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.

Adhesion of Candida albicans, but not Candida krusei, to salivary statherin and mimicking host molecules.

The aim of the present study was to identify salivary molecules affecting adhesion of Candida albicans and Candida krusei to salivary pellicles and epithelial cells. Strains of C. albicans (GDH18, GDH3339, CA1957, ATCC 28366 and ATCC 10321), but not C. krusei (strains ATCC 14243 and Ck9), bound to saliva-coated hydroxyapatite and buccal epithelial cells. Parotid saliva fractions containing statherin, glycosylated proline-rich proteins (PRP) and as yet unidentified components mediated adhesion of strain GDH18; Fuc alpha 1-2Gal beta 1-4Glc partly inhibited the adhesion to those fractions not containing statherin. Pure statherin, but not PRP-1, mediated dose-dependent adhesion of C. albicans strain GDH18 to hydroxyapatite beads. Candida isolates (GDH18, GDH3339 and CA1957) bound somewhat more avidly to statherin/saliva relative to ATCC strains 28366 and 10321, while the opposite was true for adhesion to buccal epithelial cells. Adhesion of C. albicans strain GDH18 to saliva-coated hydroxyapatite and buccal epithelial cells was completely (93%) and partly (43%) blocked by statherin-specific immunoglobulin G (IgG) antibodies, respectively. Control IgG antibodies did not block Candida adhesion. Blockage of Candida adhesion to epithelial cells also occurred with Fuc alpha 1-2Gal beta 1-4Glc (49%) and N-acetylglucosamine (38%), while statherin specific IgG antibodies in combination with Fuc alpha 1-2Gal beta 1-4Glc almost completely eliminated Candida adhesion (79%). In addition, statherin in solution blocked the adhesion of strain GDH18 to epithelial cells by inducing aggregation of Candida cells.

Strains of Actinomyces naeslundii and Actinomyces viscosus exhibit structurally variant fimbrial subunit proteins and bind to different peptide motifs in salivary proteins.

Oral strains of Actinomyces spp. express type 1 fimbriae, which are composed of major FimP subunits, and bind preferentially to salivary acidic proline-rich proteins (APRPs) or to statherin. We have mapped genetic differences in the fimP subunit genes and the peptide recognition motifs within the host proteins associated with these differential binding specificities. The fimP genes were amplified by PCR from Actinomyces viscosus ATCC 19246, with preferential binding to statherin, and from Actinomyces naeslundii LY7, P-1-K, and B-1-K, with preferential binding to APRPs. The fimP gene from the statherin-binding strain 19246 is novel and has about 80% nucleotide and amino acid sequence identity to the highly conserved fimP genes of the APRP-binding strains (about 98 to 99% sequence identity). The novel FimP protein contains an amino-terminal signal peptide, randomly distributed single-amino-acid substitutions, and structurally different segments and ends with a cell wall-anchoring and a membrane-spanning region. When agarose beads with CNBr-linked host determinant-specific decapeptides were used, A. viscosus 19246 bound to the Thr42Phe43 terminus of statherin and A. naeslundii LY7 bound to the Pro149Gln150 termini of APRPs. Furthermore, while the APRP-binding A. naeslundii strains originate from the human mouth, A. viscosus strains isolated from the oral cavity of rat and hamster hosts showed preferential binding to statherin and contained the novel fimP gene. Thus, A. viscosus and A. naeslundii display structurally variant fimP genes whose protein products are likely to interact with different peptide motifs and to determine animal host tropism.

Actinomyces naeslundii genospecies 1 and 2 express different binding specificities to N-acetyl-beta-D-galactosamine, whereas Actinomyces odontolyticus expresses a different binding specificity in colonizing the human mouth.

