David P. Stanton

A Review of the Salivary Proteome and Peptidome and Saliva-derived Peptide Therapeutics

Saliva is a glandular secretion that is vital in the maintenance of healthy oral tissues. In this review we outline the high abundance salivary proteins, summarise the status of the salivary proteome and peptidome, the genetic origin and recognised functions of these proteins, the diseases associated with salivary disorders, and the emerging saliva-derived peptide therapeutics. Different proteomic approaches have reported the identification of over 1,300 proteins in saliva. However there are fewer than 100 high abundance proteins, identified by multiple methods including, two-dimensional polyacrylamide gel electrophoresis and HPLC combined with mass spectrometry. Analysis of the genes coding for the salivary proteins demonstrated a non-uniform chromosomal distribution with chromosome 4 having the largest proportion of genes expressed in salivary glands. Several diseases are associated with salivary disorders including Sjögren’s syndrome, Prader-Willi syndrome, dental caries and stress related disorders. Saliva as a diagnostic medium for various biochemical tests has provided a non-invasive and accessibility advantage over other more regularly tested body fluids such as blood and urine. To-date the emerging saliva-based therapeutics include artificial salivas and antimicrobial agents based on histatins and mucins.

Porphyromonas gingivalis Cysteine Proteinase Inhibition by κ-Casein Peptides

ABSTRACT Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The Arg-specific (RgpA/B) and Lys-specific (Kgp) cysteine proteinases of P. gingivalis are major virulence factors for the bacterium. In this study κ-casein(109-137) was identified in a chymosin digest of casein as an inhibiting peptide of the P. gingivalis proteinases. The peptide was synthesized and shown to inhibit proteolytic activity associated with P. gingivalis whole cells, purified RgpA-Kgp proteinase-adhesin complexes, and purified RgpB proteinase. The peptide κ-casein(109-137) exhibited synergism with Zn(II) against both Arg- and Lys-specific proteinases. The active region for inhibition was identified as κ-casein(117-137) using synthetic peptides. Kinetic studies revealed that κ-casein(109-137) inhibits in an uncompetitive manner. A molecular model based on the uncompetitive action and its synergistic ability with Zn(II) was developed to explain the mechanism of inhibition. Preincubation of P. gingivalis with κ-casein(109-137) significantly reduced lesion development in a murine model of infection.

Acidogenic potential of soy and bovine milk beverages

UNLABELLED Soy beverages are water extracts of whole soybeans and are often promoted as a healthy alternative to bovine milk. Little analysis has been carried out to determine the effects of soy beverages on oral health, especially their potential acidogenicity. OBJECTIVES The aim of this study was to determine the potential acidogenicity of a range of soy and bovine milk beverages. METHODS In vitro acid production by Streptococcus mutans was measured in soy and milk beverages at a constant pH of 6.5 or 5.5, as was the fall in pH over a 10 min period. The acid buffering capacity and calcium and phosphate concentrations (total and soluble) of the beverages were also determined. RESULTS The rate of acid production by S. mutans in the milk beverages was five to six times lower at pH 6.5 than in the soy beverages and three to five times lower at pH 5.5. Whilst the pH fall in the presence of S. mutans over 10 min was negligible in the milk beverages there was a significant decrease in pH in the soy beverages. This was also reflected in the lower buffering capacity of the soy beverages. The levels of soluble calcium in the soy beverages were lower than those in the milk beverages although total calcium contents were similar. CONCLUSIONS Soy beverages have a higher potential acidogenicity than bovine milk beverages. CLINICAL SIGNIFICANCE STATEMENT Patients consider soy beverages to be a healthy, low cariogenic alternative to other beverages, including bovine milk. This study shows that soy beverages have a higher potential acidogenicity than bovine milk and therefore may have a greater potential cariogenicity.

Effect of calcium phosphate addition to fluoride containing dental varnishes on enamel demineralization.

