Inhibition of adhesion-promoting activity of a human salivary protein which promotes adhesion of Streptococcus mutans JBP to hydroxyapatite.

The adhesion-promoting proteins (APP) (molecular mass approx. 300 kDa), which promote adhesion of Streptococcus mutans JBP (serotype c) to hydroxyapatite, were isolated from human submandibular-sublingual (SMSL) saliva by gel filtration on a Trisacryl GP2000 M column. The effects of hexoses, pentoses, methyl-pentoses, hexosamines, N-acetylhexosamines, a basic amino acid, polyamines and ammonium chloride on the bacterial adhesion-promoting activity of the APP were examined. Galactosamine, mannosamine, L-lysine, spermine, putrescine, and ammonium chloride inhibited the adhesion-promoting activities of the APP. The other sugars, including the N-acetylhexosamines, were without effect. Thus, compounds containing a primary amino-group appear to have a specific inhibitory effect on adhesion of S. mutans JBP to APP adsorbed onto hydroxyapatite, an activity which is lost if the amino-group is acetylated.

Adsorbed salivary acidic proline-rich proteins contribute to the adhesion of Streptococcus mutans JBP to apatitic surfaces.

Experimental pellicles formed on hydroxyapatite (HA) beads from parotid or submandibular saliva promoted the adhesion of Streptococcus mutans JBP cells to a greater extent than did pellicles prepared from buffer, human plasma, or serum. The nature of the salivary components responsible was studied by the preparation of pellicles from fractions of parotid saliva obtained by chromatography on Trisacryl GF 2000 columns. Two groups of fractions promoted attachment of the organism. Components migrating in the high-molecular-weight mucin fraction were most effective, but a later-eluting fraction also possessed adhesion-promoting activity. Subfractionation of the latter material indicated that the adhesion-promoting activity was associated with the acidic proline-rich proteins (PRPs). Pellicles prepared from 10-20-micrograms/mL solutions of pure PRP-1 were effective in promoting attachment of S. mutans JBP cells. PRP-3 was less effective, while human salivary statherin, fibrinogen, fibronectin, type 1 collagen, and the amino-terminal tryptic peptide derived from PRP-1 were ineffective. The quantities of 150-residue and 106-residue PRPs and of statherin, which became incorporated into experimental pellicles prepared from saliva, were estimated with use of radiolabeled protein tracers. The data obtained suggest that these proteins compete for similar binding sites on HA, and that their ratios in saliva would therefore influence the quantity of the larger PRPs that become incorporated into the pellicle. Such competition may contribute to the variability observed in the adhesion-promoting activities of different saliva samples.

Differential adsorption and chemical affinities of proteins for apatitic surfaces.

Studies are reviewed which identify the interacting groups involved in protein adsorption onto hydroxyapatite. Thus, carboxyl and phosphoester groups bind to calcium sites in the adsorbent, and basic groups bind to phosphate sites. Detailed adsorption studies have been performed to quantitate some of these interactions. An adsorption model, based on the Langmuir adsorption isotherm, adequately fitted the data from experiments using selected amino acids, bovine albumin and two human salivary proteins. Adsorption parameters (affinities and maximum number of sites) were obtained for several apatitic adsorbents, with affinities increasing considerably in the series hydroxy- (HA), fluorhydroxy- (FHA) and fluorapatite (FA). A modest increase in the number of sites was also noted. The change in adsorption behavior, with increasing fluoride content, was attributed to a reduction in the surface free energy of the adsorbent, with a concomitant decrease in the interaction of the adsorbent with water, and a consequent enhancement of the adsorption bond. It is suggested that this effect may play a role in the cariostatic effect of fluoride. Unusual structural aspects of the salivary proteins are discussed in relation to their adsorption behavior, and the molecular segments responsible for binding to the adsorbent tentatively identified.

Polymorphism of submandibular-sublingual salivary proteins which promote adhesion of Streptococcus mutans serotype-c strains to hydroxyapatite.

Previously, we showed that human submandibular-sublingual (SMSL) salivas contain one or more proteins, Mr circa 300,000 daltons, which specifically promote adhesion of Streptococcus mutans serotype-c strains to hydroxyapatite. Also, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the adhesion-promoting proteins (APPs) exhibit heterogeneity. The aims of the present study were to determine whether APPs are generally present in human SMSL salivary secretions and to characterize the noted heterogeneity. Acid-stimulated SMSL saliva samples were obtained from 54 Japanese subjects, and Mr values were obtained by SDS-PAGE. APPs were present in all saliva samples examined, though at significantly different concentrations. The APPs occurred as either single (20 subjects) or double bands (34 subjects), with a mean Mr (88 bands) of 297 kD and a range of 248-338 kD. A plot of the frequency distribution of the APPs according to Mr showed a trimodal distribution, with mean Mr values, standard deviations, and ranges for the three groups being 265 (S.D., 6.9; range, 248-278), 293 (S.D., 6.7; range, 280-305), and 320 (S.D., 7.0; range, 310-338) kD. Variations of Mr within groups may be attributed to experimental variation, although microheterogeneity cannot be excluded. Differences between groups can best be explained in terms of three polymorphic proteins, with low (L), intermediate (I), and high (H) Mr values. Six phenotypes were detected with L, I, H, LI, LH, and IH Mr bands. A Hardy-Weinberg analysis showed that the phenotype data fit a single-gene, three-alleles model.(ABSTRACT TRUNCATED AT 250 WORDS)

Human salivary acidic proline-rich protein polymorphisms and biosynthesis studied by high-performance liquid chromatography.