A total of 102 strains of Actinomyces were isolated from teeth, buccal mucosa and tongue in eight individuals. The isolates were characterized by multivariate statistical analyses of phenotypic characteristics, serotyping and binding to beta-linked galactosamine (N-acetyl-beta-D-galactosamine) and acidic proline-rich protein structures. Based on these characteristics, isolates were classified into three major groups: (i) Isolates of Actinomyces naeslundii genospecies 2 were the dominant species on teeth and buccal mucosa and bound commonly to N-acetyl-beta-D-galactosamine (63 of 63 isolates) and acidic proline-rich proteins (63 of 63 isolates), regardless of tissue origin. They all exhibited a N-acetyl-beta-D-galactosamine binding specificity signified by N-acetyl-beta-D-galactosamine-inhibitable coaggregation with the streptococcal strains LVG1, GVE1, 24892 and MPB1; (ii) Isolates of A. naeslundii genospecies 1 were prevalent on teeth in certain individuals and bound commonly to N-acetyl-beta-D-galactosamine (20 of 20 isolates), but less commonly to acidic proline-rich proteins (5 of 20 isolates). They all possessed another N-acetyl-beta-D-galactosamine specificity, i.e. N-acetyl-beta-D-galactosamine-inhibitable coaggregation with the same streptococcal strains except for strain MPB1; (iii) Isolates of Actinomyces odontolyticus, the dominant species on the tongue (17 of 19 isolates), bound commonly to unknown structures on streptococci (17 of 19 isolates) but rarely to N-acetyl-beta-D-galactosamine (2 of 19 isolates) or acidic proline-rich proteins (3 of 19 isolates). In conclusion, A. naeslundii genospecies 1 and 2 exhibit different patterns of N-acetyl-beta-D-galactosamine and acidic proline-rich protein specificities to colonize dental and buccal mucosa surfaces, whereas A. odontolyticus utilizes another specificity to colonize the tongue.

Salivary factors in caries models.

Consideration of salivary factors in caries models rarely extends beyond viewing saliva as a sink or diluent for plaque metabolic products, or as a source of buffering, for neutralizing plaque acids. In reality, saliva has a complex chemistry and a wide range of biochemical activities that may significantly affect plaque chemistry and microbiology. Thus, saliva is a major source of microbial nutrients, without which bacterial acid production is diminished. Buffering by salivary bicarbonate, and base production from urea and basic amino acids and peptides, significantly affect Stephan curves. Saliva is supersaturated with respect to basic calcium phosphate salts and contains novel inhibitors of calcium phosphate precipitation, while specific salivary proteins bind calcium. It seems important to consider if this system is reflected in plaque. Saliva, with contributions from serum and bacterial constituents, provides most of the precursors for the acquired enamel pellicle, which acts to slow demineralization during caries attack. Pellicle constituents appear to influence initial bacterial colonization of tooth surfaces and, therefore, may affect the microbial composition of plaque, but their detailed effects on plaque are poorly understood. Microbial adaptations to the anti-bacterial systems also seem important but are poorly investigated. Thus, saliva possesses an array of activities that have substantial actual or potential impact on plaque and, therefore, merit consideration for inclusion in systems intended to model dental caries.

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.

Primary structure of a novel human salivary acidic proline-rich protein.

Human salivary acidic proline-rich proteins (PRPs) form a significant fraction of the total salivary protein and fulfill several biologically important roles in the oral cavity. Five commonly occurring PRP polymorphisms, Db, Pa, PIF, Pr2 and Pr1, have been identified, their structures determined, and several uncommon polymorphisms (frequencies < 1:100) have been reported. Most PRPs occur as protein pairs, because of an unusual, limited but well-controlled post-translational cleavage. We now describe an additional uncommon polymorphism, found in the saliva of one of 127 individuals examined in a recent study, identified by high performance anion-exchange liquid chromatography. By analogy with previous terminology, we designate this protein pair as PRP-5, for the primary 150-residue polypeptide gene product, and PRP-6, for the secondary 106-residue cleavage product. Amino acid analysis of intact PRP-6 and sequence determination of PRP-6 chymotryptic peptides, residues 15-24 and 26-35, show a single difference in PRP-6, compared to the most similar, characterized PRP, PRP-4, in that residue 30 is histidine in PRP-6, rather than arginine as in PRP-4 and in all the other sequenced PRPs. This substitution may have implications for the resistance of this polymorphic variant to degradation by trypsin-like enzymes originating from the oral microflora.