BACKGROUND The aim of this study was to evaluate the ability of calcium phosphate and fluoride containing varnishes to inhibit enamel demineralization. METHODS Six varnishes were selected for analysis: (1) Enamel Pro containing amorphous calcium phosphate; (2) Clinpro White containing functionalized tricalcium phosphate (fTCP); (3) MI Varnish containing casein phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP); (4) Duraphat (first no added calcium control); (5) Profluorid (second no added calcium control); and (6) placebo (no added calcium or fluoride control). Human enamel slabs (36) were each cut into half-slabs and covered with one of the six dental varnishes to create a window. The half-slabs were then individually immersed in a polyacrylate demineralization buffer pH 4.8 for four days at 37 °C with a change of solution each day. Mineral content was determined using transverse microradiography. RESULTS All fluoride-containing varnishes significantly inhibited enamel demineralization when compared with the placebo varnish. However, out of the calcium phosphate and fluoride containing varnishes only MI Varnish, containing fluoride and CPP-ACP was superior to the fluoride-alone varnishes. MI Varnish also released the highest levels of calcium, phosphate and fluoride ions. CONCLUSIONS MI Varnish containing fluoride and CPP-ACP was superior to the other varnishes in protecting against enamel demineralization.

Streptococcus mutans biofilm disruption by κ-casein glycopeptide

UNLABELLED Caseinomacropeptide (CMP), the variably phosphorylated and glycosylated forms of the bovine milk protein fragment, κ-casein(106-169), is produced during cheese production and has been shown to have a range of antibacterial bioactivities. OBJECTIVES To characterise the biofilm disruptive component of CMP and compare its activity with the known antimicrobial agents chlorhexidine and zinc ions. METHODS Streptococcus mutans biofilms were grown in flow cells with an artificial saliva medium containing sucrose and treated with CMP and the glycosylated forms of κ-casein(106-169) (κ-casein glycopeptide, KCG). The biofilms were imaged using confocal laser scanning microscopy (CLSM) and quantified by COMSTAT software analysis. A static biofilm assay and flow cytometric analysis were used to examine the mechanism of action of chlorhexidine and a combination of KCG with the known antimicrobial agent ZnCl2 (KCG-Zn). RESULTS CLSM analysis showed that S. mutans produced robust, structured biofilms with an average thickness of 7.37μm and a biovolume of 3.88μm(3)/μm(2) substratum after 16h of incubation in the flow cell system. A single application of 10mg/mL CMP that contained 2.4mg/mL KCG significantly reduced total biofilm biovolume and average biofilm thickness by 53% and 61%, respectively. This was statistically the same as a 2.4mg/mL KCG treatment that reduced the total biovolume and average thickness by 59% and 69%, respectively, suggesting the KCG was the biofilm disruptive component of CMP. Chlorhexidine treatment (0.1%) caused similar effects in the flow cell model. KCG-Zn caused significantly more disruption of the biofilms than either KCG or ZnCl2 treatment alone. In a static biofilm model chlorhexidine was shown to work by disrupting bacterial membrane integrity whilst KCG-Zn had no effect on membrane integrity. CONCLUSIONS KCG and KCG-Zn may have potential as natural biofilm disruptive agents.

Molecular Interactions of Peptide Encapsulated Calcium Phosphate Delivery Vehicle at Enamel Surfaces

Phosphorylated peptides derived from milk caseins, known as casein phosphopeptides (CPP), self-assemble and encapsulate the calcium and phosphate mineral in the form of amorphous calcium phosphate (ACP), thus forming CPP-ACP nanocomplexes that are nontoxic and biocompatible. The biomedical application is the repair of tooth surfaces (enamel) at early stages of tooth decay. These nanocomplexes release calcium and phosphate ions to rebuild demineralised HA crystals in enamel subsurface lesions. The topical application of CPP-ACP at the tooth surface initiates a series of interactions at the enamel mineral hydroxyapatite surface and at the enamel salivary pellicle that are not well understood. In this study, we have shown that the β-casein (1-25) peptide binds reversibly to Ca2+, Mg2+, Mn2+, La2+, Ni2+, and Cd2+ metal ions. In contrast, binding to Sn2+, Fe2+, and Fe3+ ions resulted in ion-induced aggregation. The casein peptides as well as the mineral ions dissociate from the CPP-ACP complexes to adsorb to both the uncoated and saliva-coated mineral surface with the mineralisation increasing monotonically with increasing pH. Furthermore, SEM of the CPP-ACP revealed images of spherical particles surrounded by ACP mineral. In conclusion, the enamel remineralisation process involves an array of interactions between the peptide and mineral ions of the CPP-ACP delivery vehicle and the tooth enamel mineral with its salivary pellicle.