Human salivary acidic proline-rich proteins (PRPs) constitute a significant fraction of the total salivary protein and possess important biological activities. Different genetic and post-translationally processed forms of the PRPs exhibit significant quantitative variations in several of these activities, especially the modulation of salivary calcium phosphate chemistry and oral bacterial adhesion. To quantify and understand these differences, we have developed a high-performance liquid chromatography (HPLC) method to identify and measure individual PRPs in saliva. The data obtained permit the identification of PRP polymorphisms and phenotypes, the determination of the relative amounts of PRPs derived from the two loci, PRH1 and PRH2, and the measurement of the extent of post-translational cleavage of the primary polypeptide products. Substantial inter-gland and inter-individual variations were found in relative amounts of PRPs derived from the two loci (at least two-fold), and in post-translational cleavage (greater than two-fold), both of which are likely to be biologically significant. Also in this study, the presence of what appear to be minor amounts of numerous variant PRPs in glandular secretions was observed, and two uncommon PRP polymorphisms were identified in the 127 subjects studied.

Molecular basis of salivary proline-rich protein and peptide synthesis: cell-free translations and processing of human and macaque statherin mRNAs and partial amino acid sequence of their signal peptides.

Acidic proline-rich phosphoproteins and phosphopeptides are abundant components of parotid and submandibular salivary secretions in man and in the subhuman primate, Macaca fascicularis. The major acidic proline-rich proteins and the proline-rich phosphopeptide, statherin, of man and macaques have been shown to be potent inhibitors of calcium phosphate precipitation and are thought to function in the oral environment by maintaining saliva supersaturated with respect to calcium phosphate salts. Little is known about the biosynthesis of these proline-rich phosphoproteins and peptides, and the aim of the present work was to determine the structural relationship between statherin precursors and native human and macaque statherin. RNA was isolated from human submandibular gland, and poly(A+) mRNA was selected by affinity chromatography on oligo(dT) cellulose and translated in a reticulocyte lysate. Electrophoretic analysis of the translation products revealed that this mRNA directed the synthesis of a large number of polypeptides with Mrs ranging from 5000 to 70,000. Immunoprecipitates, prepared with an antiserum directed against human statherin, contained a single component with a Mr of 7800, approximately 2000 daltons larger than native statherin. Radiosequencing of the in vitro precursor of statherin in immunoprecipitates demonstrated the presence of a 19-residue signal peptide. These results suggest that statherin is derived from a unique structural gene, and does not result from proteolytic processing of a large polyprotein precursor.

Relationship between concentration of human salivary statherin and inhibition of calcium phosphate precipitation in stimulated human parotid saliva.

Human salivary secretions are supersaturated with respect to the calcium phosphate salts which form dental enamel, a property which provides important protection for the teeth. We previously proposed that statherin, a 43-residue phosphopeptide, plays a key role in this protective system by inhibiting or delaying potentially harmful precipitation of calcium phosphate salts in the salivary glands and mouth. The purpose of the present study was to determine if the concentrations of statherin in saliva, despite their wide normal range, are high enough to fulfill this function. Concentrations of statherin in stimulated human parotid saliva samples from 36 female and 32 male subjects, aged from 17 to 30 years, were determined by a single radial immunodiffusion method. Values found ranged from 3.0 to greater than 27.3 microM, with a mean value of 12.8 (S.D. +/- 5.46) microM. At concentrations below these values, statherin inhibited spontaneous precipitation of calcium phosphate salts from an assay system which was more supersaturated with respect to dicalcium phosphate dihydrate, and comparably supersaturated with respect to hydroxyapatite, than were human saliva samples. The inhibitory activities of five of the 65 stimulated parotid saliva samples assayed were greater than would be anticipated from their statherin concentrations. This unexplained discrepancy is not associated with the presence of the acidic proline-rich proteins in saliva, although these proteins also affect calcium phosphate precipitation. The results of this study show that statherin is present in stimulated human parotid saliva at concentrations and levels of activity which are consistent with its proposed biological function, and support the proposal that statherin plays a significant role in a system which provides a protective and reparative but stable environment for the teeth.

Socioeconomic status and health status: a study of males in the Canada Health Survey.

