDa) was used after isolation from a commercial preparation (Sigma) via size-exclusion chromatography.19 Native rat bone osteopontin (bOPN) was purified as described by Goldberg and Sodek20 and a MW of 37 622 g/mol determined by MALDI-TOF mass spectrometry (MALDI-TOF MS; Bruker Reflex III). Recombinant full-length rat OPN (recOPN) was expressed as a N-terminal His-tagged protein following a protocol used for recombinant bone sialoprotein.21 The purified protein was analyzed by MALDI-TOF MS and a MW of 36 100 g/mol determined. Bovine milk OPN (mOPN) was purified from bovine milk as described by Soerensen and Petersen22 and a MW of 34 106 g/mol determined by MALDI-TOF MS. Rat kidney OPN (kOPN) was extracted from the secretion of NRK (normal rat kidney) cells following a modification of the protocol developed for bone OPN.20 In brief, kOPN was purified from conditioned culture medium of confluent NRK cells and sequential chromatography on FastQ Sepharose, Superdex 200PG, and MonoQ Sepharose. The protein was pooled, dialyzed and lyophilized, and a yield of 0.15 mg/L determined. After purification of kOPN, MALDI-TOF MS analysis yielded a MW of 36 652 g/mol. The phosphate content of kOPN was determined to be 10.7 by alkaline phosphatase treatment as described by Keykhosravani et al.23 To verify identity, protein content and purity, SDS-PAGE and amino acid analysis (Alberta Peptide Institute, Edmonton, Canada) were used. For labeling polypeptides, Alexa Fluor-488 carboxylic acid (AlexaFluor-488) was obtained from Invitrogen (Canada-Molecular Probes, Burlington, Canada). 2.2. Fluorescence Labeling and Solution Preparation of Polypeptides. For fluorescence imaging, labeling of the polyelectrolytes with AlexaFluor-488 was performed as previously described.15 Amino acid analysis (Alberta Peptide Institute; University of Alberta) was carried out using norleucine as an internal standard to determine the yield of labeled peptides. The masses obtained were used to prepare aqueous stock solutions of 50 μg/mL BIF, lysozyme, THP, fetuin, albumin, bikunin, PTF1, kOPN, and poly asp35. As a control, a stock solution of the Alexa fluorochrome itself was also prepared. In addition, aqueous stock solutions of 50 μg/mL unlabeled polyelectrolyte were prepared for all additive molecules used in this study. 2.3. Crystallization Experiments. Crystallization of calcium oxalates was initiated using the method previously described,14,15 (17) Grohe, B.; Rogers, K. A.; Goldberg, H. A.; Hunter, G. K. J. Cryst. Growth 2006, 295, (2), 148-157. (18) Kretz, C. A.; Stafford, A. R.; Fredenburgh, J. C.; Weitz, J. I. J. Biol. Chem. 2006, 281, (49), 37477-37485. (19) Heiss, A.; Eckert, T.; Aretz, A.; Richtering, W.; Van Dorp, W.; Schafer, C.; Jahnen-Dechent, W. J. Biol. Chem. 2008, 283, (21), 14815-14825. (20) Goldberg, H. A. a. S., J., Purification of mineralized tissue-associated osteopontin. J. Tissue Cult. Meth. 1994, 16, 211-215. (21) Tye, C. E.; Rattray, K. R.; Warner, K. J.; Gordon, J. A.; Sodek, J.; Hunter, G. K.; Goldberg, H. A. J. Biol. Chem. 2003, 278, (10), 7949-55. (22) Soerensen, E. S. P., T. E. J. Dairy Res. 1993, 60, (4), 535-542. (23) Keykhosravani, M.; Doherty-Kirby, A.; Zhang, C.; Brewer, D.; Goldberg, H. A.; Hunter, G. K.; Lajoie, G. Biochemistry 2005, 44, (18), 6990-7003.

