M.S. Lamkin

Structural Features of Salivary Function

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.

Adsorption of Human Salivary Proteins to Hydroxyapatite: A Comparison Between Whole Saliva and Glandular Salivary Secretions:

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.

Multiple forms of statherin in human salivary secretions.

Sequential chromatography of hydroxyapatite-adsorbed salivary proteins from submandibular/sublingual secretions on Sephadex G-50 and reversed-phase HPLC resulted in the purification of statherin and several statherin variants. Amino acid analysis, Edman degradation and carboxypeptidase digestion of the obtained protein fractions led to the determination of the complete primary structures of statherin SV1, statherin SV2, and statherin SV3. SV1 is identical to statherin but lacks the carboxyl-terminal phenylalanine residue. SV2, lacking residues 6-15, is otherwise identical to statherin. SV3 is identical to SV2 but lacks the carboxyl-terminal phenylalanine. These results provide the first evidence for multiple forms of statherin which are probably derived both by post-translational modification and alternative splicing of the statherin gene.

Physiological Regulation of the Secretion of Histatins and Statherins in Human Parotid Saliva

The small salivary phosphoproteins, histatins and statherins, have important functions in the oral cavity in terms of antimicrobial actions and regulation of calcium phosphate homeostasis. Neither the effects of various physiological stimuli on their secretion nor the nature of the efferent receptor involved in the stimulus-secretion coupling has been determined previously. These aspects are important for improved understanding of the secretory control of salivary proteins and may have implications regarding the effects of specific medications on salivary constituents and oral health. The effects of graded mechanical (chewing on short and long silicone tubings) and gustatory stimulation (0.5, 1.5, and 5.0% citric acid) on the secretion of histatins and statherins were studied in the presence and absence of adrenolytic agents (n = 10). In this model, secretory rates of both proteins increased with increases in flow rate, with 5.0% citric acid representing a particularly potent stimulus. Histatin and statherin secretory rates were significantly reduced by the β 1-adrenolytic agent (histatins to 58 to 72% and statherins to 11 to 29% of that in corresponding control experiments), but not by the α 1-adrenolytic agent. Since the β1-adrenergic receptors played an important role in the stimulus-secretion coupling of these proteins, protective salivary functions in the oral cavity may be compromised during β 1-adrenolytic treatment.

Pellicle Precursor Proteins: Acidic Proline-rich Proteins, Statherin, and Histatins, and their Crosslinking Reaction by Oral Transglutaminase

Previous studies have demonstrated that whole saliva and pellicle formed in vitro from oral fluid contain covalently crosslinked salivary proteins. The purpose of this study was to determine which salivary proteins can act as substrates for transglutaminase, an enzyme responsible for the covalent crosslink reaction between a glutamine residue and a lysine residue. Transglutaminase was prepared from the pellet fraction of human whole saliva. Dansyl cadaverine (N-dansyl-1,5-diaminopentane) was used to study the reactivity of glutamine residues in acidic large and small proline-rich proteins, statherin, and the major histatins, whereas a glutamine-containing dansylated peptide was used to study the reactivity of lysine residues in these proteins. Crosslink formation was measured fluorometrically after the addition of fluorescent probe to the salivary protein substrate and transglutaminase. The covalent attachment of the fluorescent probe to salivary proteins was confirmed by SDS-PAGE. It was found that almost all of the lysines present in the acidic PRPs and statherin, and some of the lysines present in histatins, could participate in the crosslink reaction. Glutamine reactivity was also observed, but a maximum of only 14% of glutamine residues present in acidic PRPs and statherin participated in the crosslink formation. These results demonstrate that primary pellicle precursor proteins, acidic proline-rich proteins, statherin, and the major histatins are capable of undergoing crosslink reactions catalyzed by oral transglutaminase. This may enable other proteins in the oral cavity to be incorporated into the acquired enamel pellicle.

Pellicle Precursor Protein Crosslinking: Characterization of an Adduct between Acidic Proline-rich Protein (PRP-1) and Statherin Generated by Transglutaminase

Recent work with oral transglutaminase indicated that this enzyme, derived from oral epithelial cells, crosslinked pellicle precursor proteins which may be important in the formation of the acquired enamel pellicle. The purpose of this study was to investigate whether purified acidic PRP-1 can form crosslinks with statherin, and whether such a crosslink is derived from a transglutaminase-catalyzed reaction between glutaminyl and lysyl side-chains, leading to a covalent bond formation. Enzymatic reaction products were analyzed by SDS-PAGE and reverse-phase HPLC. The SDS electrophoretogram revealed a protein band with an apparent molecular weight of 32 kDa, which is consistent with the combined apparent molecular weight of acidic PRP-1 (24 kDa) and statherin (8 kDa). A reaction product isolated by HPLC was characterized by amino acid analysis, which showed a stoichiometry consistent with being an adduct composed of one molecule of acidic PRP-1 and one molecule of statherin. In negative control experiments, it could be shown that this adduct was not detected when the lysines of both substrates were modified by reductive methylation prior to the enzymatic reaction. In addition, amino acid analysis and mass spectrometry confirmed the presence of a γ-glutamyl-∈-lysine dipeptide after enzymatic hydrolysis and the absence of this dipeptide after acid hydrolysis. Analysis of the data obtained indicates that oral transglutaminase is capable of crosslinking acidic PRP-1 and statherin in vitro. In addition, this finding exemplifies the potential of post-secretory processing of salivary proteins, which may represent an additional mechanism to generate new protein species.

