Bernd Grohe

The flexible polyelectrolyte hypothesis of protein−biomineral interaction

Biomineralization is characterized by a high degree of control over the location, nature, size, shape, and orientation of the crystals formed. For many years, it has been widely believed that the exquisitely precise nature of crystal formation in biological tissues is the result of stereochemically specific interactions between growing crystals and extracellular matrix proteins. That is, the ability of many mineralized tissue proteins to adsorb to particular faces of biominerals has been attributed to a steric and electrical complementarity between periodic regions of the polypeptide chain and arrays of ions on the crystal face. In recent years, however, evidence has accumulated that many mineral-associated proteins lack periodic structure even when adsorbed to crystals. It also appears that protein-crystal interactions involve a general electrostatic attraction rather than arrays of complementary charges. In the present work, we review these studies and present some relevant new findings involving the mineral-modulating phosphoprotein osteopontin. Using molecular dynamics simulations, we show that the adsorption of osteopontin peptides to hydroxyapatite crystals does not involve a unique conformation of the peptide molecule, and that the adsorbed peptides are not aligned with rows of Ca(2+) ions on the crystal face. Further, we show that the interface between osteopontin peptides and calcium oxalate monohydrate crystals consists of peptide regions of high electronegativity and crystal faces of high electropositivity. Collectively, the above-mentioned studies suggest that interactions between mineral-modulating proteins and biologically relevant crystals are primarily electrostatic in nature, and that molecular disorder assists these proteins in forming multiple bonds with cations of the crystal face.

Role of Phosphate Groups in Inhibition of Calcium Oxalate Crystal Growth by Osteopontin

Osteopontin (OPN) inhibits the growth of calcium oxalate monohydrate (COM) and other crystal phases in a phosphorylation-dependent manner. In the present study, the role of OPN phosphate groups in adsorption to, incorporation into and inhibition of COM crystals was studied by comparing OPN isoforms differing in phosphorylation. OPN isoforms purified from rat bone (bOPN), which contains 10 phosphates, and cow milk (mOPN), which contains 25 phosphates, were compared with rat recombinant OPN (rOPN), which is not phosphorylated. Using fluorescence-labeled proteins and confocal microscopy, we show that mOPN and rOPN, like bOPN, adsorb preferentially to the edges between {100} and {121} faces of preformed COM crystals, and to a lesser extent to the {100} and {121} faces. Using scanning electron microscopy, we show that growth of COM in the presence of bOPN or mOPN results in a ‘dumbbell’ morphology, whereas crystals grown with rOPN are only slightly affected. COM crystals grown in the presence of low concentrations of fluorescence-labeled bOPN incorporate the protein into the crystal lattice. In crystals imaged in the {010} plane, incorporation of bOPN results in a cross-shaped pattern of fluorescence, consistent with preferential adsorption to {100}/{121} edges throughout the growth process.

Novel findings in patients with primary hyperoxaluria type III and implications for advanced molecular testing strategies

Identification of mutations in the HOGA1 gene as the cause of autosomal recessive primary hyperoxaluria (PH) type III has revitalized research in the field of PH and related stone disease. In contrast to the well-characterized entities of PH type I and type II, the pathophysiology and prevalence of type III is largely unknown. In this study, we analyzed a large cohort of subjects previously tested negative for type I/II by complete HOGA1 sequencing. Seven distinct mutations, among them four novel, were found in 15 patients. In patients of non-consanguineous European descent the previously reported c.700+5G>T splice-site mutation was predominant and represents a potential founder mutation, while in consanguineous families private homozygous mutations were identified throughout the gene. Furthermore, we identified a family where a homozygous mutation in HOGA1 (p.P190L) segregated in two siblings with an additional AGXT mutation (p.D201E). The two girls exhibiting triallelic inheritance presented a more severe phenotype than their only mildly affected p.P190L homozygous father. In silico analysis of five mutations reveals that HOGA1 deficiency is causing type III, yet reduced HOGA1 expression or aberrant subcellular protein targeting is unlikely to be the responsible pathomechanism. Our results strongly suggest HOGA1 as a major cause of PH, indicate a greater genetic heterogeneity of hyperoxaluria, and point to a favorable outcome of type III in the context of PH despite incomplete or absent biochemical remission. Multiallelic inheritance could have implications for genetic testing strategies and might represent an unrecognized mechanism for phenotype variability in PH.

Label-free mapping of osteopontin adsorption to calcium oxalate monohydrate crystals by tip-enhanced Raman spectroscopy.