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Grohe et al. by adding first oxalate solutions ([C2O42- ] = 1 mM or 2 mM); containing 5 mM sodium acetate and 75 mM sodium chloride) to reaction containers followed by water and calcium solutions ([Ca2þ] = 1 mM or 2 mM); containing 5 mM sodium acetate and 75 mM sodium chloride). For working under low-supersaturation conditions (LSC), final concentrations were [Ca2þ] = [C2O42-] = 1 mM, sodium acetate 10 mM and sodium chloride 150 mM. The final concentrations under high-supersaturation conditions (HSC) were [Ca2þ] = [C2O42-] = 2 mM, sodium acetate 10 mM and sodium chloride 150 mM. The supersaturation for COM (calculated as σ = 1/2 ln [aCa 3 aOx/KSP], via the approximation of the ionic strength24,25) was either 2.86 (LSC) or 3.57 (HSC) and that for COD either 2.31 (LSC) or 3.02 (HSC), using solubility products KSP for COM (KSP;COM =2.2410-9 M2) or COD (KSP;COD = 6.76  10-9 M2) at 37 C.26 Crystallization of CaOx was investigated either in the presence or in the absence of polyelectrolytes and the precipitates studied by scanning electron microscopy. For crystallization, 1 mL aliquots of LSC or HSC solution (with or without polyelectrolyte) were added to wells of tissue-culture plates (24-well, FALCON) containing freshly cleaved mica disks (diameter: 9.5 mm). If polyelectrolyte was added to the wells, the volume of water was correspondingly reduced. Following incubation at 37 C for 30 min, the mica disks were rinsed with water and air-dried. For studying the adsorption behavior of polyelectrolytes to COM crystals, 200 μL aliquots of LSC solutions were added to glassbottomed polystyrene dishes. Following nucleation, crystals were grown for 3 h. Each dish was then placed on the heated (37 C ( 0.2 C) stage of a confocal microscope. Following addition of fluorescent-labeled polyelectrolytes (20 μL aliquots of 1 μg/mL polypeptide) to the solution adsorption to the crystals was allowed for a period of 45 min prior to imaging. The final pH of all reaction solutions was between 6.65 and 6.75; even high polyelectrolyte concentrations (up to 500 μg/mL) did not significantly change the pH. The concentration ranges of each protein (OPN, THP, lysozyme, bikunin, PTF1, albumin, fetuin) studied were similar to or higher than those found in human urine.2,9,27-32 2.4. Microscopy, Imaging, and Data Processing. Scanning electron microscopy (SEM; Carl Zeiss, LEO 1540XB) and scanning confocal microscopy/fluorescence imaging (SCM/FI; Carl Zeiss, Zeiss LSM 410) were performed as previously described.15 To study fluorescence intensities of fluorescent protein along crystal faces, images were analyzed by measuring intensity along edges corresponding to each crystal face, and the background intensity subtracted. The intensities for each crystal face were linked to obtain the fluorescence intensity around the perimeter of an individual crystal. The resulting data were used to plot graphs of relative fluorescence intensity versus the fractional perimeter (normalized to 1) for (a) each optical section of each crystal, (b) a mean for all optical sections of each crystal, and (c) the average for all optical sections of all measured crystals, representing the adsorption profile of a given molecule. Only the latter is shown in this work (Figure 3). For more details please refer to Taller et al.15 The polyelectrolyte-affected rate of CaOx precipitation was determined by plotting the precipitated volume of control samples (24) Weaver, M. L.; Qiu, S. R.; Hoyer, J. R.; Casey, W. H.; Nancollas, G. H.; De Yoreo, J. J. ChemPhysChem 2006, 7, (10), 2081-2084. (25) Davis, C. W., Ion Associoation; Butterworths: London, U.K., 1962. (26) K€onigsberger, E.; and K€onigsberger, L.-C. J. Pure Appl. Chem. 2001, 73, (5), 785-797. (27) Shiraga, H. M., W.; VanDusen, W. J.; Clayman, M. D.; Miner, D.; Terrell, C. H.; Sherbotie, J. R.; Foreman, J. W.; Przysiecki, C.; Nielson, E. G.; and Hoyer, J. R. Proc. Nat. Acad. Sci. U. S. A. 1992, 89, 426-430. (28) Kumar, S.; Muchmore, A. Kidney Int. 1990, 37, (6), 1395-1401. (29) Houser, M. T. Clin. Chem. 1983, 29, (8), 1488-1493. (30) Yamasaki, F.; Shinkawa, T.; Watanabe, M.; Mizota, M. Pflugers ArchivEur. J. Physiol. 1996, 433, (1-2), 9-15. (31) Bezeaud, A.; Guillin, M. C. Br. J. Hamaetol. 1984, 58, (4), 597-606. (32) Ix, J. H.; Chertow, G. M.; Shlipak, M. G.; Brandenburg, V. M.; Ketteler, M.; Whooley, M. A. Nephrol., Dial., Transplant. 2006, 21, (8), 2144-2151.

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Article and precipitated volumes of samples formed in the presence of effectors against the effector concentration used. For calculating volumes, COM and COD dimensions were measured from SEM micrographs. For COM, Æ001æ and Æ010æ dimensions were measured from {100}-nucleated crystals; Æ001æ and Æ100æ dimensions were measured from crystals nucleated from {010} faces. The Æ010æ and Æ100æ dimensions represent the measured distances between the {010} and {100} faces, respectively; the Æ001æ dimension represents the distance between the ends of the crystal along the c axis. Volumes of COM precipitate were calculated and normalized as follows: mean area of the {100} face  mean Æ010æ dimension  number of crystals/mm2 of mica. For COD, dimensions were measured from crystals nucleated from all orientations. The data from each crystal (e.g., widths, angles, heights) were entered in a calculation routine33 and the volume determined. Overall volumes of COD precipitate were calculated and normalized as follows: mean volume of COD crystals  number of crystals/mm2 of mica. All values given are mean ( standard deviation using 3-10 microscopic fields per calculated volume, with numbers of replicates between 35 and 270 crystals per microscopic field. To test if sample values are significantly different from corresponding controls, one-way ANOVA and Dunnett multiple-comparison tests were carried out.

2.5. Calculation of Protein Net Charge and Hydrophilicity. Protein net charges were calculated based on amino acid sequences, pKA values of individual amino acids, and the peptide end groups (N, C terminus) as well as posttranslational modifications such as phosphorylation, sulfation, and glycosylation.34,35 Using the Henderson-Hasselbalch equation at the experimental pH of 6.75, the charge associated with individual amino acids and end termini could be calculated and their contribution to the net polypeptide charge summed over the entire sequence.36 To account for charges associated with phosphate, sulfate and sialic acid groups at the given pH, net charges of -1.5, -1, and -1 was allocated to each group, respectively, based on their pKA values.35,37 The charges of all posttranslational modifications of a polypeptide were then added to the net charge calculated for the backbone of that polypeptide. Although this approach does not take into account, for example, 3D structures of polypeptides or effects of the local polypeptide environment, this approximation allows at least for a determination of relative magnitudes of polypeptide net charges at our working pH of 6.75. The hydrophilicity of polypeptides was calculated on the basis of an empirical hydrophilicity scale for individual amino acids elaborated by Hopp and Woods,38 The scale was derived by using amino-acid solubility data in aqueous, nonaqueous and mixed solvents to determine the change of free energy, dG (kJ/mol), via the van’t Hoff equation for the transfer of amino acids from an aqueous to a nonaqueous phase. The results are hydrophilicity values that are positive for hydrophilic amino acids and negative for hydrophobic amino acids. For posttranslational modifications (phosphate, sulfate, and sugar groups), no hydrophilicity data are available. Based on the scale developed by Hoop and Woods, we assigned phosphate and sulfate groups the same value as aspartic and glutamic acids (3). As glycosylation is believed to generate an even higher hydrophilicity, we assigned each sugar group (oligosaccharide chain) a value of 4. Calculating the hydrophilicity in this way leads to values that predict the relative hydrophilicity of polypeptides rather than providing absolute values. This approach does not take into account the secondary (33) Data were entered into a calculation routine. Available at htt://www. mathe-formeln.de (accessed 2008. (34) Protein data (species, sequences etc.) were found at the Swiss-Prot/ TrEMBL database (ExPASy (Expert Protein Analysis System) Proteomics Server. Avaialable at http://ca.expasy.org (accessed 2008. (35) Stryer, L., Biochemistry. 3rd ed.; Freeman: New York, 1988; p 1090. (36) Tanford, C. a. K., J.G. J. Am. Chem. Soc. 1957, 79, (20), 5333-5339. (37) De Bruyn, P. P. H.; Michelson, S.; and Becker, R. P. J. Cell Biol. 1978, 78. (38) Hopp, T. P.; and Woods, K. R. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, (6), 3824-3828.