Salivary cystatin SA-III, a potential precursor of the acquired enamel pellicle, is phosphorylated at both its amino- and carboxyl-terminal regions☆

Cystatin SA-III was purified from human submandibular/sublingual glandular secretions by adsorption to hydroxyapatite, gel filtration chromatography, and reversed-phase HPLC. The amino acid sequence of its amino-terminus was deduced by sequential Edman degradation and found to be identical to the first 10 residues of cystatin HSP-12. The purified protein was digested with endoproteinase Asp-N and the digestion products were subjected to fast atom bombardment mass spectroscopy. m/z values corresponding to 12 peptides were aligned to the sequence of cystatin S preceded by the eight-residue amino-terminal peptide detected in HSP-12. This process resulted in the assignment of peptides corresponding with 118 out of the 121 amino acid residues predicted from the nucleotide sequence for cystatin SA-III. In order to align several peptides, it was necessary to substitute four residues of phosphoserine for four residues of serine. Fast atom bombardment mass spectrometry and additional Edman degradation procedures localized the phosphate moieties to Ser-3, Ser-99, Ser-112, and Ser-116. This is the first report of the structure of cystatin SA-III deduced by amino acid sequencing techniques and indicates the sites of phosphoserine within the molecule. Based on these assignments, cystatin SA-III is unique among salivary proteins in that it possesses phosphate groups at its amino-terminus as well as its carboxyl-terminus.

New in vitro Model for the Acquired Enamel Pellicle: Pellicles Formed from Whole Saliva Show Inter-subject Consistency in Protein Composition and Proteolytic Fragmentation Patterns

The present investigation was undertaken to investigate the variability of proteins in whole saliva which adsorb to hydroxyapatite and are thus likely to be precursors of the acquired enamel pellicle. Whole-saliva proteins from 18 subjects were absorbed to hydroxyapatite, and the gel filtration patterns of released proteins revealed four major peaks and three minor peaks eluting between the major peaks. Amino acid analysis indicated that minor peaks contained fragments of proteins in major peaks, and this was confirmed by sequence analysis. Major peaks comprised 95% and minor peaks comprised 5% of protein absorbed to hydroxyapatite, suggesting a limited proteolytic capacity of whole saliva. HPLC elution patterns of components in minor peaks suggested that proteolysis is not totally random but is an orderly and consistent process. These studies suggest that whole saliva may be suitable as a model system for the investigation of post-secretory modifications of salivary proteins important for the formation of the acquired enamel pellicle.

Three forms of BRP-2 (bone resorptive proteins) from human cancer ascites fluid and their relationship to human serum alpha-2 HS-glycoprotein.

SummaryTwo new forms of BRP-2, a previously described bone resorptive protein, were purified from ascites fluids obtained from patients with hypercalcemia and metastatic bone cancer. The apparent molecular weights of BRP-2 and of these two proteins were 52,000, 48,000, and 46,000, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The three proteins have essentially the same amino acid compositions but differ with respect to their carbohydrate moieties. The amino-terminal amino acid sequences of the three glycoproteins were identical to each other as well as to human serum α2HS-(human serum) glycoprotein. The relationship of the three forms of BRP-2 to α2HS was also established immunochemically. The ascites proteins, as well as α2HS, on a molar basis, were approximately one-tenth as potent as bovine parathyroid hormone fragment (1–34) in their abilities to stimulate calcium release from bonein vitro. This study describes for the first time a possible function for human serum α2HS.

Salivary Secretions: Narcolepsy and Central Nervous System Stimulants

In an ongoing attempt to develop a model to study the influence of various diseases and drugs on saliva, we studied persons with narcolepsy treated with central nervous system stimulants. The aim was to study the secretion of salivary proteins in narcolepsy in the presence and absence of central nervous system stimulants. For this purpose, two proteins synthesized in acinar cells, acidic proline-rich proteins (PRPs) and statherin, were selected. Persons with narcolepsy treated with central nervous system stimulants only were included, n = 12, ages 14 to 68, seven females. Citric-acid-stimulated parotid and submandibular/sublingual saliva samples were collected from these persons during medication, after a drug-free period of one week, at least two weeks after the drug had been re-introduced, and from a matched healthy control group. PRP and statherin concentrations were determined by means of reversed-phase and anion exchange chromatography, respectively. Both concentration and output of statherins and PRPs were increased in persons with narcolepsy receiving central nervous system stimulants compared with healthy control individuals. When the drug was withdrawn, salivary flow rates were not influenced. In contrast, withdrawal of the drug led to a significantly decreased secretion of PRPs and statherin. The reduced protein secretion may reflect decreased adrenergic activation in narcolepsy, to be reversed by treatment with central nervous system stimulants. It can be concluded that measurements of both salivary fluid and salivary proteins may be necessary for an overall evaluation of the effects of a given drug or disease on salivary secretion.