Acquired Enamel Pellicle Research Articles

Proteomic Evaluation of Acquired Enamel Pellicle during In Vivo Formation

Acquired enamel pellicle (AEP) is a protein film that forms on the enamel surface of teeth by selective adsorption of proteins and peptides present in the mouth. This protein film forms the interface between enamel and the damage oral biofilm, which modulates the attachment of bacteria found in oral biofilm. The overall goal of this study was to gain insight into the biological formation of the human in vivo AEP. This study hypothesized that AEP is created by the formation of successive protein layers, which consist of initial binding to enamel and subsequent protein-protein interactions. This hypothesis was examined by observing quantitative and qualitative changes in pellicle composition during the first two hours of AEP formation in the oral cavity. Quantitative mass spectrometry approaches were used to generate an AEP protein profile for each time-point studied. Relative proteomic quantification was carried out for the 50 proteins observed in all four time-points. Notably, the abundance of important salivary proteins, such as histatin 1, decrease with increasing of the AEP formation, while other essential proteins such as statherin showed constant relative abundance in all time-points. In summary, this is the first study that investigates the dynamic process to the AEP formation by using proteomic approaches. Our data demonstrated that there are significant qualitative and quantitative proteome changes during the AEP formation, which in turn will likely impact the development of oral biofilms.

Acidulated Phosphate Fluoride Application Changes the Protein Composition of Human Acquired Enamel Pellicle

We evaluated, by proteomic analysis, whether the chemical changes provoked on enamel by acidulated phosphate fluoride (APF) application alter the protein composition of acquired enamel pellicle. Enamel slabs, pretreated with distilled water (negative control), phosphoric acid (active control) or APF solution, were immersed in human saliva for pellicle formation. The adsorbed proteins were extracted and analyzed by liquid chromatography-mass spectrometry/mass spectrometry. Fifty-six proteins were identified, 12 exclusive to APF and 11 to phosphoric acid. APF decreased the concentration of histatin-1, but increased the concentration of S100-A9, which is confirmed by immunoblotting. The findings suggest that APF application changes the acquired enamel pellicle composition.

Proteome and Peptidome of Human Acquired Enamel Pellicle on Deciduous Teeth

Understanding the composition and structure of the acquired enamel pellicle (AEP) has been a major goal in oral biology. Our lab has conducted studies on the composition of AEP formed on permanent enamel. The exhaustive exploration has provided a comprehensive identification of more than 100 proteins from AEP formed on permanent enamel. The AEP formed on deciduous enamel has not been subjected to the same biochemical characterization scrutiny as that of permanent enamel, despite the fact that deciduous enamel is structurally different from permanent enamel. We hypothesized that the AEP proteome and peptidome formed on deciduous enamel may also be composed of unique proteins, some of which may not be common with AEP of permanent enamel explored previously. Pellicle material was collected from 10 children (aged 18–54 months) and subjected to mass spectrometry analysis. A total of 76 pellicle proteins were identified from the deciduous pellicle proteome. In addition, 38 natural occurring AEP peptides were identified from 10 proteins, suggesting that primary AEP proteome/peptidome presents a unique proteome composition. This is the first study to provide a comprehensive investigation of in vivo AEP formed on deciduous enamel.

Pub: Probing the Interaction of Human Salivary Alpha-Amylase and Amylase Binding Protein A(AbpA) of Streptococcus gordonii