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Figure 1. Growth habit of calcium oxalates in the absence of effectors. (a) Scanning electron micrograph (SEM) of COM crystals grown under low-supersaturation conditions (LSC; [Ca2þ] = [C2O42-]=1 mM), viewed from a Æ100æ direction (top) and a Æ010æ direction (bottom). (b) SEM of COM grown under LSC and imaged from an oblique angle view. (c) SEM of COM crystals grown under high-supersaturation conditions (HSC; [Ca2þ] = [C2O42-] = 2 mM), viewed from a Æ010æ direction (top) and a Æ100æ direction (bottom). Note that crystals exhibit features (steps and pits) containing angles characteristic of COM grown at LSC. (d) SEM of COM and COD crystals grown under HSC. COM is viewed from a Æ010æ direction (top), while the dihydrate phase is viewed parallel to the Æ001æ direction (X). structure of polypeptides, which can change the hydrophilicity by exposing more hydrophilic amino acid groups to the solvent. However, Hopp and Woods38 successfully used this procedure to predict antigenic determinants in protein sequences and, recently, Elhadj et al.39 successfully correlated polypeptide-induced step growth of calcite with hydrophilicity data predicted by applying this method.

3. Results 3.1. Effect of Supersaturation on COM and COD Growth Habit. Calcium oxalates were grown on mica substrates and visualized by SEM. Figure 1 shows COM and COD crystals formed under control conditions (no additive). Under low-supersaturation conditions, COM crystals are well-faceted monoclinic penetration twins with {010}, {100}, and {121} faces developed (Figure 1a,b). Under high-supersaturation conditions, COM and COD are formed. COM crystals have rough and “spiky” surfaces (Figure 1c), whereas COD generates “starfish” shapes (Figure 1d), a well-known morphology of dihydrates grown under these conditions.40 Neither calcium oxalate trihydrate nor an amorphous phase was ever observed. 3.2. Adsorption of Polyelectrolytes to COM Crystals. For studying adsorption of polyelectrolytes to faces of COM by SCM/FI, crystals were pregrown on glass-bottomed dishes at low-supersaturation conditions (no additive added). The crystals nucleated either from a {100} or an {010} face. Their growth habits were similar to those developed on mica substrates under the same conditions for SEM studies. To specify the crystal faces with which the polyelectrolytes interact, each labeled molecule was added to solutions containing pregrown COM and imaged by SCM/FI. Crystals and adsorption profiles were scanned with helium-neon (HeNe, λ = 632.8 nm; red) and krypton-argon (KrAr, λ = 488 nm; green) lasers every 0.5-1.0 μm along the microscopic z-axis (Figure 2). Each scan represents an optical (39) Elhadj, S.; De Yoreo, J. J.; Hoyer, J. R.; Dove, P. M. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, (51), 19237-19242. (40) Wesson, J. A. W., E. Scan. Microsc. 1996, 10, (2), 415.

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Figure 2. Confocal micrographs of Alexa-labeled polyelectrolytes adsorbed to COM crystals nucleated from {100} and {010} faces. (a,b,i) PTF1. (c, d, j) albumin. (e,f,k) kOPN. (g, h, l) bikunin. The scale bar in panel (l) applies to all panels. Please refer to the text for details of protein adsorption. Panels a-h are optical sections taken either (a-f) approximately halfway between the {010} “bottom” and the {010} “top” face or (g,h) at the crystal-glass interface of crystals nucleated from a {010} face. Panels a, c, e, and g are combined red (HeNe laser, λ = 632.8 nm; illuminated crystal) and green (KrAr laser, λ = 488 nm; illuminated protein) false color images; panels b,d,f and h are green images converted to gray scale (to gain more contrast), corresponding to a, c, f, and h, respectively. Note the formed aggregates at the glass and the crystal ({100}, {121} faces) shown in panel h. Panels i - l are optical sections of the upper half (i and k: near the top; j: at the top) of crystals nucleated from a {100} face or (l) from the top a {010}-nucleated crystal. Note: There is no fluorescence detected at the {010} face (panel l). Shown are green-channel images converted to gray scale.

section of a crystal and the fluorescence profile surrounding the crystal, shown as composite (SCM/FI, red/green) or as fluorescence image (FI, black/white). Optical sections through {010}nucleated crystals are bounded by two {100} side faces and four {121} end faces, whereas the perimeter of an optical section through {100}-nucleated crystals is composed of two {010} side faces and four {121} end faces. Please note that in neither case are the {121} faces parallel to the microscopic z-axis. In addition, scans at the very top of {010}-nucleated and {100}-nucleated crystals visualize the uppermost {010} and {100} faces, respectively. Combined SCM/fluorescence and fluorescence-alone images of PTF1 (a,b,i), albumin (c,d,j), kidney OPN (e,f,k) and bikunin (g,h,l) are presented in Figure 2. Panels a-h show optical sections taken either (a-f) approximately halfway between the “bottom” and “top” faces or (g,h) at the crystal-glass interface of crystals nucleated from an {010} face. Panels i-k represent optical sections of the upper half (i and k: near the top; j: at the top) of crystals nucleated from a {100} face, whereas an optical section from the top of a {010}-nucleated crystal is shown in panel l. For PTF1, fluorescence appears to be most intense at the {100}/{121} edges, but also occurs at {100} faces (Figure 2a,b). In contrast, albumin Langmuir 2009, 25(19), 11635–11646

