Chemical synthesis of phosphoseryl-phosphoserine, a partial analogue of human salivary statherin, a protein inhibitor of calcium phosphate precipitation in human saliva

Human salivary secretions are supersaturated with respect to basic calcium phosphates but spontaneous precipitation of these salts from saliva, or surface-induced precipitation of calcium phosphates onto dental enamel, does not normally occur. This unexpected stability has been attributed to the inhibitory activities of two kinds of salivary phosphoproteins: statherin and the acidic, proline-rich phosphoproteins (PRP). Investigation of the structure-function relationships of statherin, the most potent inhibitor of primary (spontaneous) and secondary (seeded) precipitation of calcium phosphate salts in human saliva has been limited to studies of peptide segments obtained from the native peptide by specific proteolysis. Solid phase peptide synthesis (SPPS) is a useful and potentially more flexible alternative. Phosphoserine residues (positions 2 & 3) play critically important roles in the precipitation-inhibition activities of statherin, but SPP synthesis of these phosphorylated peptides is precluded because of the instability of phosphoserine residues in the presence of HF. Thus, this peptide was synthesized by solution-phase methods. The dipeptide possessed substantial inhibitory activity in assays for inhibition of both primary and secondary precipitation of calcium phosphate salts, but was not as active as either N-terminal tryptic hexapeptide of statherin or intact statherin. Syntheses of other model phosphorylated peptides are underway to expand the structure-function relationships.     

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.     

The primary structures of six human salivary acidic proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f).

Human glandular salivary secretions contain several acidic proline-rich phosphoproteins (PRPs). These proteins have important biological functions related to providing a protective environment for the teeth, and appear to possess other activities associated with modulation of adhesion of bacteria to oral surfaces. These functions and activities depend on the primary structures of the PRPs. Previously determined amino acid sequences of two 150-residue molecules, PRP-1 and PRP-2, and two related 106-residue proteins, PRP-3 and PRP-4, indicated that residue 4 was Asn in PRP-1 and PRP-3, and Asp in PRP-2 and PRP-4, and position 50 was Asn in all four proteins. Recent data from cDNA sequence studies and further structural studies, however, showed that the previously proposed sequences cannot be completely correct. The present work has shown that the protein previously designated as PRP-1 actually consisted of two positional isomers, PIF-s, which has Asn and Asp at positions 4 and 50 respectively, and authentic PRP-1, which has the reverse arrangement. The same isomerism is present in the smaller proteins, PIF-f and PRP-3. Since the isomeric pairs have identical compositions and charges, their presence was not previously detected. Also, by using a more highly purified preparation, it has been found that position 50 in PRP-2 and PRP-4 is Asp, rather than Asn previously reported. These new findings for the six PRPs define their complete primary structures, which are now consistent with those proposed for PRP-1 and PIF-s from cDNA data, and are also consistent with the chromatographic and electrophoretic behaviours of the six PRPs and their derived peptides. These corrected structures are important for understanding the biological functions and activities of these unusual proteins.     

Intraoral lubrication of PRP-1, statherin and mucin as studied by AFM

The aim of this paper was to elucidate the mechanisms behind salivary lubrication with special emphasis on the lubricity of three key components of the pellicle, viz human acidic proline-rich protein 1 (PRP-1), human statherin and bovine submaxillary mucin (BSM). The lubricating properties of the proteins have been assessed by means of colloidal probe atomic force microscopy, and are discussed in relation to their adsorption behaviour. To various extents, the proteins investigated all showed a lubricating effect when adsorbed to silica surfaces. For comparable concentrations, PRP-1 was found to have a more pronounced lubricating effect than BSM, which in turn showed a higher lubricity than statherin. The relative lubricity is in accordance with previously reported relative adsorbed amounts of the three proteins, within the investigated concentration interval. It is concluded that PRP-1 has the highest lubricating capacity as a pure fraction among the preparations investigated, and that the lubricating effect of PRP-1 as a pure fraction is notably large as compared to the lubricity of human whole saliva.     