Amylase binding protein A (AbpA) of Streptococcus gordonii serves as a major receptor for human salivary α-amylase (HSAmy), the predominant enzyme in human salivary secretions, to bind to the bacterial cell surface. On enamel surfaces, the binding of AbpA to HSAmy renders S. gordonii act as a scaffold for other oral bacteria to attach to the acquired enamel pellicle leading to complex bacterial communities and invasion of host tissues. While the role of AbpA in adhesion, starch metabolism and biofilm formation in influencing the ecology of the oral biofilms has been established, the structure function relationships of AbpA are yet to be defined. Since distally located aromatic residues of HSAmy are involved in the interaction with AbpA, we hypothesized that AbpA might use separate structural regions to bind to HSAmy. To test this, several deletion mutants of AbpA were constructed and studied to correlate the effect of deletions in binding to HSAmy in the following: 1) their secondary structural features through circular dichroism studies; 2) their ability to alter the capacity of HSAmy to hydrolyze starch and several oligosaccharides after complex formation and 3) their binding to HSAmy using surface plasmon resonance spectroscopy. Our results showed that a) AbpA does not bind at the active site of salivary α-amylase; b) both the N-terminal region (24-56 residues) and a central region (residues 124-165) are required for binding to HSAmy; and c) the C-terminal end residues (166- 195) are not necessary for binding. These results clearly show that AbpA binding to HSAmy encompasses distinct and distal regions of the structure.

Effect of Dialyzed Saliva on Human Enamel Demineralization

Background: Saliva is supersaturated with respect to calcium and phosphate ions. Salivary ions may well play a role in the subsequent adsorption of proteins and consequently in the formation of the acquired enamel pellicle. Among several biological functions, the enamel pellicle forms a selectively permeable barrier that regulates demineralization processes. Aim: The aim of this study was to evaluate the importance of salivary proteins when adsorbed on enamel surface and the resultant protective effect against demineralization without the presence of salivary ions. Methods: Enamel surfaces were coated with whole saliva, parotid saliva, dialyzed whole saliva or dialyzed parotid saliva (molecular weight cutoff 1 kDa). Adsorption was allowed to proceed for a period of 2 h. Enamel specimens were then washed with deionized water and immersed into a demineralization solution of pH 4.5 for 12 days. This solution was used to measure the amount of calcium and phosphate released from enamel specimens after the demineralization period. Results: All coated specimen groups showed a significantly higher protection than those not coated with any type of saliva. In addition, undialyzed saliva (whole saliva and parotid saliva) was more effective in protecting the enamel against demineralization than dialyzed saliva. Conclusion: The present investigation indicates that the ionic composition of saliva can amplify the demineralization protection effect by reducing acid-induced enamel demineralization. Moreover, a protective effect of salivary proteins without presence of ions was demonstrated in this study.

New Insights into the Composition and Functions of the Acquired Enamel Pellicle

The acquired enamel pellicle (AEP) is a thin acellular film that forms on tooth surfaces upon exposure to the oral environment. It consists predominantly of salivary proteins, but also includes non-salivary-derived proteins, carbohydrates, and lipids. Since it is the interface between teeth and the oral environment, the AEP plays a key role in the maintenance of oral health by regulating processes including lubrication, demineralization, and remineralization and shaping the composition of early microbial flora adhering to tooth surfaces. Knowledge of the 3D structure of the AEP and how that correlates with its protective functions may provide insight into several oral pathological states, including caries, erosion, and periodontal disease. This review intends to update readers about the latest discoveries related to the formation, ultrastructure, composition, and functions of the AEP, as well as the future of pellicle research, with particular emphasis on the emerging role of proteomic and microscopy techniques in oral diagnosis and therapeutics.

Quantitative Proteomic Analysis of the Effect of Fluoride on the Acquired Enamel Pellicle