Inhibition of calcium phosphate precipitation by human salivary statherin: structure-activity relationships.

Previous studies of human statherin showed the active region for inhibition of secondary calcium phosphate precipitation (crystal growth) to reside in the highly charged amino-terminal one-third of this molecule, and the neutral tyrosine-, glutamine- and proline-rich carboxy-terminal two-thirds of the molecule is required for maximal inhibition of primary (spontaneous) precipitation. The purpose of the present study was to define more clearly the activities of these different molecular segments of statherin with respect to the two kinds of inhibitory activities. Peptides from statherin were prepared by specific proteolysis using trypsin, endoproteinase Arg-C, and activated factor X to produce the amino-terminal hexa-, nona- and decapeptides, respectively, and carboxypeptidase-A was used to obtain a peptide extending from residue 1 to about residues 32-37. The peptides were purified by anion exchange and gel filtration chromatography, and characterized and quantified by amino-acid analysis. Serially diluted samples of statherin and derived peptides were assayed to determine the concentrations, giving a standard 50% inhibition of precipitation (C50%) in assay systems designed for this purpose using polyaspartate as a standard. Results are expressed as (C50% statherin)/(C50% peptide). For inhibition of primary precipitation, these values were peptide(1-6), 0.20; peptide(1-9), 0.15; peptide(1-31/35), 0.24. For inhibition of secondary precipitation, the values were peptide(1-6), 3.8; peptide(1-9), 2.8; peptide(1-10), 1.9; peptide(1-32/37), 1.5. These quantitative findings show that maximum inhibition of primary precipitation by statherin requires the entire molecule. Thus, removal of a relatively small segment of its carboxy-terminal region results in a substantial reduction in inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Adhesive properties of strains of Fusobacterium nucleatum of the subspecies nucleatum, vincentii and polymorphum.

This study surveyed some adhesive properties of strains of Fusobacterium nucleatum representative of the 3 recently defined groups or subspecies that could relate to their colonization and virulence. With one exception, F. nucleatum strains agglutinated sheep erythrocytes, but the quantity of bacteria required and the sensitivity of the hemagglutination reactions to inhibition by 0.05 M galactose or arginine varied between strains, and did not exhibit clear-cut correlations with subspecies. Neuraminidase treatment of erythrocytes generally enhanced the hemagglutinating activity of most strains, but trypsin treatment had no effect. Strains of F. nucleatum also attached in moderate numbers to buccal epithelial cells. Treatment of the epithelial cells with neuraminidase or with trypsin increased the numbers of all Fusobacterium strains that attached. Treatment of hydroxyapatite (HA) beads with submandibular or parotid saliva also promoted the adhesion of all strains of F. nucleatum studied. Treatment of HA with human serum or albumin produced a selective effect. Adhesion of some strains was promoted by serum and albumin treatment, and that of other strains was unaffected. Adhesion of all strains of F. nucleatum was enhanced to statherin-treated HA, whereas HA treated with salivary proline-rich protein-1 did not foster F. nucleatum attachment. Three of 4 strains of the subspecies vincentii, and each of 2 polymorphum strains studied exhibited strong adhesion to HA treated with either human type I or type IV collagen. However, only 1 of 5 strains of the subspecies nucleatum bound well to collagen-treated HA.(ABSTRACT TRUNCATED AT 250 WORDS)

Delineation of a segment of adsorbed salivary acidic proline-rich proteins which promotes adhesion of Streptococcus gordonii to apatitic surfaces.

