Crystallization of Calcium Oxalates Is Controlled by Molecular Hydrophilicity and Specific Polyanion-Crystal Interactions

Abstract

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.

Citrate Modulates Calcium Oxalate Crystal Growth by Face-Specific Interactions

“…An affinity of citrate for the {100} face is not surprising as this face is the most Ca 2+ rich [Jung et al, 2004] and the preferred site of interaction for acidic peptides and proteins Langdon et al, 2009]. The predicted interaction of citrate with the {010} face of COM is more surprising as this face is the least preferred by acidic proteins and peptides Taller et al, 2007;Grohe et al, 2009;Langdon et al, 2009]. Also, the SEM and SCIM data reported above indicate that citrate interacts with COM faces in the order {100} 1 {121} 1 {010}.…”

“…In previous studies, we have used confocal microscopy, scanning electron microscopy (SEM) and molecular dynamics (MD) to study interactions between polyelectrolytes and COM crystals Taller, 2007;Grohe et al, 2009;Hunter et al, 2009]. In general, these interactions are dominated by electrostatic forces.…”

Osteopontin Signals through Calcium and Nuclear Factor of Activated T Cells (NFAT) in Osteoclasts

“…The recombinant protein was purified by nickel affinity chromatography and fast protein liquid chromatography. The purified protein was subjected to amino acid analysis and selected samples were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, giving a relative molecular mass of 36,100 (26). OPN in which Arg-Gly-Asp (RGD) was mutated to Lys-Ala-Glu (KAE) (OPN-KAE) was prepared using the QuikChange™ Site-directed Mutagenesis System (Stratagene).…”

Enhanced osteogenesis of bone morphology protein-2 in 2- N ,6- O -sulfated chitosan immobilized PLGA scaffolds

“…As a result, surface hydrophilicity of S-PLGA scaffold was improved by 26SCS immobilization. It is also reported that scaffold with a hydrophilic surface is more favorable for cell attachment and proliferation [25] as well as the binding of some proteins [32].…”

“…2B) consisted of three components which were centered at ∼289 eV, ∼287 eV and ∼285 eV and attributed to ester/carboxylic bonds (O C O), ether bonds ( C O ) and carbon-hydrogen bonds ( C H ), respectively [26,32]. Only a slight variation could be seen in chemical bonds groups, indicating aminolysis induced small changes on scaffolds surface (Table S1, supporting information).…”