The acquired enamel pellicle (AEP) is a thin film formed by the selective adsorption of salivary proteins onto the enamel surface of teeth. The AEP forms a critical interface between the mineral phase of teeth (hydroxyapatite) and the oral microbial biofilm. This biofilm is the key feature responsible for the development of dental caries. Fluoride on enamel surface is well known to reduce caries by reducing the solubility of enamel to acid. Information on the effects of fluoride on AEP formation is limited. This study aimed to investigate the effects of fluoride treatment on hydroxyapatite on the subsequent formation of AEP. In addition, this study pioneered the use of label-free quantitative proteomics to better understand the composition of AEP proteins. Hydroxyapatite discs were randomly divided in 4 groups (n = 10 per group). Each disc was exposed to distilled water (control) or sodium fluoride solution (1, 2 or 5%) for 2 hours. Discs were then washed and immersed in human saliva for an additional 2 hours. AEP from each disc was collected and subjected to liquid chromatography electrospray ionization mass spectrometry for protein identification, characterization and quantification. A total of 45 proteins were present in all four groups, 12 proteins were exclusively present in the control group and another 19 proteins were only present in the discs treated with 5% sodium fluoride. Relative proteomic quantification was carried out for the 45 proteins observed in all four groups. Notably, the concentration of important salivary proteins, such as statherin and histatin 1, decrease with increasing levels of fluoride. It suggests that these proteins are repulsed when hydroxyapatite surface is coated with fluoride. Our data demonstrated that treatment of hydroxyapatite with fluoride (at high concentration) qualitatively and quantitatively modulates AEP formation, effects which in turn will likely impact the formation of oral biofilms.

Impact of Acquired Enamel Pellicle Modification on Initial Dental Erosion

The acquired enamel pellicle that forms on the tooth surface serves as a natural protective barrier against dental erosion. Numerous proteins composing the pellicle serve different functions within this thin layer. Our study examined the effect of incorporated mucin and casein on the erosion-inhibiting potential of the acquired enamel pellicle. Cyclic acidic conditions were applied to mimic the erosive environment present at the human enamel interface during the consumption of soft drinks. One hundred enamel specimens were prepared for microhardness tests and distributed randomly into 5 groups (n = 20) that received the following treatment: deionized water, humidity chamber, mucin, casein, or a combination of mucin and casein. Each group was exposed to 3 cycles of a 2-hour incubation in human saliva, followed by a 2-hour treatment in the testing solution and a 1-min exposure to citric acid. The microhardness analysis demonstrated that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. The addition of individual proteins did not statistically impact the function of the pellicle. These data suggest that protein-protein interactions may play an important role in the effectiveness of the pellicle to prevent erosion.

Histatin 1 Resists Proteolytic Degradation when Adsorbed to Hydroxyapatite

Histatins are salivary proteins that exhibit a high affinity for hydroxyapatite and contribute to the acquired enamel pellicle. Previous studies have observed that, despite the high proteolytic activity in saliva, significant numbers of histatin molecules in acquired enamel pellicle are intact. Our working hypothesis was that histatins are less susceptible to proteinases present in saliva when adsorbed on the hydroxyapatite. To test this premise, we incubated histatin 1 with hydroxyapatite and human whole saliva. Proteolytic products of this incubation were then characterized by PAGE, HPLC, and mass spectrometry. This study shows for the first time that binding to hydroxyapatite confers intact histatin 1 with resistance to proteolytic degradation.

Reduced statherin expression in labial glands of diabetic subjects

Many oral diseases are associated to diabetes and, correspondingly, the salivary composition is strongly altered in subjects affected by this metabolic disorder with respect to healthy ones. Periodontal disease, tooth demineralization, caries, and oral infections, which frequently trouble diabetics, are generally ascribed to changes in some secretory protein expression. We focused our attention on the expression of the phosphoprotein statherin, which seems to play important roles in maintaining the buccal health, in human salivary glands. Recently, we reported the ultrastructural localization of this protein in human major and minor salivary glands and a significant reduction of its expression in submandibular glands of diabetic patients versus normal ones. In this study, we compare the subcellular immunoreactivity of statherin in diabetic and non diabetic labial glands. Surgical samples were fixed with a glutaraldehyde and paraformaldehyde mixture, embedded in Epon, and treated for immunogold histochemistry using a polyclonal antibody specific for statherin. Specific immunoreactivity was revealed in serous cells, where gold particles were deposited onto a lot of secretory vesicles and tubules diffused throughout the cytoplasm, Golgi apparatus and RER. The gold particles were counted and the labelling density was expressed as number of gold particles /μm2. The results obtained with diabetic samples were then compared with those in the controls. Statistical analysis showed a significant reduction of statherin expression in the diabetic versus normal glands. These data suggest that statherin secretion is affected by diabetes not only in major but also in minor salivary glands, supporting the belief that scarce amounts of this protein could be partly responsible for oral disturbances. Since the best known statherin functions are related to the formation of the acquired enamel pellicle and inhibition of calcium salt precipitation, reduced amounts could determine defects in the pellicle and in calcium metabolism, thus supplying inadequate protection to the teeth.