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Figure 3. Averaged intensity (arbitrary units) plots of polyelectrolyte fluorescence of multiple COM crystals nucleated from a {010} face. Each plot shown in a, b, or c represents the average intensity of multiple optical sections around the perimeters of five COM crystals nucleated from a {010} face. The gray bars represent the error in the position of the edges between {100} and {121} faces. For more details please refer to section 2.4 and Taller et al.15.

appears to adsorb somewhat more uniformly (Figure 2c,d,j) and, of relevance, albumin was also detected at glass surfaces (not shown). For kidney OPN (kOPN), fluorescence is concentrated mainly at the edges between {100} and {121} faces (Figure 2e,f,k), an outcome also found for bone- and milk-derived OPN and the prokaryotic-expressed recombinant OPN.7,15 Interestingly, there appears to be no significant adsorption of kOPN to {100} faces (Figure 2k). For bikunin, it is evident that the protein is adsorbing to the glass surface and all faces and edges of the crystal, except to the {010} face (Figure 2h,l). It also appears that labeled bikunin is aggregating into small particles (Figure 2h). However, there are no such particles found on {010} faces of crystals (Figure 2l) suggesting an aggregation on the crystal and glass surfaces rather than an aggregation in solution with subsequent sedimentation. Addition to COM of Alexa-labeled bikunin fragment (BIF), a small basic molecule (∼1.0 kDa) or lysozyme (also basic, ∼14.3 kDa) did not result in detectable adsorption (not shown). As a control, imaging was also performed on COM crystals to which AlexFluor-488 alone had been added. No adsorption to COM was observed (not shown). Our adsorption studies also included Alexa-labeled THP and fetuin. However, an apparent aggregation of these proteins upon incubation with COM crystals occurred, with no specific proteincrystal interaction observed. These findings are not surprising considering that earlier studies have reported aggregation under certain conditions (see section 1). To confirm the observed differences in adsorption behavior of PTF1 and albumin, intensity profiles of several optical sections were generated and averaged for each of five crystals labeled with polypeptide. These five intensity profiles were averaged again and plotted versus the fractional perimeter as shown in Figure 3. For PTF1, high relative intensity (RI) (up to ∼28 arbitrary units (AU)) values were measured for regions corresponding to the four Langmuir 2009, 25(19), 11635–11646

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edges between {100} and {121} faces (Figure 3a). The RI drops to near zero in the groove regions between the {121} faces implying poor adsorption, whereas RI values of ∼7.5 along the {100} faces is indicative of significant adsorption. The intensity profile for albumin (Figure 3b) is similar to that found for PTF1 ({100}/{121} edges: RI ∼22 AU; along {100} faces: RI ∼5 AU) except that fluorescence is also significant along the {121} faces and the groove regions (RI ∼10 AU) between them. For purposes of comparison, adsorption of poly asp35 was also studied. Resulting intensity profiles reveal no clear association of poly asp35 with particular edges or faces (Figure 3c). There may be a higher affinity of the molecule to {100}/{121} edges and {121} faces, but the general intensity range is relatively small (1520 AU). This outcome is very different from that found for the smaller poly asp10, which exhibits selective adsorption to {121} faces.15 3.3. Precipitation of COM and COD in the Presence of Polyelectrolytes. Calcium oxalate crystals formed in the presence or absence of polyelectrolytes were visualized by SEM. For some macromolecules used in this study, the effects on nucleation and crystal growth were small (e.g., albumin, PTF1); for other polypeptides, however, dramatic effects on precipitation reactions were found (poly asp, OPN, etc.). 3.3.1. Polyelectrolytes with Minor Effects on the Volume of Precipitate. In Figure 4a,b, the precipitation behavior of COM and COD is shown for bikunin, PTF1, and albumin. At LSC and 10 μg/mL polyelectrolyte (Figure 4a), only bikunin shows significant effects on the volume of COM precipitation. With respect to COD, there was no significant alteration in volume of crystal with these proteins. At HSC and 20 μg/mL polyelectrolyte added (Figure 4b), there were no significant effects on the volume of COM precipitation, although all effectors significantly reduced the volume of COD precipitated. Of note is the observation that bikunin promoted COM formation and totally abolished COD, a finding potentially related to a bikunin-mediated generation of COM nucleation sites on substrate surfaces (see also section 3.2, Figure 2h,l). Panels c-e in Figure 4 show scanning electron micrographs of calcium oxalate crystals grown at LSC and in the presence of 10 μg/mL bikunin, albumin, and PTF1, respectively. Images indicate that bikunin inhibits COM growth along the Æ100æ dimension and forms thinner crystals than those of controls (compare Figure 4c and Figure 1a,b). In contrast, growth in the presence of albumin (Figure 4d) and PTF1 (Figure 4e) caused no significant difference in shape and growth dimensions. Panels f and g show growth at HSC and in the presence of 20 μg/mL PTF1 and bikunin, respectively. While PTF1 shows no significant effect on shapes and crystal dimensions (compare to control, Figure 1c, d), addition of bikunin resulted in a dense distribution of COM crystals in three size categories (see Table 1), presumably a result of an increase in nucleation rate. The crystals formed are either small or medium-sized penetration twins like those of LSCcontrols, or rough-surfaced crystals comparable to COM of HSC-controls (see Figure 1). COM crystals are occasionally accompanied by COD, the sizes and shapes of which are similar to those found in HSC controls (compare Figure 4g with Figure 1d). In contrast to bikunin, crystal growth in the presence of AlexaFluor-488, lysozyme, BIF, THP, albumin, or fetuin showed no significant effects on precipitation kinetics or crystal growth, compared to COM or COD of HSC controls (not shown). 3.3.2. Polyelectrolytes with Significant Effects on the Volume of Precipitate. Figure 5 represents the precipitation behavior of calcium oxalates grown in the presence of increasing DOI: 10.1021/la901145d