The Interactions of CPP-ACP with Saliva

The repair of early dental caries lesions has been demonstrated by the application of the remineralisation technology based on casein phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP–ACP). These complexes consist of an amorphous calcium phosphate mineral phase stabilised and encapsulated by the self-assembly of milk-derived phosphopeptides. During topical application of CPP–ACP complexes in the oral cavity, the CPP encounters the enamel pellicle consisting of salivary proteins and peptides. However the interactions of the CPP with the enamel salivary pellicle are not known. The studies presented here reveal that the predominant peptides of CPP–ACP complexes do interact with specific salivary proteins and peptides of the enamel pellicle, and provide a mechanism by which the CPP–ACP complexes are localised at the tooth surface to promote remineralisation.

Fluoride content of tank water in Australia

BACKGROUND The aims of this study were to: (1) analyse the fluoride content of tank water; (2) determine whether the method of water collection or storage influenced fluoride content; and (3) survey participant attitudes towards water fluoridation. METHODS Plastic tubes and a questionnaire were distributed through dentists to households with water tanks in Victoria. A midstream tank water sample was collected and fluoride analysed in triplicate using ion chromatography RESULTS All samples (n = 123) contained negligible amounts of fluoride, with a mean fluoride concentration of <0.01 ppm (range: <0.01-0.18 ppm). No statistically significant association was found between fluoride content and variables investigated such as tank material, tank age, roof material and gutter material. Most people did not know whether their tank water contained fluoride and 40.8% preferred to have access to fluoridated water. The majority thought fluoride was safe and more than half of the respondents supported fluoridation. Fluoride content of tank water was well below the optimal levels for caries prevention. CONCLUSIONS People who rely solely on tank water for drinking may require additional exposure to fluoride for optimal caries prevention.

Antibacterial efficacy of casein-derived peptides against Enterococcus faecalis

BACKGROUND The aim of this study was to test a casein peptide in its glycosylated form (kappa-casein glycopeptide, KCGP) and its non-glycosylated form (kappa-casein peptide, KCP) for antibacterial efficacy against Enterococcus faecalis in planktonic and biofilm cultures. METHODS E. faecalis strain JKD 15036 was exposed to different concentrations of KCGP and KCP in a 96-well culture plate. The effect of the peptides on the growth of E. faecalis in planktonic culture was monitored by measuring optical density over 7 hours. Biofilm formation was measured after 24 hours using a crystal violet assay. All experiments were performed in triplicate. RESULTS KCGP and KCP inhibited growth of E. faecalis in planktonic culture with no significant difference in activity between the peptides. KCGP at 0.16% w/v was significantly better at inhibiting E. faecalis biofilm formation than KCP at the same concentration and significantly better than NaOCl at 1.0% w/v. CONCLUSIONS KCGP effectively inhibited E. faecalis biofilm formation and may have potential to augment the efficacy of traditional antiseptic agents.

Physico-chemical Characterisation of the Processes Involved in Enamel Remineralisation by CPP-ACP

Casein phosphopeptides derived from tryptic digests of milk caseins spontaneously assemble with calcium and phosphate ions at high pH to form casein phosphopeptide-amorphous calcium phosphate complexes (CPP-ACP). These complexes have been shown to be able to repair lesions in tooth enamel (biohydroxyapatite – HA) both in vitro and in vivo (specifically white spot lesions in the early stages of tooth decay). In order to better understand the processes involved in enamel remineralisation, the chemical equilibria between the CPP and calcium and phosphate ions as a function of pH were investigated. Furthermore, a thin-enamel slab technique was developed with enhanced sensitivity to monitor the diffusion of radio-opaque ions into individual lesions over a period of days to weeks.