Evidence of Intact Histatins in the in vivo Acquired Enamel Pellicle

Understanding the composition and function of the acquired enamel pellicle (AEP) has been a major goal in oral biology. The aim of this study was to test the hypothesis that intact histatins are part of the in vivo AEP and that histatins after adsorption to HA have effects on in vitro enamel demineralization. This is the first study demonstrating the presence of intact histatins in vivo in the AEP. The in vitro experiments show that all naturally occurring histatins in the AEP have the potential to provide some level of protection against acid injury.

Small molecular weight proteins/peptides present in the in vivo formed human acquired enamel pellicle

Objective The aim of this study was to investigate the type and the nature of peptides present in the in vivo formed human acquired enamel pellicle. Design Pellicle material was collected from 10 volunteers and subjected to sample preparations consisting of centrifugal filtration using a 10 kDa molecular weight cut-off membrane and high-resolution gel filtration chromatography. The fractions containing peptides <10 kDa obtained by both methods were analyzed by LC–ESI-MS/MS. Results 78 natural pellicle peptides with molecular weights ranging from 766.9 Da to 3981.4 Da were identified originating from 29 different proteins. Conclusions The number of peptides present in acquired enamel pellicle appears to be large and this is likely to enhance the functional spectrum of this protein film. The presence of small peptides in pellicle may be functionally important since structure/function studies of many salivary proteins have shown that specific domains within these native proteins retain or even exhibit enhanced biological activities. The data present the basis for determining the precise function of these pellicle peptides and for gaining insights into the role pellicle plays in the oral cavity.

Structure-function relationships in human salivary α-amylase: role of aromatic residues in a secondary binding site

AbstractHuman salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (i) hydrolysis of starch; (ii) binding to hydroxyapatite; and (iii) binding to bacteria (e.g. viridans streptococci). Oral bacteria utilize the starch hydrolyzing activity of HSAmy to derive their nutrients from dietary starch. Localized acid production by bacteria, through the metabolism of maltose generated by HSAmy, can lead to the dissolution of tooth enamel, a critical step in dental caries formation. HSAmy is a component of the acquired enamel pellicle and is used by Streptococcus gordonii to colonize the oral cavity. Although the active site of HSAmy for starch hydrolysis is well characterized, the regions responsible for the bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one of the secondary saccharide-binding sites harboring the aromatic residues W316 and W388, may play an important role in bacterial binding. To test this hypothesis, the aromatic residues W316 and W388 were mutated to alanine. The wild type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch binding and bacterial binding. Our results clearly showed that (i) the mutants W316A and W388A were not impaired in starch binding or bacterial binding; (ii) mutation of aromatic residues at these sites does not alter the overall conformation of the molecule; and (iii) the hydrolytic activity of the enzyme is unaffected against starch as substrates but reduced significantly against oligosaccharides.