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Figure 4. Crystallization of COM and COD in the presence of weakly interacting macromolecules. (a, b) Crystallization of COM and COD under (a) low-supersaturation conditions (LSC; [Ca2þ] = [C2O42-] = 1 mM) and under (b) high-supersaturation conditions (HSC; [Ca2þ] = [C2O42-] = 2 mM), in the presence of 10 μg/mL effector at LSC or 20 μg/mL effector at HSC. Effectors: bikunin, PTF1, albumin. (c-e) Scanning electron micrographs of COM grown under LSC using 10 μg/mL of (c) bikunin, (d) albumin, (e) PTF1, and COM and COD grown under HSC using 20 μg/mL of (f) PTF1, (g) bikunin. Note the high amount of COM in (g) suggesting that bikunin increases the nucleation rate of COM, under the given conditions. The scale bar in panel d also applies to panels c, e, and f. #P < 0.05, *P < 0.01 - significantly different from corresponding control (Calculated by Dunnett’s multiple comparison test). Table 1. Size Distribution of COM Crystals Precipitated in the Presence of Bikunin COM size category

Æ001æ dimension [ μm]

shape

4.05(4) ( 0.60(2) small penetration twins 8.12(3) ( 1.28(4) medium penetration twins a b 17.15(9) ( 1.82(2) large spiky habits a Morphologies like those of LSC-controls. b Habits similar to those of HSC-controls. a

concentrations of poly asp10, poly asp35, or poly glu10. All of these polyelectrolytes decrease the volume of precipitated COM drastically, both at LSC (Figure 5a) and at HSC (Figure 5c). Small concentrations of poly asp35 are sufficient to completely abolish COM formation at LSC (0.056 μM; 2 μg/mL) and inhibit it by more than 50% at HSC (0.27 μM; 10 μg/mL). Both poly asp10 and poly glu10 show similar tendencies but at higher molar concentrations, and with poly asp10 being more potent than poly glu10. Interestingly, to achieve a similar volume decrease of precipitated COM at HSC requires similar mass of the effectors, but 3-4 times higher molar concentrations of poly asp10 (0.87 μM; 8 μg/mL) than poly asp35 (0.27 μM; 10 μg/mL). With respect to COD (at LSC), a biphasic dose-response relationship is noted for both poly asp10 and poly asp35 (Figure 5b). It is readily apparent that at lower concentrations (0-1.0 μM), where poly asp inhibits COM formation, the generation of COD correspondingly increases, whereas at higher concentrations (>0.06-1.0 μM) the precipitation of COD is increasingly inhibited. The relative effects of poly glu10 on COM and COD formation are similar to that described for poly asp, although the former peptide is less potent in inhibiting COM formation. At HSC, the precipitated COD volume was not significantly changed using poly asp10, poly asp35 or poly glu10 (not shown; for control, see Figure 4b). The precipitation of COM in the presence of OPN isoforms was performed at LSC (Figure 6a) and at HSC (Figure 6c). Under both conditions, phosphorylated OPN (bOPN, kOPN, mOPN) causes a large decrease of precipitated volume, whereas recOPN shows no significant effect. At LSC, the highly phosphorylated mOPN appears to be a stronger inhibitor than the more 11640 DOI: 10.1021/la901145d

Æ010æ dimension [ μm]

Æ100æ dimension [ μm]

1.16(2) ( 0.111(9) 2.75(0) ( 0.28(8) 4.09(4) ( 0.38(6)

1.12(6) ( 0.15(0) 2.33(5) ( 0.22(4) 6.73(6) ( 0.37(9)

moderately phosphorylated bOPN or kOPN, whereas at HSC the three phosphorylated isoforms resulted in similar precipitated volumes; concentrations of ∼0.14 μM phosphorylated OPN resulted in a ∼40% reduction of COM formation. In comparison, the use of poly asp35, a poly carboxylate with a similar molecular mass, requires the addition of ∼0.21 μM to reach this level (see Figure 5c). The formation of COD at LSC is shown in Figure 6b. As above for the synthetic polypeptides, a dose-response relationship is observed for all OPN isoforms. Increasing OPN concentrations resulted in a significantly increased volume of COD precipitates, while COM formation was strongly inhibited. However, a decrease of COD formation at higher effector concentrations is not indicated. At HSC, formation of COD was not affected significantly by any OPN isoform used (not shown, for control see Figure 4b). 3.3.2.1. Polyelectrolyte Effects on COM Formation. The effects of the polyelectrolytes on crystal shapes are shown in Figure 7. Crystallization was performed at either LSC or HSC. Using poly asp or poly glu under LSC, the generation of COM was observed only occasionally (poly asp35: 0.028 μM, 1 μg/mL; poly glu10: 0.098 μM, 1 μg/mL; see Figure 5a). Under HSC, however, precipitation of COM is not totally inhibited. The crystals formed are either roughly orthorhombic, with highly faceted faces, if grown in the presence of 0.78 μM poly glu10 (8 μg/mL, Figure 7a), or they appear to be porous and oval-shaped in crosssection, if using 0.65 μM poly asp10 (6 μg/mL, Figure 7b). Addition of 0.226 μM (8 μg/mL) poly asp35 generates COM crystals that appear to be a mixture of the habits induced by poly asp10 and poly glu10 (Figure 7c); this finding correlates with the Langmuir 2009, 25(19), 11635–11646

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Figure 5. Crystallization of COM and COD in the presence of poly asp and poly glu. (a,b,c) Crystallization of COM and COD under (a,b) LSC and under (c) HSC. Effectors: poly asp35, poly asp10 and poly glu10. Mass concentrations used at LSC: 1, 2 μg/mL of all polypeptides; 5, 10 μg/mL of poly asp35. Polypeptide addition at HSC: 2, 8, 11 μg/mL of poly glu10; 6, 8 μg/mL of poly asp10; 5, 8, 10, 15 μg/mL of poly asp35. #P{010} faces. RecOPN, kOPN, and bOPN have, however, a much greater specificity for the {100}/{121} edges than does the highly phosphorylated mOPN. Hunter et al. proposed that phosphate groups on osteopontin may be required for efficient interaction with the Ca2þ-rich {100} face, but not for interaction with the crystal edges.7 It is proposed that all these processes are based on electrostatic interactions. The nonphosphorylated protein PTF1 (Figure 2a,b; 3a), on the other hand, adsorbs to COM with a similar profile to that of mOPN,7 suggesting that charge distribution may be also critical. In addition, recent studies have shown that the 10-kDa poly asp10 adsorbs preferentially to {121} faces of COM,15 which are less Ca2þ-rich than {100} faces,16 proposing a process based on both (54) Xu, Y. B.; Carr, P. D.; Guss, J. M.; Ollis, D. L. J. Mol. Biol. 1998, 276, (5), 955-966. (55) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153-203.

