Dentin Phosphophoryn Research Articles

Development of a monoclonal antibody against dentin phosphophoryn: a tool to study odontoblastic activity.

The role played by phosphophoryn, one of the major noncollagenous proteins of dentin extracellular matrix, in the mineralization process has not been fully characterized. The purpose of our work was to produce monoclonal antibodies (MAbs) against dentin phosphophoryn and to test their reactivity with primary culture of odontoblasts. Dentin phosphophoryn (DPP) was extracted after the mechanical dissociation of teeth and dialyzed against guanidine and EDTA solutions followed by CaC1(2) precipitation. These extracts were characterized by SDS-PAGE and staining with Coomassie blue and Stains-All. After immunization of mice with these extracts, we produced MAb 7G4, which reacted with dentin phosphophoryn as revealed by Western blot. MAb 7G4 reactivity was tested against a primary culture of pig odontoblasts, revealing filaments specifically stained by the anti-DPP antibody. This antibody will be of great interest to study the mineralization process and dental pulp reaction after capping with various calcium phosphate materials.

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”.

Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization

Acidic phosphorylated proteins have been shown to be prominent constituents of the extracellular matrix of bone and dentin. The acidic phosphoproteins of bone contain more glutamic acid than aspartic acid and a lower serine content than either. On the other hand, the major dentin acidic phosphoproteins, phosphophoryns, have been defined as aspartic acid- and serine-rich proteins, with a lesser content of glutamic acid. Both sets of phosphoproteins have been implicated as key participants in regulating mineralization, but it has been difficult to unify their mechanisms of action. We have now identified, by cDNA cloning, a new serine-rich acidic protein of the dentin matrix, AG1, with a composition intermediate between the bone acidic proteins and dentin phosphophoryns. AG1 has numerous acidic consensus sites for phosphorylation by both casein kinases I and II. Immunochemical and organ culture biosynthetic studies show that AG1 is present in phosphorylated form at low levels in the dentin matrix. If fully phosphorylated, AG1 would bear a net charge of -175/molecule of 473 residues. AG1 contains single RGD integrin binding and N-glycosylation sequences. The overall picture that emerges is that of a matrix-associated acidic phosphoprotein, with a potentially high calcium ion binding capacity, present at levels compatible with a regulatory role in dentin mineralization.

Affinity of bone sialoprotein and several other bone and dentin acidic proteins to collagen fibrils.

Bone and dentin contain several kinds of mineral-binding proteins and cell-attachment proteins. The authors examined the affinity of these proteins to type I collagen, a major matrix protein of the tissue. Bone sialoprotein (BSP), bone Gla protein (BGP), bone small proteoglycan II (PG II), osteonectin (ON), and dentin phosphophoryn (DPP) were labeled with fluorescein isothiocyanate and incubated with reconstituted type I collagen fibril. DPP, BGP, BSP, and PG II were absorbed significantly to the collagen fibril at physiological ionic strength with dissociation constants of 10(-6)-10(-7) M. BSP and PG II enhanced the fibrillogenesis of collagen. These acidic proteins can affect the surface properties of collagen fibril, and BSP, having the cell-attachment sequence Arg-Gly-Asp, possibly mediates interaction between collagen fibril and cells.

The in vitro phosphorylation of the native rat incisor dentin phosphophoryns.

Phosphophoryns are the major non-collagenous proteins of the mineralized matrix of rat incisor dentin. Nearly half the phosphophoryn residues are serines, and 85-90% of these are phosphorylated. Since phosphorylation may be important for phosphophoryn function, it was of interest to identify the kinase(s) responsible for catalyzing their phosphophorylation. Rat osteosarcoma (ROS) 17/2.8 osteoblast-like cells were selected as the enzyme source. Native rat incisor phosphophoryns (RIPP-I, II, III) were not substrates for any of the ROS 17/2.8 messenger-dependent kinases but were phosphorylated by membrane-associated endogenous messenger-independent kinases. These were resolved chromatographically and identified as casein kinase (CK) I and II by elution properties and immunoblotting with a CKII antibody. The CKI preferentially used RIPP-III as substrate, while CKII preferred RIPP-I and II. Heparin at 100 and 500 ng/assay and NaCl at 0.25-0.4 M inhibited phosphorylation of the RIPP by CKI and CKII in parallel. At 10 mM spermine, phosphorylation of RIPP-I and II by CKII, and of RIPP-III by CKI were inhibited, but phosphorylation of RIPP-III by CKII was enhanced. Purified sea star oocyte CKII demonstrated the same substrate specificity and spermine concentration shift as the ROS 17/2.8 CKII. These data show that osteoblast-like cells are a rich source of membrane-bound CKI and CKII activity. The different patterns of phosphorylation of RIPP-I, II, and III further show that they are distinct synthetic products of the odontoblast.