Salivary flow patterns and the health of hard and soft oral tissues

This nonsystematic review summarizes the effects of saliva on some of the diseases affecting the hard and soft oral tissues. Saliva enters the mouth at several locations, and the different secretions are not well-mixed. Saliva in the mouth forms a thin film, the velocity of which varies greatly at different sites. This variation appears to account for the site specificity of smooth-surface caries and supragingival calculus deposition. Saliva protects against dental caries, erosion, attrition, abrasion, candidiasis and the abrasive mucosal lesions seen commonly in patients with hyposalivation. These effects are the result of saliva's being a source of the acquired enamel pellicle; promoting the clearance of sugar and acid from the mouth; being supersaturated with respect to tooth mineral; containing buffers, urea for plaque base formation, and antibacterial and antifungal factors; and lubricating the oral mucosa, making it less susceptible to abrasive lesions. For optimal oral health, people should keep food and liquids in the mouth as briefly as possible. The most important time for toothbrushing is just before bedtime, because salivary flow is negligible during sleep and the protective effects of saliva are lost. Chewing sugar-free gum or sucking on sugar-free candies stimulates salivary flow, which benefits hard and soft oral tissues in many ways.

Intrinsic enzymatic crosslinking and maturation of the in situ pellicle

Aim The acquired enamel pellicle is a proteinaceous layer formed on all solid substrata exposed to the oral cavity. It has been supposed that the pellicle undergoes maturation after protein adsorption. The aim of the present study was to investigate enzyme activities with an impact on intrinsic maturation processes in in situ formed pellicles. Methods Bovine enamel specimens were exposed to the oral cavity in six subjects to allow in situ pellicle formation over 3, 30 and 120 min. The slabs were fixed on the buccal and palatal surfaces of individual splints fixed with silicone impression material. After rinsing with deionised water, the pellicle samples were tested fluorimetrically for transglutaminase, protease and elastase activity. Phosphatase activities were tested photometrically. Separate samples were used for each of the enzymes tested. Results Transglutaminase was detected in in situ pellicle (16.7 ± 21.2 mU/cm 2) as was alkaline phosphatase activity (0.87 ± 0.99 mU/cm 2). For both enzymes, there was no correlation of enzyme activities with time or localisation of pellicle formation. Acidic phosphatase- and protease-activities were not detectable. Only traces of elastase activity were found in 57% of the samples. Conclusion Transglutaminase and phosphatase activity are detectable within in situ pellicle. Enzymatic crosslinking and dephosphorylation appear more important for intrinsic maturation of the acquired enamel pellicle than proteolysis.

Conformational Changes in Salivary Proline-Rich Protein 1 upon Adsorption to Calcium Phosphate Crystals

Conformational analyses of PRP1, a proline-rich acidic salivary protein and major component of the acquired enamel pellicle, have been carried out in solution and upon binding to two enamel prototypes, hydroxyapatite (HA) and carbonated hydroxyapatite (CHA), using Fourier transform infrared spectroscopy (FTIR) in attenuated total reflection (ATR) mode. We have shown for the first time that, in solution, large portions of PRP1 adopt the hydrated polyproline type II (PPII) helical structure in addition to the random coil structure, with the maximum absorbance of the amide I band around 1620 cm(-1). Upon binding to HA or CHA, the protein undergoes significant conformational changes, loosing a considerable portion of hydrated PPII and random coil domains with a shift in the maximum absorbance to 1666 cm(-1), indicating that a large fraction of the protein is composed of beta turns. A small fraction of PPII in a calcium-bound or anhydrous form (approximately 1642 cm(-1)) was also observed in the HA- and CHA-bound proteins, which could play a role in protein-mineral interactions. The conformational changes in PRP1 adsorbed on CHA and HA were similar in nature; however, these changes were greater in the protein bound to HA. Interestingly, these results are in agreement with protein adsorption data that show that less protein is adsorbed onto CHA than onto HA. Our results demonstrate that binding to apatitic mineral surfaces leads to major conformational changes in PRP1, which might reflect the expulsion of water and the formation of protein-mineral and/or protein-protein interactions in the adsorbed layer.