Determination of the post-translational modifications of salivary acidic proline-rich proteins

Human salivary acidic proline-rich proteins were analyzed by electrospray-ion trap mass spectrometry and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All acidic-PRP isoforms share a common N-terminal region, which contains a pyroglutamic acid residue at the N-terminus, and two phosphorylation sites on Ser 8 and 22. At the same time, HPLC-MS spectra revealed isoforms of PRP-1 and PRP-3 having a different number of phosphoserine residues, namely, a mono-phosphorylated form of PRP-1 and PRP-3 and a tri-phosphorylated form of PRP-1. The analysis of the masses of tryptic digests suggested that the third phosphate residue should be located on Ser 17. Another protein with a mass of 30,923 amu was detected along the HPLC pattern and MS data of its tryptic digest suggested that it corresponds to the dimer of Pa, the isoform of PRP-1 with a substitution Arg-Cys at 103 position. Finally, structural identification is pending for another post-translational modification of acidic-PRP that provides an increase of 111-114 amu.     

The coupling of RP-HPLC and ESI-MS in the study of small peptides and proteins secreted in vitro by human salivary glands that are soluble in acidic solution

The aim of this study was the development of a method based on the coupling of RP-HPLC and ESI-MS for identifying and quantifying proteins and peptides secreted by human salivary glands in vitro. Salivary gland specimens, obtained from informed patients undergoing surgical resection, were incubated in an optimized medium. Incubation media of glandular specimens, selected on the basis of cytomorphological and ultrastructural analysis, were investigated by HPLC-MS. Several salivary peptides/proteins, previously recognized in human whole saliva, were searched for along the chromatogram by the selected ion monitoring (SIM) strategy. Analysis of the incubation media of parotid glands revealed the presence of basic PRPs PC, PD, PH, IB-1, II-2, and acidic PRP-1 and PRP-3 in all of the investigated samples. Basic PRPs PB and PA, acidic PRPs, and cystatins SN and S1 were detected in all of the incubation media of submandibular glands, whereas histatin 1 was detected in only one sample. Moreover, the method allowed detection of some post-translational derivatives of known salivary proteins, as well as of several previously unidentified small peptides. The present method represents a sensitive and powerful instrument to detect peptides and proteins secreted by human salivary glands in vitro.     

Determination of the human salivary peptides histatins 1, 3, 5 and statherin by high-performance liquid chromatography and by diode-array detection

A reversed-phase high-performance liquid chromatography (HPLC) method with diode-array detection for the quantification of several human salivary peptides is described. Sample pretreatment consisted of the acidification of whole saliva by phosphate buffer. This treatment produced precipitation of mucins, α-amylases and other high-molecular-mass salivary proteins, simultaneous inhibition of intrinsic protease activities and reduction of sample viscosity. Direct HPLC analysis by diode-array detection of the resulting acidic sample allowed one to quantify histatin 1, histatin 3, histatin 5, statherin, as well as uric acid, in normal subjects. Moreover, the groups of peaks pertaining to proline-rich proteins and cystatins were tentatively identified. The method can be useful in assessing the concentration of salivary peptides from normal subjects and from patients suffering oral and/or periodontal diseases.     

Salivary proline-rich proteins

Summary Proline-rich proteins are major components of parotid and submandibular saliva in humans as well as other animals. They can be divided into acidic, basic and glycosylated proteins. The primary structure of the acidic proline-rich proteins is unique and shows that the proteins do not belong to any known family of proteins. The proline-rich proteins are apparently synthesized in the acinar cells of the salivary glands and their phenotypic expression is under complex genetic control.The acidic proline-rich proteins will bind calcium with a strength which indicates that they may be important in maintaining the concentration of ionic calcium in saliva. Moreover they can inhibit formation of hydroxyapatite, whereby growth of hydroxyapatite crystals on the tooth surface in vivo may be avoided. Both of these activities as well as the binding site for hydroxyapatite are located in the N-terminal proline-poor part of the protein. Little is known about the functions of the glycosylated and basic proline-rich proteins.     

Different isoforms and post-translational modifications of human salivary acidic proline-rich proteins

The human salivary acidic proline-rich proteins (aPRPs) complex was investigated by different chromatographic and mass spectrometric approaches and the main aPRPs, namely PRP-1, PRP-2 and PIF-s (15,515 amu), Db-s (17,632 amu) and Pa (15,462 amu) proteins, were detected. All these isoforms are phosphorylated at Ser-8 and Ser-22 and have a pyroglutamic moiety at the N-terminus. Apart from Pa, all the other aPRPs undergo a proteolytic cleavage at Arg-106 residue (Arg-127 in Db-s protein), that generates the small PC peptide (4371 amu) and PRP-3, PRP-4, PIF-f (11,162 amu) and Db-f (13,280 amu) proteins, all of which were detected. With regard to the Pa protein, the main form detected was the dimeric derivative (Pa 2-mer, 30,922 amu) originated by a disulfide bond involving Cys-103 residue. Besides these known isoforms, several previously undetected aPRP derivatives were found (in minor amounts): (i) the triphosphorylated derivatives of PRP-1/PRP-2/PIF-s and Db-s, showing the additional phosphate group at Ser-17; (ii) the mono-phosphorylated forms at either Ser-22 or Ser-8 of PRP-1/PRP-2/PIF-s, PRP-3/PRP-4/PIF-f, Db-s and Db-f; (iii) a nonphosphorylated form of PRP-3/PRP-4/PIF-f; (iv) the triphosphorylated and diphosphorylated forms of Pa 2-mer. Moreover, minor quantities of PRP-3/PRP-4/PIF-f lacking the C-terminal Arg (11,006 amu), and of Pa 2-mer lacking the C-terminal Gln (30,793 amu) were found. By this approach the different phenotypes of PRH1 locus in 59 different subjects were characterized.     