Domain structure and sequence distribution in dentin phosphophoryn

Phosphophoryn (PP) is a protein unique to the mineralized matrix of dentin. It also has a unique composition, with aspartic acid and phosphoserine comprising greater than 85% of all amino acid residues. Because of this unique composition and high content of phosphoserine, it has been difficult to apply direct peptide sequencing procedures effectively. However, to understand its function, and to prepare suitable probes for screening cDNA libraries, some sequence distribution information is required. To this end, using bovine (b) and rat incisor (ri) PPs, partial mild acid hydrolysis has been used to cleave at the aspartic acid residues and generate free amino acids and small peptides. The nature of the released amino acids and peptides has been determined. Peptides have also been generated by limited digestion with trypsin. Some of the peptides have been purified by h.p.l.c. techniques and sequenced. About 90% of the bPP and riPP were resistant to trypsin, and the large resistant fragment was sharply depleted of the non-aspartic acid and non-phosphoserine [(P)Ser] residues. All peptides isolated were acidic, but the remaining residues (other than aspartic acid and serine) appeared to be collected in regions flanking the trypsin-resistant core. These data show directly the presence of regions [Asp]n, [(P)Ser]m and [Asp-(P)Ser-Asp]k as prominent sequence features. A domain structure model is proposed.

Concentration-dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth

The effect of dentin phosphophoryn on hydroxyapatite formation and growth was studied in an in vitro gelatin gel diffusion system. Phosphophoryn, in low concentrations (0.010–1 μg/ml) promoted de novo hydroxyapatite formation; at a higher concentration (100 μg/ml) in the same system, the dentin matrix protein inhibited hydroxyapatite growth. Similar inhibition of hydroxyapatite growth was seen in solution. The intact phosphophoryn was not essential for either inhibition of seeded growth or promotion of mineralization, since the formic acid degraded protein was comparably effective. Transmission electron microscopy of the precipitates formed at 7 days showed no significant differences in crystallite size distribution in the presence and absence of phosphophoryn. However there was a dose-dependent decrease in the number of mineral clusters formed in the presence of increasing amounts of phosphophoryn, suggesting inhibition of secondary nucleation. These data provide support for the postulated ‘multifunctional’ role of the dentin phosphoprotein in the mineralization process.

Self-association of calcium and magnesium complexes of dentin phosphophoryn.

Self-association of rat dentin phosphophoryn in the presence of calcium and magnesium ions was examined by chemical cross-linking and electron microscopy. Highly phosphorylated phosphophoryn (HP) binds a maximum of 1.33 calcium ions or 1.07 magnesium ions per organic phosphate residue at pH 7.4-8.0. The Ca-HP complexes are predominantly linear when the calcium content of the complex is less than about 65% of the saturation level. At higher calcium levels, the protein has a folded conformation, and transient protein-protein interactions occur. The equilibrium mixture of monomers and oligomers is predominantly monomeric unless the protein is saturated with calcium. The saturated Ca-HP complex forms discrete high molecular weight particles about 25 nm in diameter. The particles are electrically neutral and generally occur in clusters. Mg-HP complexes appear predominantly linear by electron microscopy at all concentrations of bound magnesium up to about 99% of the saturation level; however, protein-protein interaction is measurable when the magnesium content is as little as 65% of the saturation level. At saturation, Mg-HP complexes form high molecular weight particles which are negatively charged. Because of the negative charge, these particles form a stable colloidal suspension and have a rather stellate configuration.

Binding of calcium and phosphate ions to dentin phosphophoryn.

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 by an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-) ion, and for each PO4(3-) ion bound, the protein binds about 1.5 additional Ca2+ ions. Therefore, the protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. Empirically the binding of calcium and phosphate can best be described as a function of a neutral ion activity product in which 2.5-10% of the phosphate is HPO4(2-). The stoichiometry of the bound clusters is similar to that of amorphous calcium phosphate, and it is clear that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution. About 15% of the bound phosphate is buried in protected domains, and it is stable with respect to dissociation for extended periods in phosphate-free calcium buffers. The buried mineral maintains the protein in an aggregated state even at calcium ion concentrations which are too low for the aggregation of unmineralized HP. In vivo HP should be ineffective in the nucleation of a crystalline mineral phase, if it is secreted in a mineralized aggregated state similar to casein and the bivalve phosphoprotein.

Binding of calcium and phosphate ions to dentin phosphophoryn.

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 using an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-)ion, and for each PO4(3-)ion bound, the protein binds about 1.5 additional Ca2+ ions. The protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. The stoichiometry of the bound clusters is similar to amorphous calcium phosphate, indicating that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution.

A monoclonal antibody against dentin phosphophoryn recognizes a bone protein(s) appearing at the beginning of ossification.

