Saliva plays an important role in the maintenance of oral health by exhibiting multiple host defense functions. These include homeostatic processes, lubrication, antimicrobial activity, and the control of demineralization/remineralization of teeth. Biochemical studies of saliva and salivary secretions established that specific salivary proteins are responsible for these defense functions. Because some of these salivary proteins have been characterized extensively, including their primary structures, it has become feasible to explore their structure/function relationships. Acidic proline-rich proteins (PRPs), for example, exhibit high affinity to hydroxyapatite, inhibit crystal growth of calcium phosphate salts from solutions supersaturated with respect to hydroxyapatite, bind calcium ions, and interact with several oral bacteria on adsorption to hydroxyapatite. Statherins, histatins, and cystatins also exhibit affinities to mineral surfaces, inhibit calcium phosphate precipitation, and play a role in maintaining the integrity of teeth. Furthermore, histatins exhibit both antibacterial and antifungal activities. Approaches to identifying the functional domains of these salivary proteins include functional assays of enzymatically digested proteins and peptides, synthetic peptides and peptide analogues, and chemically modified proteins as well as biophysical studies of native proteins or peptides. Such studies have demonstrated that the fungicidal activities of histatins reside in the middle portion of the polypeptide chain, whereas the hydroxyapatite binding domains of PRPs and statherin reside in the phosphorylated amino-terminal regions. Identification of functional domains is vital in understanding the mechanisms of action and this information can be exploited in the development of therapeutic agents.
The protein compositions of in vitro pellicles formed from whole saliva and parotid and submandibular secretions were determined by use of synthetic hydroxyapatite as a model for dental enamel. The adsorbed and unadsorbed protein fractions were analyzed by amino acid analysis and both anionic and cationic discontinuous polyacrylamide gel electrophoresis. For further characterization of the in vitro pellicle, the adsorbed fractions were subjected to gel filtration on Sephadex G-100 and reversed-phase chromatography on C18 columns. Amylase, acidic and glycosylated proline-rich proteins, statherins, and histatins were identified in the parotid-derived pellicle. Detailed analysis of the statherin-containing fractions resulted in the observation of several statherin-like proteins. The use of cationic gel electrophoresis allowed for the identification of histatin 3 and histatin 5, which have not been previously detected in pellicle formed in vitro. The protein composition of submandibular-derived pellicle was similar to that of parotid-derived pellicle except for the presence of cystatins and the absence of glycosylated proline-rich proteins. In contrast, in vitro pellicle derived from whole saliva exhibited a vastly different composition, consisting primarily of amylase, acidic proline-rich proteins, cystatins, and proteolytically-derived peptides. The results indicate that acidic phosphoproteins as well as neutral and basic histatins from pure secretions selectively adsorb to hydroxyapatite, whereas in whole saliva some of these proteins are proteolytically degraded, dramatically changing its adsorption pattern.
Proline-rich proteins (PRPs), histatins, and statherin are salivary proteins that exhibit high affinities for hydroxyapatite surfaces. In vitro experiments with parotid submandibular/sublingual or whole saliva have shown these proteins to adsorb selectively to tooth surfaces. This investigation focuses on the histo-morphological identification of PRPs, histatins, and statherin in acquired enamel pellicles. Synthetic hydroxyapatite or bovine enamel were exposed to glandular secretions, and whole saliva and pellicle precursor proteins were identified immunohistologically by electron microscopy. Results obtained by back-scattered scanning electron microscopy showed these proteins to be present in pellicles. Pellicles displayed a distinct structure consisting of a sponge-like meshwork of microglobules. Interconnections between structural elements were identified in submandibular/sublingual and whole saliva pellicles only. Transmission electron microscopy of pellicles formed on bovine enamel surfaces revealed a tendency for preferential localization of precursor proteins within the protein film. Since the data showed the presence of pellicle precursors in pellicles derived both from glandular secretions and from whole saliva, it is likely that PRPs, histatins, and statherin are integral components of acquired enamel pellicles in vivo.
Salivary proteins bind to enamel surfaces and hydroxyapatite in a highly selective manner. Numerous studies have identified these proteins as primarily proline-rich proteins, cystatins, statherin, and histatins. Previously, the hydroxyapatite-binding potential of these proteins had been characterized in systems consisting of singly purified protein and adsorbent. The purpose of this study was to investigate the adsorption of each protein in the presence of complete salivary secretion. Proteins, shown to adsorb to hydroxyapatite, were purified, biotinylated, and added back to the remaining proteins to form a series of reconstituted secretions. The adsorption of each biotinylated protein in the reconstituted secretion to hydroxyapatite was then measured as a function of time. Results indicated that three different adsorption patterns occur. A simple hyperbolic pattern is characteristic of amylase, glycosylated proline-rich protein (PRG), and cystatin. A faster adsorption process is observed for PRP-3, PRP-4, PIF-f, and statherin. A more complex pattern, exhibiting a rapid phase followed by a slower phase, is characteristic of PRP-1, PRP-2, PIF-s, and histatins. These results suggest that there are different adsorption processes involved in the binding of salivary proteins to hydroxyapatite. Two possible mechanisms are direct adsorption of protein to hydroxyapatite and indirect adsorption of protein by interacting with other proteins already bound to hydroxyapatite.
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