Bovine Dentin Phosphophoryn Research Articles

Protein dynamics of bovine dentin phosphophoryn

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and o-phosphoseryl [Ser(P)] residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentin. The elucidation of the structure-function relationship of phosphophoryn has been a challenge because of its high-molecular weight, high negative charge, repetitive sequence, and lability. We have used the dynamic behavior of the (1)H NMR signal at 600 MHz to provide insight into the molecular dynamics of phosphophoryn. Our results indicate that phosphophoryn is a molecule of uniformly high mobility, thus belonging to a recently identified class of intrinsically disordered proteins that are characterized by sequences of low complexity and rich in polar and charged residues. The significance of our results is that phosphophoryn, because of its uniform nature has the potential to be replaced by biomimetic synthetic peptide analogs that together with amorphous calcium phosphate may lead to the development of novel, nontoxic, apatite-based dental restorative materials.

Association of bovine dentine phosphophoryn with collagen fragments

Bovine dentine phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser(P)) residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentine. Phosphophoryn was purified from bovine dentine using EDTA extraction, Ca(2+) precipitation, anion exchange and size exclusion chromatography. The purified protein migrated on SDS-PAGGE as a single band. The protein was dephosphorylated using a chelex alkaline dialysis procedure, repurified using anion exchange and size exclusion chromatography and then subjected to cleavage with trypsin. The digest was subjected to reversed-phase HPLC and analysed by Q-TOF mass spectrometry. The only non-trypsin peptides that could be identified were two collagen Type I alpha2 peptides whose sequence was determined by fragmentation analysis. The association of collagen fragments with highly purified phosphophoryn suggests that the EDTA extraction method yields BDP that is strongly bound to collagen fragments. This association now helps explain discrepancies in molecular weight and amino acid composition data for various phosphophoryn preparations compared with the same data calculated from the C-terminal extension of mouse, rat and human dentine sialophosphoprotein (DSPP) gene products. Analysis of the mutation pattern of the clinical disorder Osteogenesis Imperfecta within the region enclosed by the identified collagen fragments reveals that phosphophoryn associates with a segment of collagen that is crucial for structure and/or function.

D-Aspartic acid in bovine dentine non-collagenous phosphoprotein

In tooth dentine, owing to its slow metabolism after its formation, racemized and transformed d-aspartic acid remains in the tissue and accumulates with age. However, no dentinal proteins which contain d-aspartic acid have been identified. In this study, a non-collagenous phosphoprotein was purified from bovine dentine. Its molecular mass was about 130 kDa and its amino acid composition was very similar to that of bovine dentine phosphophoryn. The purified protein contained a large proportion of aspartic acid residues and some of them were stereoinverted from the l-isomer to the d-isomer. The d-/ l-aspartic acid ratio of dentine non-collagenous phosphoproteins purified from 8-month-old fetal, postnatal and 1-year-old bovine first incisors showed that the stereoinversion tended to increase with age. These results suggest that the purified non-collagenous phosphoprotein is a candidate for the protein in dentine containing d-aspartic acid.

N-terminal sequence analysis of bovine dentin phosphophoryn after conversion of phosphoseryl to S-propylcysteinyl residues.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser[P]) residues, is synthesized by odontoblasts and is believed to be involved in matrix-mediated biomineralization of dentin. We have purified BDP, using selective precipitation and ion exchange chromatography, from an EDTA soluble dentin extract and converted the Ser(P) residues to S-propylcysteinyl residues that are stable to Edman degradation, facilitating the determination of the amino acid sequence of the N-terminal 38 residues. After the initial Asp-Ser(P)-Pro-Asn-Ser(P)-Ser(P)-Asp-Glu-Ser(P)-Asn-Gly-, the sequence contained the repeated motifs Asp-Ser(P) and Asp-Ser(P)-Ser(P). Purified BDP migrated as a single band on gradient SDS-PAGE with an apparent molecular weight of 156 kDa. This value was consistent with the molecular weight of the dephosphorylated protein of 105 kDa determined by means of MALDI mass spectrometry.