Decalcified and nondecalcified sections of fetal bovine tibia were stained immunohistochemically with a monoclonal antibody against dentin phosphophoryn. In the epiphyseal portion of the long bone, osteoblasts, osteocytes and the bone matrix were stained, but chondrocytes and the cartilage matrix were not. Similar staining was observed in the epiphyseal and diaphyseal portions of bones. These findings suggest that a protein(s) with the same epitope as phosphophoryn may be synthesized and secreted by osteoblasts at the beginning of ossification and may be involved in mineralization of bone tissue. On Western blots of proteins extracted from fetal bovine bone, the antibody reacted with two bands of molecular weights of about 71,000 and 63,000. These proteins and antibody(s) to the proteins may be useful for detection of the phenotype of osteogenesis.

Two classes of dentin phosphophoryns, from a wide range of species, contain immunologically cross-reactive epitope regions

An immunological species comparison, using a monospecific rabbit polyclonal antibody directed against rat incisor alpha-phosphophoryn, has been undertaken to assess the similarity in epitope regions among various dentin phosphophoryns (PP) that were prepared from human, monkey, bovine, ovine, and echinoderm teeth. Dentin extracellular matrix proteins were extracted with a standard method using 0.5 M EDTA in the presence of enzyme inhibitors. Final phosphophoryn purification was performed on DEAE ion exchange HPLC. Cross-reactivity of the polyclonal antibody was examined by enzyme-linked immunosorbant assay (ELISA) and dot-blot. The results of this investigation demonstrate a cross-reactivity of the rat-alpha-phosphophoryn antibody (anti-RIPP) with at least one phosphophoryn component in each dentin studied, indicating the existence of similar antigenic determinants among these proteins. It would seem that these epitope regions have been strongly conserved since the epitope region is also present in the phosphoprotein of echinoderm teeth. No cross-reactivity was found with phosvitin (a phosphoserine-rich phosphoprotein), rat serum albumin, bovine serum albumin, or collagen type IV. However, a new and distinct second cross-reactive phosphophoryn, not calcium ion-precipitable, was found in the EDTA insoluble fraction from the teeth. These results indicate that dentin phosphophoryns are specific phenotypic markers for odontoblast expression. Because of the species cross-reactivity, the polyclonal anti-RIPP antibody may be a useful probe in studying the distribution of phosphophoryns in other species, such as human teeth.

Difference in noncollagenous matrix composition between crown and root dentin of bovine incisor.

A comparative study of the organic matrix in crown dentin and root dentin of the bovine incisor has been performed utilizing biochemical analyses and histochemical stainings. Root dentin contained only half the amount of dentin phosphophoryn present in crown dentin. Composition of noncollagenous matrix other than phosphophoryn was also suggested to be different between crown and root dentin. These data indicate that odontoblasts forming root dentin may secrete a noncollagenous matrix having a composition different from that of crown dentin, and that crown and root dentin may be separate in nature. The distinct compositions of noncollagenous matrix may reflect the difference in the mineralization process between crown and root dentin.

Increase of dentin phosphophoryn with dentin formation.

Dentin phosphophoryn was quantified on bovine and rabbit dentin at three developmental stages. Phosphophoryn was extracted from teeth with 0.6M HCl, and quantified as optical density on DEAE-cellulose chromatogram or as phosphoserine content. Bovine phosphophoryn showed progressive increase with formation of dentin. Matrix-associated phosphophoryn was also quantified as phosphoserine content in insoluble dentin residue which was extracted with 6 M urea after decalcification. This fraction increased with formation of dentin both in bovine and rabbit dentin. Phosphophoryn is thought to be related to the later stage of dentin formation.

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.

Histological distribution of phosphophoryn in normal and pathological human dentins

Dentin phosphophoryn is a highly phosphorylated protein which has a hydrophilic character but is not soluble in dilute acetic acid. A histochemical method was developed for staining this protein with Stains-all in situ utilizing those chemical properties. We have succeeded in detecting the presence of this protein in circumpulpal orthodentin of human permanent and deciduous teeth, but not in mantle dentin, secondary dentin and reparative dentin. Phosphophoryn staining was also absent in the dentin of dentinogenesis imperfecta (DI) Type II, a genetic disorder of dentin formation. From these results, it is suggested that phosphophoryn is synthesized and secreted only by physiologically-differentiated odontoblasts and that the mineralization processes of mantle, secondary, reparative and DI dentins may be different from that of circumpulpal orthodentin.

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.

Identification of Dentin Phosphophoryn Localization by Histochemical Stainings

Phosphophoryn, the most abundant of the dentin non-collagenous proteins, has been considered to be related in function to the mineralization process. In the present study, identification of dentin phosphophoryn localization was attempted using newly developed, precautionary histological methods by which phosphophoryn was retained in the sections during the specimen preparation and stained selectively in situ. Phosphophoryn was found to be present widely in all of the calcified dentin except the mantle dentin, the external, first-formed portion of dentin, but was not found in the predentin, the inner, uncalcified layer of dentin. These results indicate that phosphophoryn is apparently related to the mineral phase of calcified dentin and that the mineralization process of mantle dentin, which is formed before the odontoblasts are fully differentiated, may be different from that of circumpulpal dentin.