Expression and characterization of human salivary statherin from Escherichia coli using two different fusion constructs.

Saliva is a supersaturated solution with respect to hydroxyapatite, the main inorganic component of tooth enamel. Several acidic phosphoproteins are present in saliva which allow the supersaturated state to be maintained without random crystallization occurring. Statherin is the only salivary protein currently known to inhibit both the primary and secondary precipitation of hydroxyapatite in the supersaturated environment of saliva. To identify the residues of statherin that are necessary to control biomineralization, a recombinant form of human statherin was produced from Escherichia coli using a yeast intein fusion construct. The primary structure of the recombinant statherin was characterized by SDS-PAGE, N-terminus sequencing, MALDI mass spectrometry, and amino acid analysis and found to have the expected values relative to human-derived statherin. The secondary structure of the recombinant statherin was investigated by circular dichroism spectroscopy, which revealed the predominant presence of random coil in phosphate-buffered saline solution, with a higher propensity toward alpha helicity in 100% TFE. This increase in helicity in 100% TFE was also found in statherin that was synthesized by solid-phase synthesis. These results demonstrate that human statherin can be produced in a recombinant form which behaves comparably to the natural form.     

Immunological comparison of proline-rich proteins from human and primate parotid secretion.

Antisera raised in response to proline-rich proteins purified from parotid secretions of man and the primate Macaca fascicularis were employed to investigate the interrelationships of these proteins by immunodiffusion, immunoelectrophoresis and the combined use of disc gel acrylamide electrophoresis with radial immunodiffusion. The major human proline-rich proteins, PRP I, PRP II, PRP III and PRP IV as well as several minor proline-rich proteins cross-react with antiserum to PRP I or PRP III. Similarly primate parotid saliva contains several components cross-reacting with antiserum directed against a purified primate proline-rich protein, MPRP. Antiserum to PRP I or PRP III cross-reacted with MPRP and primate parotid saliva protein, whereas antiserum to MPRP cross-reacted only with human parotid saliva protein and not with the isolated human proline-rich proteins. The immunological relationships of these salivary proline-rich proteins within and between species suggest their origin from a common precursor molecule.     

Group A streptococcus adheres to pharyngeal epithelial cells with salivary proline-rich proteins via GrpE chaperone protein

Group A Streptococcus pyogenes (GAS) is an important human pathogen that frequently causes pharyngitis. GAS organisms can adhere to and invade pharyngeal epithelial cells, which are overlaid by salivary components. However, the role of salivary components in GAS adhesion to pharyngeal cells has not been reported precisely. We collected human saliva and purified various salivary components, including proline-rich protein (PRP), statherin, and amylase, and performed invasion assays. The GAS-HEp-2 association ratio (invasion/adhesion ratio) and invasion ratio of GAS were increased significantly with whole human saliva and PRP, while the anti-PRP antibody inhibited the latter. GAS strain NY-5, which lacks M and F proteins on the cell surface, was promoted to cohere with HEp-2 cells by whole human saliva and PRP. The 28-kDa protein of GAS bound to PRP and was identified as GrpE, a chaperone protein, whereas the N-terminal of GrpE was found to bind to PRP. A GrpE-deficient mutant of GAS strain B514Sm, TR-45, exhibited a reduced ability to adhere to and invade HEp-2 cells. Microscopic observations showed the GrpE was mainly expressed on the surface of the cell division site of GAS. Furthermore, GrpE-deficient mutants of GAS and Streptococcus pneumoniae showed an elongated morphology as compared with the wild type. Taken together, this is the first study to show an interaction between salivary PRP and GAS GrpE, which plays an important role in GAS infection on the pharynx, whereas the expression of GrpE on the surface of GAS helps to maintain morphology.     