In the ectopic biomineralization of calcium oxalate kidney stones, the competition between calcium oxalate monohydrate (COM) formation and its inhibition by the phosphoprotein osteopontin (OPN) plays a key role in COM stone-forming processes. To get more insights into these processes, tip-enhanced Raman spectroscopy (TERS) was used to provide surface-specific information about the adsorption of OPN to faces of COM crystals. In TERS, the surface plasmon resonance of a metallic AFM tip is locally excited when the tip is placed in the optical near-field of a laser focused on the crystal surface. Excitation of this localized surface plasmon resonance allows the enhancement of the Raman signal as well as the improvement of the spatial resolution beyond the diffraction limit of the light. As TERS works label free and noninvasively, it is an excellent technique to study the distribution of adsorbed proteins on crystal faces at the submicrometer scale. In the present work, we generated Raman intensity maps indicating high spatial resolution and a distinct variation in relative peak intensities. The collected TERS spectra show that the OPN preferentially adsorbs to edges and faces at the ends of COM crystals (order: {100}/{121} edge > {100} face > {100}/{010} edge ≈ {121}/{010} edge > {010} face) providing also relevant information on the inhibition of crystal growth. This study demonstrates that TERS is an excellent technique for detailed investigations of biomolecules adsorbed, layered, or assembled to a large variety of surfaces and interfaces.

Mechanism of inhibition of calcium oxalate crystal growth by an osteopontin phosphopeptide

Osteopontin (OPN) inhibits the nucleation and/or growth of several biominerals, including hydroxyapatite (HA) and calcium oxalate monohydrate (COM), and is thought to function in the prevention of soft-tissue calcification. In previous studies, pOPAR, a peptide corresponding to amino acids 65–80 of rat bone OPN (pSHDHMDDDDDDDDDGD), was shown to be a potent inhibitor of HA crystal growth. We now show that formation of COM in the presence of this peptide results in plate-shaped crystals with rounded ends and scalloped {100} faces. Measurement of crystal dimensions revealed that the pOPAR inhibits growth of COM faces in the order {100} > {121} > {010}. Crystal growth inhibitors are believed to act by adsorbing to growth steps, sites at which lattice-ion addition is energetically favoured. To test this hypothesis, we performed molecular dynamics (MD) simulations of pOPAR adsorption to {121} steps on a {100} face and {121} steps on an {010} face. In the former case, the peptide adsorbs to the {100} (terrace) plane in preference to the {121} (riser) plane; in the latter, the peptide adsorbs to the {121} (riser) plane in preference to the {010} (terrace) plane. These studies represent the first use of MD to study the interaction between an inhibitor and crystal steps. Contrary to the prevailing belief that crystal growth inhibitors adsorb to both lattice planes of a step, we show that pOPAR interacts preferentially to either the terrace or the riser, depending on which is more cationic.

Phosphorylation of Osteopontin Peptides Mediates Adsorption to and Incorporation into Calcium Oxalate Crystals

Phosphorylated peptides of osteopontin (OPN) have been shown to inhibit the growth of the {100} face of calcium oxalate monohydrate (COM). The inhibitory potency has been shown to be dependent on the phosphate content of the peptide. The purpose of this study is to better understand the means by which phosphate groups promote crystal growth inhibition by OPN peptides. Peptides of rat bone OPN 220–235 peptides have been synthesized with zero (P0), 1 (P1) or 3 (P3) phosphate modifications. COM crystals were grown in the presence of 0.1–10 μg of P0, P1 or P3. P0 incorporation into COM crystals was evident at 10 μg/ml of peptide, whereas the phosphorylated peptides P1 and P3 were incorporated at all tested concentrations. At 5 μg/ml of P3, COM crystals exhibited a ‘dumbbell’ morphology. To study the peptide-mineral interaction, surface frequency plots were constructed from molecular dynamics simulations of OPN peptide adsorption. Carboxylate and phosphate groups were found to adsorb in specific orientations to the COM {100} surface. In conclusion, it appears that the phosphate groups on OPN peptides are capable of interacting with the COM {100} surface. This interaction appears to increase the adsorption energy of the peptide to the surface, thus enhancing its inhibitory potency.

Peptides of Matrix Gla Protein Inhibit Nucleation and Growth of Hydroxyapatite and Calcium Oxalate Monohydrate Crystals

Matrix Gla protein (MGP) is a phosphorylated and γ-carboxylated protein that has been shown to prevent the deposition of hydroxyapatite crystals in the walls of blood vessels. MGP is also expressed in kidney and may inhibit the formation of kidney stones, which mainly consist of another crystalline phase, calcium oxalate monohydrate. To determine the mechanism by which MGP prevents soft-tissue calcification, we have synthesized peptides corresponding to the phosphorylated and γ-carboxylated sequences of human MGP in both post-translationally modified and non-modified forms. The effects of these peptides on hydroxyapatite formation and calcium oxalate crystallization were quantified using dynamic light scattering and scanning electron microscopy, respectively. Peptides YGlapS (MGP1-14: YγEpSHEpSMEpSYELNP), YEpS (YEpSHEpSMEpSYELNP), YGlaS (YγESHESMESYELNP) and SK-Gla (MGP43-56: SKPVHγELNRγEACDD) inhibited formation of hydroxyapatite in order of potency YGlapS > YEpS > YGlaS > SK-Gla. The effects of YGlapS, YEpS and YGlaS on hydroxyapatite formation were on both crystal nucleation and growth; the effect of SK-Gla was on nucleation. YGlapS and YEpS significantly inhibited the growth of calcium oxalate monohydrate crystals, while simultaneously promoting the formation of calcium oxalate dihydrate. The effects of these phosphopeptides on calcium oxalate monohydrate formation were on growth of crystals rather than nucleation. We have shown that the use of dynamic light scattering allows inhibitors of hydroxyapatite nucleation and growth to be distinguished. We have also demonstrated for the first time that MGP peptides inhibit the formation of calcium oxalate monohydrate. Based on the latter finding, we propose that MGP function not only to prevent blood-vessel calcification but also to inhibit stone formation in kidney.

