P.F. Riley

Anticariogenicity of Calcium Phosphate Complexes of Tryptic Casein Phosphopeptides in the Rat

Casein phosphopeptides (CPP) stabilize calcium phosphate through the formation of casein-phosphopeptide amorphous calcium-phosphate complexes (CPP-CP). The ability of CPP-CP to reduce caries activity was investigated by use of specific-pathogen-free rats inoculated with Streptococcus sobrinus. The animals consumed a defined cariogenic diet free of dairy products. Solutions (100 pL) of the CPP-CP (0.1, 0.2, 0.5, 1.0% w/v) were applied to the animals molar teeth twice daily. Other groups of animals received solutions containing 500 ppm F, the non-phosphorylated peptides of a casein tryptic digest (0.5% w/v), or the calcium-phosphate complex of a synthetic octapeptide, Ac-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe, corresponding to the common sequence in the CPP. The CPP-CP significantly reduced caries activity in a dose-response fashion, with 1.0% CPP-CP producing 55% and 46% reductions in smooth surface and fissure caries activity, respectively, being similar to that of 500 ppm F. The anticariogenic effects of CPP-CP and fluoride were additive, since animals receiving 0.5% CPP-CP plus 500 ppm F had significantly lower caries activity than those animals receiving either CPP-CP or fluoride alone. The tryptic digest of casein with the phosphopeptides selectively removed showed no anticariogenic activity. The synthetic octapeptide-calcium phosphate complex significantly reduced caries activity, confirming that this calcium-phosphate-stabilizing portion of the casein phospho-peptides is associated with anticariogenicity. The CPP-CP did not significantly affect the level of S. sobrinus in fissure plaque. The proposed mechanism of anticariogenicity for CPP-CP complexes is that they substantially increase the level of calcium phosphate in plaque, depressing enamel demineralization and enhancing remineralization.

The analysis of multiple phosphoseryl-containing casein peptides using capillary zone electrophoresis

Multiple phosphoseryl-containing sequences of peptides and proteins stabilize amorphous calcium phosphate at neutral and alkaline pH and have been implicated in the nucleation/regulation of biomineralization. In an approach to analyze these peptides using capillary zone electrophoresis (CZE) we have attempted to relate the absolute electrophoretic mobility of various casein phosphopeptides to their physicochemical properties. Multiple phosphoseryl-containing peptides were selectively precipitated from enzymic digests of sodium caseinate and further purified using RP-HPLC and anion-exchange fast protein liquid chromatography. Purified fractions were then analyzed by CZE. Absolute electrophoretic mobilities of 13 peptides were determined by measurement of migration times relative to that of a neutral marker, mesityl oxide. A linear relationship (r2 = 0.993) was obtained between absolute electrophoretic mobility and q/M(r)2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M(r) is the molecular mass. M(r)2/3 is a measure of the surface area of a sphere that has a volume proportional to the M(r) of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. As absolute electrophoretic mobility is influenced by charge and size CZE can be used to monitor peptide phosphorylation, dephosphorylation, deamidation and truncation. This technique therefore would be suitable for quantitative analysis of peptide substrates in kinase and phosphatase studies. In conclusion CZE is a rapid and efficient technique for the resolution of multiple phosphoseryl-containing peptides from enzymic digests of casein.

Protein Dissimilation by Human Salivary-sediment Bacteria

Proteins of known composition and structural characteristics were incubated (1.0 mglmL) with re-suspended salivary sediment (2.5% vl v) in a lactate-salt medium with an initial pH of 5.2 for two hr at 37°C. Hydrolysis of the proteins was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Hydrogen ion, amines, and ammonia were measured by use of a combined pH electrode, high performance liquid chromatography, and glutamate dehydrogenase, respectively. Of the proteins studied, the caseins αs1, β, and K and the histones H1 and H3 were extensively hydrolyzed by the salivary-sediment bacteria. The hydrolysis of these proteins was attributed to their relative lack of tertiary (folded) structure. The only amine detected was the polyamine putrescine arising from the catabolism of arginine following the hydrolysis of the arginine-rich histone H3. None of the other proteins extensively hydrolyzed by salivary sediment, although containing arginyl and lysyl residues, served as substrates for putrescine or cadaverine production. Pre-hydrolysis of the arginine-rich histone H3 and poly-L-arginine with trypsin resulted in a marked increase in putrescine produced, suggesting that the salivary-sediment proteolytic activity was not trypsin-like. Incubation of salivary-sediment bacteria with the caseins and the histone H3 resulted in an increase in ammonium ion concentration and an associated decrease in hydrogen ion concentration. The increase in ammonium ion concentration not attributed to arginine hydrolysis was correlated with the content of glutaminyl plus asparaginyl residues of the proteins. The results suggest that amido nitrogen, in the form of glutaminyl and asparaginyl residues of salivary and dietary proteins, is a potential source of nitrogen for oral bacteria and may also play a role in plaque pH homeostasis.

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.

Kinetics of Hydroxyapatite Dissolution during Incubation with Sucrose and Streptococcus mutans and Its Application to Cariogenicity Testing

The kinetics of acid production from sucrose fermentation by Streptococcus mutans has been investigated. The rate of acid production, expressed as nmoles H+ produced per minute per milligram bacterial protein, was shown to be saturated at a sucrose concentration of 2 mM, a concentration below the taste threshold. Similarly the rate of calcium release from hydroxyapatite when added to the same system, expressed as nmoles Ca++ released per minute per milligram bacterial protein, was also saturated at a sucrose concentration of 2mM. The Michaelis constant (Km) for both processes was 0.2 mM sucrose. These results indicate that the rate-limiting step in this in vitro system was the production of organic acids by fermentation. In vitro tests to determine the cariogenicity of foods containing saturating sucrose concentrations are of limited value and the low Km value obtained here shows that most foods would contain saturating sucrose concentrations. Hence, as part of a cariogenicity test these foods should be diluted to allow comparison at non-saturating sucrose concentrations. Further, the low Km value could help explain the poor correlation reported between sucrose intake and caries incidence.

Synthesis and Characterisation of a Multiphosphorylated Phosphophoryn Repeat Motif; H-[Asp-(Ser(P))2]3-Asp-OH

Protein phosphorylation is a critical mechanism in the regulation of cellular biochemical pathways and phosphopeptides can play an important role in determining function. However, the use of phosphopeptides especially multiphosphorylated peptides is hampered by their low abundance, difficulty in isolation from biological samples and in their chemical synthesis. Here we describe methodologies for the Fmoc synthesis, purification and mass spectral analysis of the multiphosphorylated sequence H-[Asp-(Ser(P))2]3-Asp-OH from phosphophoryn a protein involved in dentine mineralization. Critical steps in the synthesis of phosphophoryn using Fmoc-Ser(PO3Bzl,H)-OH as the building block were double acylation steps for each residue, alternating HBTU and HATU as the acylating agents and synthesis on a chlorotrityl resin which was essential for complete removal of the benzyl-side chain protecting groups. The synthetic phosphophoryn was only effectively purified by anion exchange and size exclusion chromatography as both alkaline and acid buffers failed to aid in purification by reversed phase HPLC. MALDI-TOF analysis of phosphophoryn was achieved with good sensitivity (20xa0fmol/ml) and resolution using the DNA matrix 3-hydroxypicolinic acid, whereas typical protein/peptide matrices failed to provide mass spectra. The synthetic phosphophoryn peptide was found to bind calcium, binding 6xa0mol of calcium per mole of peptide. In conclusion the methodology described here can be easily adopted for the synthesis and analysis of a wide variety of multiphosphorylated peptides.