The relationships between education/occupation/income and health status have been well documented in the international epidemiological and sociological literature for many years, however, specific studies on the subject are scarce in Canada. Even when relationships have been demonstrated, the reasons for these relationships are much debated. This study presents an analysis of the relationship between socioeconomic status (SES) and health status. The study is based on analysis of data from a sample of nearly 2000 male principal income earners from the 1978 Canada Health Survey. Firstly, is there a relationship between an individual's SES and health status in Canada? Secondly, what aspects of SES--education, occupational status, and/or income--are most important? Thirdly, what are the possible explanations of the observed relationship? That is, is it possible to disaggregate the relationship and thereby infer possible causal mechanisms? The findings indicated a direct positive relationship between SES and health status, i.e. the higher an individual's SES, the better that person's health. The major exception to this was the SES/fitness relationship. In this instance, the higher the SES, the lower the level of fitness. Though age was an important control variable as SES, fitness and illness are age related, the findings relating SES to the health measures remained even when age was controlled for. Of the three SES measures, income was consistently the best correlate of health status. Occupational status showed the most inconsistent relationships with health status. The findings supported both the social causation and social selection hypotheses. That is, social position can have an effect on health status (social causation), while health status can affect one's social position (social selection).

Role of cryptic receptors (cryptitopes) in bacterial adhesion to oral surfaces.

Progress in characterizing the receptors that promote bacterial attachment to teeth and oral epithelial cells has suggested that hidden molecular segments may frequently be involved. Such cryptic receptors, referred to as 'cryptitopes', may become exposed by several mechanisms. Hidden segments of salivary acidic proline-rich proteins evidently become exposed when the molecules undergo a conformational change as they adsorb to apatitic mineral. Adhesins of Actinomyces viscosus and certain other prominent dental plaque bacteria are able to bind to these cryptitopes, and this enables these organisms to bind to proline-rich proteins on apatitic surfaces while avoiding interactions with these proteins in solution. Cryptitopes may also become exposed as a result of enzymatic action. Thus, several bacteria, including Fusobacterium nucleatum, Eikenella corrodens, A. viscosus, A. naeslundii and Bacteroides intermedius, have adhesins that bind to galactosyl receptors which become exposed after treatment with neuraminidase. Similarly, the adhesion of some Gram-negative bacteria, such as Bact. gingivalis, is enhanced when tissue surfaces are treated with certain proteases, or lysosomal enzymes derived from human polymorphonuclear leucocytes. It seems likely that elevated levels of enzymes present in gingival fluid as sequelae of poor oral hygiene and gingivitis may generate cryptitopes for potentially periodontopathic bacteria, and thereby contribute to modulation of the gingival flora.

Partial characterization of a human submandibular/sublingual salivary adhesion-promoting protein.

Human submandibular/sublingual saliva contains a protein that promotes adhesion of Streptococcus mutans JBP serotype-c to spheroidal hydroxyapatite in vitro. A high molecular-weight (250,000-300,000 Da) adhesion-promoting protein (APP) was purified by Trisacryl 2000 M gel-filtration chromatography and gel electroelution before it was partially characterized. Lectin blotting identified that the terminal carbohydrates include N-acetyl glucosamine-beta 1-4-N-acetylglucosamine, galactose and galactose-beta 1-3-N-acetyl galactosamine. Antibodies to APP demonstrated no difference in the immunoreactive pattern of APP from saliva of caries-active or caries-resistant individuals belonging to four different ethnic groups: Asian, African-American, Hispanic or Caucasian. No immunological similarities to salivary mucins or parotid agglutinins were detected by Western blotting using immuno-cross-reactivity as a criterion. APP appears to be a unique protein found in submandibular/sublingual saliva. Understanding such a protein could help prevent S. mutans attachment to the enamel surface.

Purification and characterization of a rabbit salivary protein, a potent inhibitor of crystal growth of calcium phosphate salts.

Human saliva is supersaturated with respect to basic calcium phosphate salts but is stabilized by specific macromolecules that inhibit calcium phosphate precipitation. One of the families of inhibitory proteins in human and monkey saliva is the acidic proline-rich proteins. The purpose of this study was to isolate and characterize inhibitors of calcium phosphate precipitation from rabbit parotid saliva. Saliva was fractionated by immunoaffinity chromatography and anion exchange chromatography. Individual fractions were assayed for their ability to inhibit calcium phosphate crystal growth and the fraction associated with the inhibition was purified by repeated anion exchange chromatography, preparative gel electrophoresis and electroelution. A major (APRP) and two minor proteins (AM1, AM2) that were inhibitory were purified. APRP is an acidic proline-rich phospho-glycoprotein and a very potent inhibitor of secondary crystal growth of calcium phosphate as it was active at a concentration of 2 x 10(-8) M in a standard assay. The N-terminal sequence of one APRP was EYENLDGSLAATQNDDD?Q and a clostripain fragment of APRP had the following N-terminal sequence PQHRPPRPGGH-????SPPP?GN???PPP. Although the N-terminal segment of APRP does not resemble that of proline-rich proteins, alignment of the clostripain fragment with the repeat region of such proteins from rat, mouse, monkey and man revealed a high degree of similarity, indicating a structural relationship with the proline-rich protein family.