Salivary peptidomics

In recent years, there has been an increased interest in the study of saliva. This bodily fluid contains a vast number of protein species, the salivary peptidome, of low molecular weight, comprising approximately 40-50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Owing to the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related to sample collection, time of analysis, sample preservation and treatment are necessary for the successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400-600 are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of epithelial cells. Proteolysis events are the main supply for the peptidome and considerable efforts have been made to identify the resulting fragments, the cleavage sites and the involved proteases. The salivary proteins more prone to proteolysis are proline-rich proteins (PRPs; acidic PRPs and basic PRPs), statherin, histatins and P-B peptide. Gln-Gly cleavages are largely associated with PRP classes, while Tyr-Gly cleavages are related to histatin 1 and to the P-B peptide. The interest in saliva has been growing for clinical purposes, as it is an alternative sample to other traditional bodily fluids, such as blood or urine, since it involves an easy and noninvasive collection. In fact, apart from its usefulness as a source of information for the prognosis, diagnosis and treatment of oral diseases, such as Sj?gren's syndrome, gum disease, tooth decay or oral cancer, saliva might also be seen as a potential tool to the diagnosis of systemic diseases. Owing to the enormous amount of previously discovered salivary peptide species, in this article, we attempt to harmonize the nomenclature, following International Union of Pure and Applied Chemistry recommendations.     

A comparison of salivary pH in sympatric wild lemurs (Lemur catta and Propithecus verreauxi) at Beza Mahafaly Special Reserve, Madagascar

Chemical deterioration of teeth is common among modern humans, and has been suggested for some extinct primates. Dental erosion caused by acidic foods may also obscure microwear signals of mechanical food properties. Ring-tailed lemurs at the Beza Mahafaly Special Reserve (BMSR), Madagascar, display frequent severe tooth wear and subsequent tooth loss. In contrast, sympatric Verreaux's sifaka display far less tooth wear and infrequent tooth loss, despite both species regularly consuming acidic tamarind fruit. We investigated the potential impact of dietary acidity on tooth wear, collecting data on salivary pH from both species, as well as salivary pH from ring-tailed lemurs at Tsimanampesotse National Park, Madagascar. We also collected salivary pH data from ring-tailed lemurs at the Indianapolis Zoo, none of which had eaten for at least 12 hr before data collection. Mean salivary pH for the BMSR ring-tailed lemurs (8.098, n=41, SD=0.550) was significantly more alkaline than Verreaux's sifaka (7.481, n=26, SD=0.458). The mean salivary pH of BMSR (8.098) and Tsimanampesotse (8.080, n=25, SD=0.746) ring-tailed lemurs did not differ significantly. Salivary pH for the Indianapolis Zoo sample (8.125, n=16, SD=0.289) did not differ significantly from either the BMSR or Tsimanampesotse ring-tailed lemurs, but was significantly more alkaline than the BMSR Verreaux's sifaka sample. Regardless of the time between feeding and collection of pH data (from several minutes to nearly 1 hr), salivary pH for each wild lemur was above the "critical" pH of 5.5, below which enamel demineralization occurs. Thus, the high pH of lemur saliva suggests a strong buffering capacity, indicating the impact of acidic foods on dental wear is short-lived, likely having a limited effect. However, tannins in tamarind fruit may increase friction between teeth, thereby increasing attrition and wear in lemurs. These data also suggest that salivary pH varies between lemur species, corresponding to broad dietary categories.     

The composition of enamel salivary films is different from the ones formed on dental materials

This study utilized two-dimensional gel electrophoresis (2DE) to illustrate the compositional differences between in vitro salivary conditioning films (denoted pellicles) formed on human enamel as well as on the dental materials titanium and poly(methyl methacrylate). The salivary pellicles were formed by immersing each surface in individual tubes containing small volumes of freshly collected whole saliva. Saliva remaining in the tubes after the pellicle formation for 2 h was visualized by means of 2DE and silver staining. The results showed that the protein patterns in 2DE of the liquid phase of saliva left after the exposure to the respective surfaces, regarding proteins <100 kDa in size, were different depending on the surface used. Several protein groups and/or individual proteins were shown to be distinct for each surface used.     