Mineral induction by immobilized phosphoproteins

Dentin phosphoproteins are thought to have a primary role in the deposition of mineral on the collagen of dentin. In this study we determined the type of binding between collagen and phosphoproteins necessary for mineral formation onto collagen fibrils and whether the phosphate esters are required. Bovine dentin phosphophoryn or phosvitin from egg yolk were immobilized on reconstituted skin type I collagen fibrils by adsorption or by covalent cross-linking. In some samples the ester phosphate was removed from the covalently cross-linked phosphoproteins by treatment with acid phosphatase. All samples were incubated at 37°C in metastable solutions that do not spontaneously precipitate. Reconstituted collagen fibrils alone did not induce mineral formation. The phosphoproteins adsorbed to the collagen fibrils desorbed when the mineralization medium was added, and mineral was not induced. The mineral induced by the cross-linked phosphoproteins was apatite, and the crystals were confined to the surface of the collagen fibrils. With decreasing medium saturation the time required for mineral induction increased. The interfacial tensions calculated for apatite formation by either phosphoprotein cross-linked to collagen were about the same as that for phosphatidic acid liposomes and hydroxyapatite. This similarity in values indicates that the nucleation potential of these highly phosphorylated surfaces is about the same. It is concluded that phosphoproteins must be irreversibly bound to collagen fibrils for the mineralization of the collagen network in solutions that do not spontaneously precipitate. The phosphate esters of phosphoproteins are required for mineral induction, and the carboxylate groups are not sufficient.

Phosphophoryn, an "acidic" biomineralization regulatory protein: conformational folding in the presence of Cd(II).

The divalent cation-induced protein folding properties of the template macromolecule, bovine dentine phosphophoryn (BDPP), have been examined by 1H/31P/13C/113Cd-nmr spectroscopy. Cd(II) was employed, exploiting the sensitivity of 113Cd-nmr to ligand-binding interactions and kinetics. Cation binding was studied over the stoichiometric range of 0-50: 1 Cd(II): protein (mole ratio), well below the range of Cd(II) concentration required to induce protein precipitation. The stepwise titration of divalent cation-depleted phosphophoryn at pH 7.2 in H2O/D2O with 113CdCl2 revealed that (PSer)n, (PSerAsp)n, and (Asp)n polyelectrolyte cation-binding domains undergo two major transitions in their secondary and tertiary structures: the first transition, occurring between 1:10 and 1:1 Cd(II): protein stoichiometry, and the second, between 10:1 and 50:1. By monitoring the amide NH intensities, 31P-nmr chemical shift, and 13C Asp-C, resonances, it was concluded that Cd(II) ions exhibit a binding-site preference for polyelectrolyte cation-binding domains, in the order (PSer)n > (PSerAsp)n > (Asp)n This preference correlates with the degree of negative charge density for each sequence motif. Accompanying the backbone conformational transitions at the polyelectrolyte regions were conformational transitions in the flanking hinge domains, indicating that the hinge domains participate in the folding of the phosphophoryn molecule as divalent cation binding occurs at the polyelectrolyte domains. We were unsuccessful in detecting phosphophoryn-bound Cd(II) species by 113Cd-nmr because of chemical exchange modulation. However, using a smaller 21-residue peptide mimetic of phosphophoryn, we have observed three stoichiometric-dependent 113Cd resonances that differ in terms of the oxoanion coordination number. Our observation of multiple Cd(II) species in the presence of the peptide supports our contention that Cd(II) has many chemically distinct coordination sites on phosphophoryn, each in multiple equilibria with H2O, Cl-, and side-chain oxoatoms.

Phosphophoryn, a biomineralization template protein: pH-dependent protein folding experiments.

The protein folding behavior of a polyelectrolyte protein, bovine dentine phosphophoryn (BDPP), in the pH range of 1.82-11.0 has been investigated. One- and two-dimensional nmr spectroscopy has been utilized to obtain proton spin assignments for amino acid residues in D2O and in H2O. One-dimensional 31P-nmr experiments verify the existence of three separate classes of O-phosphoserine (PSer) resonances in BDPP (alpha, beta, chi), representing three distinct PSer residue populations at pH 6.94. By means of pH titration and 1H-nmr, five populations of Asp residues can be identified. Three of these populations exhibit secondary inflection points on their pH titration curves that correspond to an observed pKa of 6.17-6.95. The presence or absence of secondary inflection points for Asp populations and the 31P-nmr chemical shift dispersion for the three PSer residue populations indicate that BDPP may be comprised of homologous (Asp-Asp)n. (PSer-PSer)n, and heterologous (PSer-Asp)n sequences arranged into polyelectrolyte cluster regions. The pH titration also revealed that certain populations of Ser, Gly, and Pro residues in BDPP exhibit pH-dependent resonance frequency shifts. The "apparent" pKa for the transition points of these frequency shifts corresponds to either the pK1a of Pser monophosphate ester and/or the pKa of Asp COOH group of BDPP polyelectrolyte regions. On the basis of these transition points, we can assign four types of Ser, Gly, or Pro-containing "intervening" regions in BDPP, based on their sensitivity to protonation and deprotonation events occurring at (Asp)n, (PSer)n, or (PSer-Asp)n anionic cluster regions that flank the intervening regions. Our 1H-nmr experiments also reveal that BDPP assumes a folded conformation at low pH. As the pH increases, this conformation undergoes several unfolding transitions as the BDPP molecule assumes more open conformations in response to increased electrostatic repulsion between polyelectrolyte anionic regions in the protein. These folding-unfolding transitions are mediated by the intervening regions, which act as "hinges" to allow the polyelectrolyte regions to fold relative to one another.