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structural alterations of the molecule during adsorption and electrostatic interactions. Adsorption to {010} faces of COM is observed less frequently. This is not surprising, as {010} faces exhibit a surface richer in C2O42- than {100} and {121} faces,13,16 a structural condition that provokes only small attractive forces for acidic polyelectrolytes in the Stern layer. In fact, we observed some {010} adsorption using albumin and poly asp35 and, in an earlier study, poly glu10,15 which might be based on structural alterations and desolvation during adsorption of these large and/ or relatively hydrophilic polyelectrolytes (see Table 2). Similarly, Qiu et al. reported adsorption of kidney OPN to {010}.56 In general, however, the {010} face is the least-preferred binding site. We demonstrate here that kOPN and PTF1 adsorb poorly to {010}, a finding that was also noted for bOPN, recOPN, mOPN, poly asp10, and OPN220-235 peptides in previous studies.7,14,15 Moreover, AFM force measurements in the presence of mOPN showed that carboxylate-functionalized tips interact with COM faces in the order of {100} > {121} > {010}.57 4.2. Precipitation of Calcium Oxalates in the Presence of Polyelectrolytes. Precipitation in the presence of polyelectrolytes was found to be strongly controlled by the total hydrophilicity of the polyelectrolyte. No or weak effects on precipitation were characteristic of hydrophobic and aggregated molecules, but also of polyelectrolytes with a total hydrophilicity in the range of 200-850 kJ/mol and net negative charges below -35. These molecules (including recOPN) showed adsorption but did not significantly affect precipitation reactions. However, acidic polyelectrolytes (kOPN, bOPN, mOPN, poly glu10, poly (56) Qiu, S. R.; Wierzbicki, A.; Orme, C. A.; Cody, A. M.; Hoyer, J. R.; Nancollas, G. H.; Zepeda, S.; De Yoreo, J. J., Molecular modulation of calcium oxalate crystallization by osteopontin and citrate. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, (7), 1811-1815. (57) Sheng, X.; Jung, T.; Wesson, J. A.; Ward, M. D. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, (2), 267-272.

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asp10, poly asp35) with total hydrophilicities >850 kJ/mol and net negative charges > -60 affected precipitation processes considerably. The inhibitory effects of highly hydrophilic polypeptides are apparently due to a direct interaction with the crystal rather than complexation of Ca ions. For example, the highest OPN concentrations used here are sufficient to bind only 0.03% of the Ca present.58 4.2.1. Polyelectrolytes with Small Effects on Precipitation. In the presence of bikunin, precipitation of COM was significantly increased, particularly at LSC, and COD formation at HSC was strongly reduced (Figure 4a-c,g). Taken together with our demonstration of nonspecific adsorption (Figure 2h,l), this suggests that dissolved bikunin adsorbs to the glass surface, generates nucleation sites for COM and catalyzes the crystallization process. After local assembly of the crystalline phase, which occurs at higher frequencies than under control conditions (increased nucleation rate), unhindered growth, preferentially from the {100} face, results in the typical morphology of penetration twins. This process also prevents COD formation. However, these findings are in contrast to results reported in the literature. Bikunin proteins have been shown to inhibit crystallization and aggregation of calcium oxalates.2,8,59,60 The reasons for the conflicting data might be a result of the reaction environment. Lack of bikunin in the solution bulk after nucleation might have led to noninhibition of COM precipitation. Significant effects of albumin and PTF1 on COM precipitation were not observed. However, addition of either protein decreased the formation of COD at HSC (Figure 4b). Our studies confirm results showing that albumin binds to calcium oxalates but does not inhibit crystal growth.5,61,62 It has been reported that albumin promotes nucleation,63,64 which could not be confirmed in this study. It is postulated that the function of both PTF1 and albumin is the coating of crystals to prevent crystal aggregation and adhesion to epithelial cell surfaces. The adsorption patterns imply, however, that higher concentrations of PTF1 than albumin are needed for this task. This hypothesis is supported by recent studies demonstrating that calcium oxalate aggregation is inhibited by albumin in a concentration-dependent manner.63,65,66 Considering the findings above, it appears that proteins with a total hydrophilicity in the range of 200-850 kJ/mol and net negative charges between -9 and -35 are within a “proteincharacteristics window” where polyelectrolytes may adsorb but not significantly affect precipitation reactions in the urinary tract. 4.2.2. Polyelectrolytes Showing Significant Effects on Precipitation. Those polyelectrolytes of stronger acidity or, more precisely, of higher net negative charge and total hydrophilicity, significantly inhibit growth and precipitation of COM (Figures 5-7). They also showed distinct adsorption to edges (58) Chen, Y.; Bal, B. S.; Gorski, J. P. J. Biol. Chem. 1992, 267, (34), 24871-24878. (59) Medetognon-Benissan, J.; Tardivel, S.; Hennequin, C.; Daudon, M.; Drueke, T.; Lacour, B. Urol. Res. 1999, 27, (1), 69-75. (60) Dean, C.; Kanellos, J.; Pham, H.; Gomes, M.; Oates, A.; Grover, P.; Ryall, R. Clin. Sci. 2000, 98, (4), 471-480. (61) Dussol, B.; Geider, S.; Lilova, A.; Leonetti, F.; Dupuy, P.; Daudon, M.; Berland, Y.; Dagorn, J. C.; Verdier, J. M. Urol. Res. 1995, 23, (1), 45-51. (62) Ryall, R. L.; Harnett, R. M.; Hibberd, C. M.; Edyvane, K. A.; Marshall, V. R. Uro. Res. 1991, 19, (3), 181-188. (63) Hess, B.; Meinhardt, U.; Zipperle, L.; Giovanoli, R.; Jaeger, P. Urol. Res. 1995, 23, (4), 231-238. (64) Cerini, C.; Geider, S.; Dussol, B.; Hennequin, C.; Daudon, M.; Veesler, S.; Nitsche, S.; Boistelle, R.; Berthezene, P.; Dupuy, P.; Vazi, A.; Berland, Y.; Dagorn, J. C.; Verdier, J. M. Kidney Int. 1999, 55, (5), 1776-1786. (65) Grover, P. K.; Moritz, R. L.; Simpson, R. J.; Ryall, R. L. Eur. J. Biochem. 1998, 253, (3), 637-644. (66) Edyvane, K. A.; Ryall, R. L.; Marshall, V. R. Clin. Chim. Acta 1986, 157, (1), 81-87.