Peptidomic analysis of human acquired enamel pellicle

Human acquired enamel pellicle is the result of a selective interaction of salivary proteins and peptides with the tooth surface. In the present work, the characterization of the peptides as well as the type of interactions established with the enamel surface was performed. Peptides from in vivo bovine enamel implants in the human oral cavity were sequentially extracted using guanidine and trifluoroacetic acid solutions and the fractions obtained were analysed by LC-MS and LC-MS/MS. Based on the LC-MS data, six phosphorylated peptides were identified in an intact form, strongly adsorbed to the enamel surface. Data from the LC-MS/MS analyses allowed us to identified 30 fragment peptides non-covalently bonded to enamel [basic proline-rich proteins, histatins (1 and 3) and acidic proline-rich protein classes]. The tandem mass spectrometry experiments showed the existence of a pattern of amide bond cleavage for the different identified peptide classes suggesting a selective proteolytic activity. For histatins, a predominance of cleavage at Arg, Lys and His residues was observed, while for basic proline-rich proteins, cleavage at Arg and Pro residues prevailed. In the case of acidic proline-rich proteins, a clearly predominance of cleavage of the Gln-Gly amide bond was evident.

Identification of Protein Components in in vivo Human Acquired Enamel Pellicle Using LC−ESI−MS/MS

The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva. The structural analysis of this integument relies on efficient pellicle harvesting and protein identification procedures. Material from three individual subjects and two pooled samples yielded the identification by LC-ESI-MS/MS of 130 pellicle proteins of which 89 were found in three or more experiments. A high intersubject consistency in pellicle composition was observed.

Acquired Enamel Pellicle and Its Potential Role in Oral Diagnostics

The acquired enamel pellicle (AEP) is a protein film with unique composition and properties, which is formed by the selective adsorption of a variety of oral fluid-derived proteins onto tooth enamel surfaces. Since events leading to caries and periodontal disease occur in close proximity to the tooth surface, pellicle constituents are likely to contain biomarkers valuable for diagnostic applications. Despite the importance of this oral structure, progress in understanding its formation and composition has been slow because of difficulties in efficient pellicle collection methods and limitations of biochemical techniques for the characterization of microgram amounts of proteins/peptides. Recent developments in both pellicle collection methods and nanoscale sensing technologies have brought the exploitation of pellicle analysis into the realm of point-of-care oral diagnostics.

Statherin and Histatin 1 Reduce Parotid Saliva-Promoted Streptococcus mutans Strain MT8148 Adhesion to Hydroxyapatite Surfaces

Small salivary phosphoproteins – statherin (ST) and histatin 1 (HT1) – are found in the acquired enamel pellicle which modulates Streptococcus mutans adhesion onto dental enamel. However, their roles in S. mutans adhesion onto enamel surfaces are still undefined. The aim of this study was to investigate whether and how ST and HT1 affect (i) S. mutans adhesion and (ii) the adsorption of S. mutans adhesion-promoting salivary proteins onto hydroxyapatite (HA) in vitro. We fractionated human parotid saliva by adsorption to HA and further by gel filtration chromatography. Adhesion of [³H]-labeled S. mutans strain MT8148 onto sintered HA plates was promoted significantly (>10-fold) by high-molecular weight glycoprotein fraction (HMWGP), but not by purified ST or HT1. More interestingly, promotion of S. mutans adhesion onto HA by HMWGP was significantly reduced by adding purified ST or HT1 to HMWGP. [³H]-labeled S. mutans adhesion on HA was positively correlated to the [¹⁴C]-labeled HMWGP adsorption onto HA, which was also reduced by the addition of purified ST and HT1. Synthetic peptides corresponding to ST and HT1 reduced the parotid saliva-promoted S. mutans adhesion. However, removal of the negative charges in the N-terminal domains of ST and HT1 diminished their inhibitory effects on S. mutans adhesion promoted by parotid saliva. We conclude that ST and HT1 competitively inhibit the adsorption of salivary HMWGP, and thereby reduce S. mutans adhesion onto HA surfaces.