Bovine dentine phosphophoryn. Conformational folding in the presence of 113Cd (II) reveals cooperativity between hinge and calcium binding domains and the presence of cation “hopping”.

Bovine dentine phosphophoryn. Conformational folding in the presence of 113Cd (II) reveals cooperativity between hinge and calcium binding domains and the presence of cation “hopping”.

Effects of dentin phosphophoryn on precipitation of calcium phosphate in gel in vitro.

In vitro precipitation of calcium phosphate was carried out using a one-dimensional double diffusion system in agar gel. Bovine dentin phosphophoryn enhanced the sharpness of the precipitation bands, although it reduced the total amount of the precipitates. Dephosphorylated phosphophoryn had no effect on the pattern of precipitates. Therefore, phosphophoryn is thought to raise the local density of nucleation in spite of its inhibitory activity on apatite formation.

Changes in interaction of bovine dentin phosphophoryn with calcium and hydroxyapatite by chemical modifications.

Dentin phosphophoryn was found to lose its precipitability with calcium ion when dephosphorylated or carboxylate-blocked, whereas precipitation of the intact protein was induced at calcium concentration as low as 2 mM. On the other hand, the adsorption capacity of phosphophoryn onto the hydroxyapatite surface increased when acidic groups were diminished by the above modifications.

Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn.

Bovine dentin phosphophoryn was iodinated with 125I, then tested for binding to native monomeric collagen, to collagen fibrils, and to gelatin. The phosphophoryn was found to bind reversibly, but specifically, to both collagen monomers and fibrils, but not to denatured collagen (gelatin). Competitive binding studies showed that bovine serum albumin, fibronectin, and bovine bone 34K glycoprotein (osteonectin) did not compete with phosphophoryn and did not inhibit its binding to collagen fibrils. Phosvitin, a phosphoserine-rich protein, did compete, but sixfold higher concentrations of phosvitin than of unlabeled phosphophoryn were required to reduce iodinated phosphophoryn binding to the same extent. Quantitative analyses of the binding showed binding to be limited to the fibril surfaces. Bound phosphophoryn enhanced the uptake of 45Ca onto collagen fiber surfaces. These data support the hypothesis that, in dentin, the phosphophoryn plays an important role in localizing the calcium binding leading to the growth of collagen-oriented calcium hydroxyapatite crystals.

Bovine dentin phosphophoryn: composition and molecular weight

The molecular weight of phosphophoryn, an acidic phosphoprotein unique to dentin matrix, has been difficult to determine because of a combination of neutral protease activities in this tissue and the intrinsic high charge density of the molecule. In this study, bovine dentin phosphophoryn (BDPP) was isolated by a procedure designed to prevent proteolysis. Bovine unerupted third molar powder was demineralized by ethylenediaminetetraacetic acid (EDTA). The EDTA-soluble phosphophoryn fraction was isolated and purified by sequential calcium chloride precipitation, gel filtration in sodium dodecyl sulfate (NaDodSO4) containing buffer, anion-exchange chromatography, and finally gel filtration in 4 M guanidine hydrochloride (4 M Gdn.HCl) buffer. Sedimentation equilibrium, sedimentation velocity, and diffusion coefficient data, viscosity studies in a high ionic strength buffer, and NaDodSO4 gradient gel electrophoresis data gave consistent results for the molecular weight of BDPP, all being in the range of 151 000-167 000. This range is much higher than any previously reported value. An anomalous behavior was observed in nongradient NaDodSO4 gel electrophoresis. Dissociative analytical gel filtration chromatography in 4 M Gdn.HCl gave a molecular weight value of 100 000. This discrepancy was resolved by studying the viscosity of BDPP in 4 M Gdn.HCl which showed BDPP does not assume a true random-chain conformation in this solvent.

The interaction of bovine dentine phosphophoryn and collagen during fibrillogenesis of collaten in vitro

Bovine dentinal phosphophoryn retards the rate of collagen self-assembly when monomeric collagen is the kinetic unit in fibriilogenesis in vitro. This inhibition is dependent on phosphorylation of the protein and affects the lag period rather than the growth phase for the formation of collagen fibrils. Treatment of the phosphophoryn with calcium markedly increases the inibitory effect. The use of several fluorescent hydrophobic probes indicates that the calcium-binding to phosphophoryn does not expose any additional interacting hydrophobic domains, thus suggesting that calcium potentiates this interaction, probably by providing a different spatial arrangement of charged groups on this polyelectrolyte, phosphophoryn.