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and/or faces of pregrown crystals (section 4.1.4). Under LSC, decreasing potency on COM precipitation occurred in the order: recOPN < kOPN, bOPN, mOPN < poly glu10, poly asp10, poly asp35, showing that strongest inhibition results from synthetic polypeptides, which have the lowest isoelectric points but not necessary the highest hydrophilicity values (see Table 2). Under HSC, however, the potency order of decreased precipitation was recOPN < poly glu10, poly asp10 < kOPN, bOPN, mOPN, poly asp35, a progression that is directly correlated to an increase of the effector hydrophilicity. We propose that the observed differences between the precipitation systems LSC and HSC are linked to the two part-processes of crystallization, nucleation and growth, and the ability of individual polypeptides to affect these processes. It appears that both the synthetic polypeptides and the phosphorylated OPN isoforms have a strong effect on the nucleation process. Both types of polyelectrolytes are potent enough to reduce crystallization of COM to very low levels. For instance, low concentrations of all synthetic polypeptides or mOPN totally abolished COM formation and, therefore, nucleation processes under LSC (Figures 5 and 6). A similar observation is reported by Wang et al.12 who show the high efficiency of phosphorylated OPN in inhibiting COM nucleation, and by Wesson et al.67 who demonstrated that poly asp and poly glu are potent inhibitors of COM nucleation. However, growth processes that appear to be more affected by hydrophilicity than nucleation are quite relevant under HSC (compared to growth under LSC), because high ion concentrations result in crystal growth over longer periods of time. During that time-span, the outer sphere of forming crystals is covered by these highly hydrophilic molecules, which are in very close proximity to the crystal surface. Growth itself, however, is affected by pinning growth steps and by hindering the diffusion of lattice-ions to the crystal surface.12,68,69 With respect to COD formation, recOPN has no significant effect at LSC or HSC. However, the addition of the phosphorylated OPN isoforms and synthetic polypeptides all resulted in a significant increase of the precipitated COD volume under LSC, but not under HSC. It is interesting to note that there is a tendency to increase COD formation by increasing the total hydrophilicity of the molecules used (Table 2). It may be the case that stronger poly acids catalyze the formation of COD (as a secondary or side reaction) or that COM formation is suppressed so vigorously that the more-soluble COD phase is allowed to form. Of physiological relevance is the fact that all native OPN isoforms, but no other urinary protein used in this study, decrease COM formation and favor the precipitation of COD, which is less adherent to cell surfaces.5,70 This outcome supports findings by Kumura et al. who report that mOPN is one of the most important polyelectrolytes in preventing crystallization in breast milk.71 Although it was shown that phosphorylation is not necessary for OPN adsorption to COM (see section 4.1.4) phosphate groups appear to be crucial for OPN to inhibit COM precipitation. It appears that not only the presence of phosphate groups, but also the overall hydrophilicity are important factors in controlling crystallization processes. Differences in inhibition are dramatic, all phosphorylated and more hydrophilic isoforms decrease the (67) Wesson, J. A.; Worcester, E. M.; Kleinman, J. G. J. Urol. 2000, 163, (4), 1343-1348. (68) Vekilov, P. G. Cryst. Growth Des. 2007, 7, (12), 2796-2810. (69) Chausov, F. F. Theor. Found. Chem. Eng. 2008, 42, (2), 179-186. (70) Martin, X.; Smith, L. H.; Werness, P. G. Kidney Int. 1984, 25, (6), 948-952. (71) Kumura, H.; Minato, N.; Shimazaki, K. I. J. Dairy Res. 2006, 73, (4), 449-453.

