Dentin Phosphophoryn Research Articles

Mineralized tissue protein profiles in the Australian form of bovine osteogenesis imperfecta.

Noncollagenous proteins and proteoglycan from the bone and dentin of affected and normal half-siblings neonatal calves with the Australian variant of bovine osteogenesis imperfecta (BOI-A) were quantitated. In contrast to a clinically similar syndrome of BOI in the progeny of a second progenitor bull identified in Texas (BOI-T), osteonectin and bone sialoprotein levels were normal in the affected BOI-A bone, and dentin phosphophoryn levels were normal in affected BOI-A teeth. However, bone proteoglycan in the affected BOI-A calves was depleted to one-third of the level found in normal siblings, a finding qualitatively similar to the depletion of bone proteoglycan in the BOI-T syndrome. These results suggest that quantitation of noncollagenous proteins and proteoglycans may be a useful technique for differentiating clinically similar syndromes of OI in man.

In vivo cleavage of dentin phosphophoryn following β elimination of its phosphoserine residues

Phosphophoryn, a unique phosphoprotein in dentin, has a polydisperse character. Keto acid was found to be present in this protein by condensation reaction with dinitrophenyl (DNP)-hydrazine and was identified to be pyruvate from the result that [ 3H]lactate was formed by the reduction of phosphophoryn with [ 3H]NaBH 4. In the case of phosvitin, incorporation of DNP-hydrazine was much less than in phosphophoryn, even after the same treatment as the extraction procedure of phosphophoryn. DNP-hydrazine incorporation was much higher in the phosphophoryn of mature dentin than in dentin of unerupted teeth. From these results, pyruvate was thought to be formed time dependently in vivo by β elimination of phosphoserine residues and subsequent cleavage of the peptide chain, which may contribute to the polydispersity of phosphophoryn.

The biosynthesis of dentin phosphophoryns by rat incisor odontoblasts in organ culture.

An in vitro system consisting of rat incisor fragments was used to study the process of dentinogenesis. In order to establish the usefulness of the organ culture, the biosynthesis and deposition of the major noncollagenous components of dentin, the phosphophoryns, were followed for specific lengths of time in culture. Three criteria were satisfied: (1) the synthesis of proteins which appeared to be chemically identical to the native proteins of dentin, (2) the accumulation of the phosphophoryns within the matrix or time, and (3) the association of the secreted proteins with the mineral phase of dentin. The synthesis of phosphophoryns was determined by using both (3H)-serine and (32P)-inorganic phosphate as precursors for synthesis of protein and post-translational modification of serine to phosphoserine. In vitro synthesized phosphophoryns were characterized by 1) their accumulation and EDTA extractability from within dentin, 2) calcium chloride precipitability, 3) elution on anion-exchange columns (DEAE cellulose and AGMP50), and 4) Mr's on SDS-PAGE and Sepharose CL-6B columns. This novel system of studying dentinogenesis provides a model with which to study the regulation of extracellular matrix protein synthesis and may be useful for revealing the effect of other agents which influence tooth development and mineralized tissue metabolism in general.

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.

Relation of Mineralization Defects in Collagen Matrices to Noncollagenous Protein Components

Hydroxyapatite crystal deposition and stabilization within the collagen matrix of bone and dentin have been linked to the presence of noncollagenous proteins (NCP). Dentinogenesis imperfecta (DI), a genetic disorder of dentin mineralization, is being studied as a model for the analysis of mineralization mechanisms. A comparative study of the NCP in normal human dentin and dentinogenesis imperfecta Type II (hereditary opalescent dentin) dentin has been performed. The proteins of each tissue were extracted and separated using a variety of techniques. The calcium-binding, highly phosphorylated protein phosphophoryn was one of the principal NCP in normal human teeth dentin, whereas there was no evidence for the presence of such a component in the DI teeth. These data imply that dentin phosphophoryn may be related in function to the mineralization process. A similar calcium-binding protein defect should be sought in the various types of osteogenesis imperfecta.

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.

Cooperativity in calcium ion binding to repetitive, carboxylate-serylphosphate polypeptides and the relationship of this property to dentin mineralization.

Analogs of phosphophoryn, a calcium-binding phosphorylated protein found in mineralized dentin, were synthesized by solid phase peptide synthesis. The dentin phosphophoryn appears to contain some sequence blocks of (Asp-PhosphoSer)n. As this sequence is difficult to synthesize, polymers of (alpha-L-Glu-L-Ser) were prepared. The 30-peptide, (alpha-L-Glu-L-Ser)15, was phosphorylated by reaction with orthophosphoric acid in the presence of trichloroacetonitrile in anhydrous dimethylsulfoxide. Calcium ion binding studies were carried out with both the 30-peptide and phosphorylated 30-peptide. The conformation of the original 30-peptide, (Glu-Ser)15, was examined, in the presence and absence of calcium ion, by circular dichroism measurements. Purified bovine phosphophoryn, previously studied by the same techniques, was partially dephosphorylated by alkaline phosphatase, and its calcium ion binding properties were determined. Dephosphorylation to 31% of the initial phosphorus content reduced the number of high affinity sites to approximately 30% of the initial value. However, the stoichiometry of binding indicated that both phosphate and carboxylate groups participate in the high affinity binding and that the binding constant was decreased only slightly. Partial phosphorylation of the 30-mer raised the calcium binding constant, Ka, from 2.1 x 10(2) to 3.3 x 10(3) M-1 and increased the amount of binding from an electrostatic equivalent number of sites to a stoichiometric equivalent number. Concomitant with binding, there was a transition from random coil to beta-like structure. These data suggest that the repetitive (Asp-PhosphoSer)n regions in phosphophoryn and the (Glu-PhosphoSer)n sequence of the synthetic polymer have special conformations which favor the unidentate binding of calcium to the carboxyl groups and phosphate groups. and which enhance the binding affinities of the carboxyl groups in such sequences in a cooperative fashion.

Some factors affecting mineralization of bone in tissue culture.

Some factors affecting mineralization of bone in tissue culture.