Cells of several strains of Streptococcus gordonii attached in much higher numbers to experimental pellicles formed from samples of submandibular or parotid saliva on hydroxyapatite (HA) beads than to buffer controls. The nature of the salivary components responsible were investigated by preparing experimental pellicles from chromatographic fractions of submandibular saliva obtained from Trisacryl GF 2000M columns. Adhesion of S. gordonii Blackburn was promoted by two groups of fractions. The adhesion-promoting activity in the first group of fractions was associated with the family of acidic proline-rich proteins (PRPs), while that of the second group is as yet unidentified. Experimental pellicles prepared by treating HA with 2 micrograms of pure 150-amino-acid-residue PRPs (PRP-1, PRP-2, and PIF-s) promoted adhesion of S. gordonii Blackburn cells to an extent comparable to that obtained with unfractionated saliva. However, pellicles prepared from a 106-residue PRP (PRP-3) were significantly less effective, and those prepared from the amino-terminal tryptic peptide (residues 1 to 30) of the PRP and the salivary phosphoprotein statherin were completely ineffective in promoting adhesion. Although adhesion of several strains of S. gordonii was promoted by adsorbed PRP-1, the adhesion of several strains of Streptococcus sanguis or Streptococcus oralis was either not affected or only weakly enhanced by this protein. S. gordonii cells bound avidly to PRPs adsorbed onto HA beads, but the streptococci did not appear to bind PRPs in solution, since concentrations of PRP as high as 200 micrograms/ml did not inhibit binding of bacterial cells to pellicles prepared from pure PRP. S. gordonii cells also attached well to PRP or a synthetic decapeptide representing residues 142 to 150 of the PRP when the peptide was linked to agarose beads. Studies with a series of synthetic decapeptides indicated that the minimal segment of PRP which promoted high levels of S. gordonii adhesion was the carboxy-terminal dipeptide Pro-Gln (residues 149 and 150).

Binding of Actinomyces viscosus to collagen: association with the type 1 fimbrial adhesin.

Treatment of hydroxyapatite (HA) with human type I or type III collagen strongly promoted adhesion of Actinomyces viscosus LY7 cells. Treatment with human type V collagen was somewhat less effective while treatment with human type IV or rat type I collagen was significantly less effective. Electron microscopic observations revealed that A. viscosus cells also attached to fibrils prepared from human type I collagen. The alpha 1 (1) polypeptide chain derived from type I collagen was ineffective in promoting binding and the alpha 2 (1) polypeptide chain exhibited moderate activity. Heat- or urea-denatured type I collagens were also ineffective in promoting binding. Mutants of A. viscosus that possess type 1 fimbriae, but not type 2 fimbriae or no fimbriae, also bound to collagen-treated HA; this suggests that the adhesin responsible was associated with type 1 fimbriae. Strains of Actinomyces israelii and Actinomyces odontolyticus also exhibited strong binding to collagen-treated HA, while Actinomyces naeslundii ATCC 12104 did not. The avidity of Actinomyces species for collagen would seem to be at least partially responsible for the high proportions of these organisms found on cemental and root tooth surfaces.

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.

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)

Binding of colloidal gold-labeled salivary proline-rich proteins to Actinomyces viscosus type 1 fimbriae.

Salivary proline-rich proteins (PRPs), which were purified from parotid saliva, were adsorbed onto 15-nm-diameter gold particles to visualize specific binding of the salivary molecules to Actinomyces viscosus type 1 fimbriae. Negatively stained preparations incubated with PRP-gold conjugates but not bovine serum albumin-gold complexes bound specifically to bacteria possessing type 1 fimbriae, A. viscosus T14V-J1 and 5519. Binding of the PRP-gold probes to strains deficient in type 1 fimbriae, i.e., strains 5951 (type 2 fimbriae only) and 147 (no fimbriae), was negligible.

Streptococcus cricetus and Streptococcus rattus bind to different segments of collagen molecules.