The scientific exploration of saliva in the post-proteomic era: from database back to basic function

The proteome of human saliva can be considered as being essentially completed. Diagnostic markers for a number of diseases have been identified among salivary proteins and peptides, taking advantage of saliva as an easy-to-obtain biological fluid. Yet, the majority of disease markers identified so far are serum components and not intrinsic proteins produced by the salivary glands. Furthermore, despite the fact that saliva is essential for protecting the oral integuments and dentition, little progress has been made in finding risk predictors in the salivary proteome for dental caries or periodontal disease. Since salivary proteins, and in particular the attached glycans, play an important role in interactions with the microbial world, the salivary glycoproteome and other post-translational modifications of salivary proteins need to be studied. Risk markers for microbial diseases, including dental caries, are likely to be discovered among the highly glycosylated major protein species in saliva. This review will attempt to raise new ideas and also point to under-researched areas that may hold promise for future applicability in oral diagnostics and prediction of oral disease.     

Mechanical characterization of in vitro-formed short-term salivary pellicle

Human saliva consists of approximately 98% water and a variety of electrolytes and proteins. Those proteins can be selectively adsorbed onto the enamel surface. The cuticular material formed on the enamel surface is termed acquired salivary pellicle (ASP), which is critical for the health of oral mucosa and teeth. The ASP is composed of a inner layer and a outer layer. The lubricating properties of ASP are closely associated with the inner layer. The aim of this research is to characterize the structural and mechanical properties of the inner layer of ASP. In this paper, enamel specimens were immersed for 1 min in human saliva. The ASP formed in vitro within 1 min was studied using a nanoindenter. The results show that the thickness of the inner layer of ASP is approximately 18 nm. Moreover, the inner layer is a heterogeneous pellicle with a gradient in density. From the surface of the inner layer to the enamel surface, the density and mechanical properties gradually increase. The research results may be helpful to extend the understanding of mechanical properties of salivary pellicle and to the oral hygiene industry for diagnose oral diseases.     

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed “shedding”, while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel “dahlite” crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.     

Amelogenins: assembly, processing and control of crystal morphology.

The remarkable properties of enamel crystals and their arrangements in an extraordinary micro-architecture are clear indications that the processes of crystal nucleation and growth in the extracellular matrix are highly controlled. The major extracellular events involved in enamel formation are: (a) delineation of space by the secretory ameloblasts and the dentino-enamel junction; (b) self-assembly of amelogenin proteins to form the supramolecular structural framework; (c) transportation of calcium and phosphate ions by the ameloblasts resulting in a supersaturated solution; (d) nucleation of apatite crystallites; and (e) elongated growth of the crystallites. Finally, during the 'maturation' step, rapid growth and thickening of the crystallites take place, which is concomitant with progressive degradation and eventual removal of the enamel extracellular matrix components (mainly amelogenins). This latter stage during which physical hardening of enamel occurs is perhaps unique to dental enamel. We have focused our in vitro studies on three major extracellular events: matrix assembly, matrix processing and control of crystal growth. This paper summarizes current knowledge on the assembly, processing and effect on crystal morphology by amelogenin proteins. The correlation between these three events and putative functional roles for amelogenin protein are discussed.     

On the Adsorption of Human Acidic Proline-rich Proteins (PRP-1 and PRP-3) and Statherin at Solid/liquid Interfaces

The objective of the present study was to investigate the adsorption of PRP-1, PRP-3 and statherin to solid surfaces in terms of dependence on concentration, the presence of electrolyte and surface wettability. Time resolved in situ ellipsometry was used to determine the adsorbed amounts and adsorption rates of pure PRP-1, PRP-3 and statherin onto pure (hydrophilic) and methylated (hydrophobized) silica surfaces. The initial film build-up was fast and plateaus were reached within 10 min at all concentrations for both types of surfaces and all proteins. The observed adsorption and calculated diffusion rates of PRP-1, PRP-3 and statherin, respectively, indicated that the initial adsorption was mass transport controlled at low concentrations. At hydrophobic surfaces, isotherm shapes and adsorbed amounts were similar for PRP-1 and PRP-3, while statherin adsorbed to a higher extent. At hydrophilic surfaces only PRP-1 adsorbed substantially, while for PRP-3 and statherin adsorbed amounts were low. The presence of Ca 2+ ions in the phosphate buffer solution increased the adsorption of statherin and PRP-3 on hydrophobic surfaces, while PRP-1 was unaffected. On hydrophilic surfaces, all three proteins adsorbed in higher amounts in NaCl, compared to CaCl 2 at similar ionic strength. It is concluded that acidic PRPs (PRP-1 and PRP-3) and statherin readily form films on a variety of materials and solution conditions, showing that their functions may be fulfilled under a wide range of conditions.