Citrate Modulates Calcium Oxalate Crystal Growth by Face-Specific Interactions

Because of its ability to inhibit the growth of calcium oxalate monohydrate (COM) crystals, citrate plays an important role in preventing the formation of kidney stones. To determine the mechanism of inhibition, we studied the citrate-COM interaction using a combination of microscopic and simulation techniques. Using scanning confocal interference microscopy, we found that addition of citrate preferentially inhibits crystal growth in <100> and, to a lesser extent, <001> directions, suggesting that citrate adsorbs to the faces of COM in the order {100} > {121} > {010}. Scanning electron microscopy showed that the resulting crystals are plate shaped, with large {100} faces and rounded ends. Molecular-dynamics simulations predicted, however, that citrate interacts with the faces of COM in a different order, i.e. {100} > {010} > {121}. Our simulations showed that citrate molecules align with the rows of Ca2+ ions on the {010} face but do not form close contacts, presumably because of electrostatic repulsion by the carboxylate groups that project from the Ca2+-rich plane. We propose that this weak interaction is responsible for citrate’s limited inhibition of COM growth in <010> directions. Overall, these findings indicate that electrostatic interactions with the Ca2+-rich faces of COM crystals are responsible for the growth-modulating properties of citrate.

Cooperation of phosphates and carboxylates controls calcium oxalate crystallization in ultrafiltered urine

Osteopontin (OPN) is one of a group of proteins found in urine that are believed to limit the formation of kidney stones. In the present study, we investigate the roles of phosphate and carboxylate groups in the OPN-mediated modulation of calcium oxalate (CaOx), the principal mineral phase found in kidney stones. To this end, crystallization was induced by addition of CaOx solution to ultrafiltered human urine containing either human kidney OPN (kOPN; 7 consecutive carboxylates, 8 phosphates) or synthesized peptides corresponding to residues 65–80 (pSHDHMDDDDDDDDDGD; pOPAR) or 220–235 (pSHEpSTEQSDAIDpSAEK; P3) of rat bone OPN. Sequence 65–80 was also synthesized without the phosphate group (OPAR). Effects on calcium oxalate monohydrate (COM) and dihydrate (COD) formation were studied by scanning electron microscopy. We found that controls form large, partly intergrown COM platelets; COD was never observed. Adding any of the polyelectrolytes was sufficient to prevent intergrowth of COM platelets entirely, inhibiting formation of these platelets strongly, and inducing formation of the COD phase. Strongest effects on COM formation were found for pOPAR and OPAR followed by kOPN and then P3, showing that acidity and hydrophilicity are crucial in polyelectrolyte-affected COM crystallization. At higher concentrations, OPAR also inhibited COD formation, while P3, kOPN and, in particular, pOPAR promoted COD, a difference explainable by the variations of carboxylate and phosphate groups present in the molecules. Thus, we conclude that carboxylate groups play a primary role in inhibiting COM formation, but phosphate and carboxylate groups are both important in initiating and promoting COD formation.

Kidney stones in primary hyperoxaluria: new lessons learnt.

To investigate potential differences in stone composition with regard to the type of Primary Hyperoxaluria (PH), and in relation to the patient’s medical therapy (treatment naïve patients versus those on preventive medication) we examined twelve kidney stones from ten PH I and six stones from four PH III patients. Unfortunately, no PH II stones were available for analysis. The study on this set of stones indicates a more diverse composition of PH stones than previously reported and a potential dynamic response of morphology and composition of calculi to treatment with crystallization inhibitors (citrate, magnesium) in PH I. Stones formed by PH I patients under treatment are more compact and consist predominantly of calcium-oxalate monohydrate (COM, whewellite), while calcium-oxalate dihydrate (COD, weddellite) is only rarely present. In contrast, the single stone available from a treatment naïve PH I patient as well as stones from PH III patients prior to and under treatment with alkali citrate contained a wide size range of aggregated COD crystals. No significant effects of the treatment were noted in PH III stones. In disagreement with findings from previous studies, stones from patients with primary hyperoxaluria did not exclusively consist of COM. Progressive replacement of COD by small COM crystals could be caused by prolonged stone growth and residence times in the urinary tract, eventually resulting in complete replacement of calcium-oxalate dihydrate by the monohydrate form. The noted difference to the naïve PH I stone may reflect a reduced growth rate in response to treatment. This pilot study highlights the importance of detailed stone diagnostics and could be of therapeutic relevance in calcium-oxalates urolithiasis, provided that the effects of treatment can be reproduced in subsequent larger studies.