Strains of Streptococcus cricetus and Streptococcus rattus exhibited striking differences in their ability to bind to different types of collagen. For example, S. cricetus AHT bound in highest numbers to hydroxyapatite (HA) treated with human type V collagen, while rat type I collagen was ineffective. In contrast, human type V collagen was least effective in promoting attachment of S. rattus LB-1, while treatment with rat or human type I collagen was effective. Adsorption of both species to human type I collagen-treated HA showed a high correlation with a Langmuir model. Estimates of adsorption parameters indicated there were greater numbers of binding sites with higher affinity for S. rattus LB-1 than for S. cricetus AHT. Treatment of HA with either the alpha 1 (1) or alpha 2 (1) polypeptide chains of collagen was effective in promoting adhesion of S. rattus LB-1 cells. In contrast, the alpha 2 (1) chain was more effective than the alpha 1 (1), chain for S. cricetus AHT. These observations indicate that S. cricetus AHT and S. rattus LB-1 cells bind to different segments of collagen molecules. Adhesion of both species was also promoted by collagen-rich fractions of human dentin.

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.

A human salivary protein which promotes adhesion of Streptococcus mutans serotype c strains to hydroxyapatite.

The aim of this study was to investigate the nature of one of the factors in human submandibular-sublingual (SMSL) saliva which promotes the adhesion of Streptococcus mutans serotype c strains to hydroxyapatite (HA) surfaces. Gel filtration chromatography of SMSL saliva on Trisacryl GF2000 gave a void volume peak which contained the major fraction of adhesion-promoting activity for S. mutans JBP to HA. Maximum adhesion-promoting activity, however, eluted slightly later than the maximum 220-nm absorbance of the void volume peak. Gel filtration of the void volume material after treatment with sodium dodecyl sulfate (SDS) gave an early-eluting larger peak followed by a smaller peak with which the adhesion-promoting activity was associated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of relatively slowly migrating material associated with the larger inactive peak, presumably mucin, and a faster-migrating band(s) associated with the smaller active peak. SDS-PAGE indicated molecular weights in the range of 300,000 to 350,000 by extrapolation from size standards. Comparison of SMSL from five individuals showed the presence of single bands or double bands associated with adhesion-promoting activity, indicating genetic polymorphism. The active material did not resemble either secretory immunoglobulin A, based on SDS-PAGE and immunoassay, or fibronectin, based on SDS-PAGE, and also differed in molecular weight from salivary mucins and salivary constituents previously reported to promote aggregation of certain oral bacteria, but a relationship to these materials cannot be excluded. This adhesion-promoting material may play a significant role in the initial colonization of tooth surfaces by S. mutans strains.

Complete primary structure of statherin, a potent inhibitor of calcium phosphate precipitation, from the saliva of the monkey, Macaca arctoides.

Human saliva, which is supersaturated with respect to basic calcium phosphate salts, is stabilized primarily by the presence of two classes of phosphoproteins, statherin and the acidic proline-rich proteins (PRP). These molecules act by inhibiting both primary (spontaneous) precipitation of calcium phosphates in saliva and secondary (surface induced) precipitation of these salts onto dental enamel. The complete amino-acid sequences of several human PRP and the N-terminal sequence of PRP from saliva of M. arctoides have been determined. Similarly, the complete sequence of statherin from human and M. fascicularis saliva is known. We now report the complete structure of statherin from the saliva of the stump-tailed monkey, M. arctoides. The structure was determined by gas-phase sequencing of intact statherin, elucidating positions 1-26, and sequencing an unpurified mixture of tryptic peptides which elucidated the remaining positions through the C-terminus (residue 42) of the molecule. This latter degradation produced an eight amino-acid overlap with that of intact statherin and was confirmed by C-terminal analysis and amino-acid composition of native statherin. The complete amino-acid sequence of M. arctoides statherin is: NH2-Asp-PSer-PSer-Glu-Glu5-Lys-Phe-Leu-Arg-Arg10 -Leu-Arg-Arg-Phe-Asp15-Glu- Gly-Arg-Tyr-Gly20-Pro-Tyr-Gln-Pro-Phe25-Val-Pro-Pro- Pro29Leu30-Tyr- Pro-Gln-Pro-Tyr35-Gln-Pro-Tyr-Gln-Pro40-Gln-Tyr-COOH This sequence differs from human statherin at positions 11, 12, 15, 16, 18, 25-27, 38-40 and from M. fascicularis statherin at positions 26 and 28.

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.