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precipitated COM volume (90-00% at LSC, ∼50% at HSC; see section 3.3.2), but recOPN does not. These observations confirm earlier studies showing that native OPN is a potent inhibitor of nucleation and growth15,72 and that recOPN adsorbs to crystals without showing any effect on their growth.7,13 Very recently, it was also shown that phosphorylation of OPN peptides increased their potency to inhibit COM crystallization.12,14 Identification of the amino acids involved in the interaction indicated, however, that predominantly the carboxylate groups of glutamic and aspartic acids are closest to crystal faces.14 For OPN, therefore, accumulating evidence suggests that phosphate groups provide a strong negative charge that electrostatically forces the molecule closer to crystal faces but that carboxy lates, perhaps for stereochemical reasons, are capable of more specific interactions with cations within the Stern layer of the crystal.7,13,15 For polypeptides completely composed of glutamic or aspartic acids (poly glu, poly asp) the driving force for close interactions can only come from the carboxylate groups. We propose that these polypeptides are electrostatically driven in very close proximity to the crystal face, where a high number of aspartic or glutamic acids interact with cations within the Stern layer of the crystal, a scenario that leads to an inhibited crystallization by blocking lattice-ion addition (perhaps stereochemically). Our studies on poly asp and poly glu give experimental support to this hypothesis. These effectors decreased the volumes of precipitated COM up to ∼100% at LSC and 50-65% at HSC (see section 3.3.2) and showed inhibitor potency for poly asp not achieved by any other polyelectrolyte used in this study. These findings confirm earlier studies that showed increasing growth inhibition of COM73 and calcium carbonates74 by increasing the number of aspartic acid residues in synthetic polypeptides. In fact, asp-rich proteins and acidic polypeptides containing repeating sequences rich in either asp or asp and glu are reported to be important for controlling crystallization of biominerals.75 4.3. Polyelectrolyte Interaction at the Solid/Liquid Interface. Hydrophilicity is a measure that describes the effects of polypeptides on water-mediated interactions. During adsorption of proteins to crystals, free energy changes reflect structural rearrangements in the protein molecule, desolvation and dehydration (in part) of the solid/liquid interface and redistribution of charged groups in the interfacial layer.53 More precisely, the change in entropy and enthalpy mirrors a reorganization process by which water accommodates the solutes, the nucleating and growing crystal and the polypeptides.39,68,76 Recently, Dimova et al.77 observed an increase of entropy in the bulk solution during polymer-calcite interactions and found strong evidence that this increase is directly correlated with the hydrophilicity of the polymer. The authors explain these findings by the release of water molecules from the interfacial layer during adsorption. Furthermore, molecular dynamics (MD) simulations suggest that (72) Worcester, E. M.; Snyder, C.; Beshensky, A. M. J. Am. Soc. Nephrol. 1995, 6, (3), 956. (73) Wang, L. J.; Qiu, S. R.; Zachowicz, W.; Guan, X. Y.; DeYoreo, J. J.; Nancollas, G. H.; Hoyer, J. R. Langmuir 2006, 22, (17), 7279-7285. (74) Elhadj, S.; Salter, E. A.; Wierzbicki, A.; De Yoreo, J. J.; Han, N.; Dove, P. M. Cryst. Growth Des. 2006, 6, (1), 197-201. (75) Addadi, L. a. W., S. Proc. Natl. Acad. Sci. U. S. A. 1985, 82, 4110-4114. (76) Navrotsky, A. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, (33), 12096-12101. (77) Dimova, R.; Lipowsky, R.; Mastai, Y.; Antonietti, M. Langmuir 2003, 19, (15), 6097-6103. (78) Makarov, V. A.; Andrews, B. K.; Smith, P. E.; Pettitt, B. M. Biophys. J. 2000, 79, (6), 2966-2974. (79) Makarov, V.; Pettitt, B. M.; Feig, M. Acc. Chem. Res. 2002, 35, (6), 376-384.

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an increase in the structuring of water at the molecular interface during such processes can produce a reduction in the rate of desolvation.78,79 Surface X-ray diffraction measurements using synchrotron radiation (SR-XRD)80,81 and MD simulations82,83 found that two or more different dense water layers can exist within the solid/liquid interface, such that density and degree of order decreased with increasing distance from the crystal surface. Hydrophilic interactions, therefore, depend on how water molecules interact with the peptides via hydrogen, polar, van der Waals, and ionic bonds, but also depend on hydrophobic parts of the peptides that present sites unfavorable for water interactions, thus restricting water restructuring.84 Therefore, in essence, hydrophilicity can be used as a proxy to describe the “ability” of a polypeptide to interact with a solid/liquid interface. A relative high hydrophilicity is, however, not the decisive factor for affecting crystal growth. Hydrophilicity is rather a mediator for effects on crystallization; it can be considered as a necessary but not as a sufficient characteristic for effects on crystal growth. The actual interference in a crystallizing system, such as in inhibiting growing steps on crystal faces, is a process caused by direct protein-crystal interactions.24,68 A dense layer of adsorbate can result in a finite number of pinned growth steps and will, at the critical surface coverage, lead to growth inhibition.69,74 These processes show relatively high binding energies and are based on electrostatic peptide-crystal interactions, which can also be stereochemical in nature.

5. Conclusions We have shown that basic, relatively hydrophobic, macromolecules have no tendency to adsorb to COM crystals, whereas more acidic macromolecules, which have higher hydrophilicities, adsorb either to all faces or preferentially to {100}/{121} edges and {100} faces of COM. However, adsorbing molecules do not necessarily cause inhibition of crystal growth or precipitation. The crucial factor in affecting crystallization is the degree of hydrophilicity and the presence of carboxylates and/or phosphate groups. A combination of high hydrophilicity and high number of carboxylates (e.g., poly asp, poly glu) or high hydrophilicity and highly phosphorylated (OPN isoforms) show a strong impact on precipitation. Weakly hydrophilic polypeptides, such as albumin or PTF1, adsorb without affecting precipitation processes. We propose that these weakly interacting molecules coat crys tals in the urinary tract and, by electrostatic repulsion, inhibit both crystal aggregation and adherence to the epithelial cell surface. Such a scenario would support processes where crystals are more easily flushed out of the nephrons in the tubular fluid stream. Acknowledgment. We gratefully acknowledge the expert technical assistance of Kari Ann Orlay and Honghong Chen. We thank Todd Simpson and Nancy Bell (Faculty of Science, Western Nanofabrication Facility), University of Western Ontario, for scanning electron microscopy. We are grateful to Jeffrey Weitz, McMasters University, Hamilton, Canada for providing prothrombin fragment, and to Willi JahnenDechent, RWTH Aachen University, Germany, for helpful (80) Arsic, J.; Kaminski, D.; Poodt, P.; Vlieg, E. Phys. Rev. B 2004, 69, (24), 245406 1-5. (81) Reedijk, M. F.; Arsic, J.; Hollander, F. F. A.; de Vries, S. A.; Vlieg, E. Phys. Rev. Lett. 2003, 90, (6), 066103 1-4. (82) Skelton, A. A.; Walsh, T. R. Mol. Simul. 2007, 33, (4-5), 379-389. (83) Yang, M. H., J.; Stipp, S. L. S. Mineral. Mag. 2008, 72, (1), 295-299. (84) Chandler, D. Nature 2005, 437, (7059), 640-647.

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discussions about fetuin A- crystal interactions. The studies reported here were supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Ontario Research and

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Development Challenge Fund and the Network for Oral Research Training and Health. S.M. thanks the CRC program of the Government of Canada, and E.S.S. thanks the Danish Dairy Research Foundation.

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