Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.     

Characterization of the human salivary basic proline-rich protein complex by a proteomic approach

Thirteen samples of human normal whole saliva were analyzed by RP-HPLC-ESI-MS and MALDI-TOF-MS to investigate the basic proline-rich protein complex. Between known basic-PRPs the P-B, P-C (or IB-8b), P-D (or IB-5), P-E (or IB-9), P-F (or IB-8c), P-H (or IB-4), IB-6, II-2, IB-1, and IB-8a glucosylated were identified, whereas the II-1, IB-7, PA, and D1-A peptides were not detected. Some detected masses not attributable to known basic-PRPs were putatively ascribed to II-2 and IB-1 nonphosphorylated, II-2 and IB-1 missing the C-terminal arginine residue, and the 1-62 fragment of IB-6, named P-J peptide. A correlation matrix analysis revealed a cluster of correlation among all the basic PRPs (apart from the P-B peptide) which is in agreement with their common parotid origin.     

Circadian Rhythms of Histatin 1, Histatin 3, Histatin 5, Statherin and Uric Acid in Whole Human Saliva Secretion

The circadian rhythms of histatins 1, 3, 5, of statherin and uric acid were investigated in whole human saliva. Histatins showed a rhythm approximately synchronous with salivary flow rate (acrophase around 5 pm), the higher amplitude pertaining to histatin 1 (about 50% of the mesor). Uric acid showed a large rhythm asynchronous with flow rate and histatin concentrations (4.4 ± 1.4 am). Statherin did not show a significant circadian rhythm on five of six volunteers. This finding confirms that the secretion route of statherin is different from that of histatins.     

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.     

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.     

Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization.

The structural domains of salivary statherin that are partly responsible for the protection and recalcification of tooth enamel were examined with respect to charge, sequence, hydrophobicity, hydrogen bonding potential, and conformation. Several fragments of statherin, 1-15 (SN15), 5-15 (SN11), 15-29 (SM15), 29-43 (SC15), 19-43 (SC25), and analogs of the N-terminal 15-residue sequence, where phosphoserines at positions 2 and 3 have been replaced by Ser (SNS15) and Asp (SNA15), respectively, were synthesized. The abilities of these fragments to adsorb at hydroxyapatite (HAP) surfaces and to inhibit its mineralization in supersaturated solutions were determined and compared with those of the whole statherin molecule, reported previously. The conformational preferences of the fragments both in aqueous and nonaqueous solutions were examined by circular dichroism. The highly charged N-terminal SN15 fragment has the greatest adsorption to HAP as compared with statherin and all other fragments. Its mineralization inhibitory activity is significantly greater than those of other fragments and comparable with that of the whole molecule. The dephosphorylated N-terminal fragment SNS15 shows a decreased tendency to adhere to and inhibit the formation of HAP, as compared with SN15. However, the substitution of Asp residues in place of phosphoserines (SNA15), restores the binding affinity and crystal growth inhibition properties, suggesting that the negative charge density at the N-terminal rather than any specific interaction of the phosphate group is important for HAP surface interactions. The C-terminal SC15 and SC25 fragments elicit a much higher affinity for HAP surface than that of the middle sequence (SM15), indicating that hydrogen bonding potential of the C-terminal sequence also contributes to the interaction of statherin with HAP. CD studies provide evidence that the N-terminal SN15 fragment has a strong tendency to adopt an ordered helical conformation, whereas the shorter N-terminal sequence, middle, and C-terminal fragments are structurally flexible and prefer to adopt scattered turn structures or unordered random conformations in organic and aqueous solutions. Collectively, the data indicate that the negative charge density, sequence (1-15), and helical conformation at the N-terminal region of statherin are important for its surface interaction with HAP.     

cDNA cloning and chromosomal localization (4q11-13) of a gene for statherin, a regulator of calcium in saliva.

On the basis of the known amino acid sequence of statherin, a human salivary protein, mixed synthetic oligonucleotides were synthesized and used to screen a cDNA library constructed from human parotid-gland mRNA. A cDNA clone coding for statherin was isolated from this library and has been completely sequenced. The cDNA represents a full-length (or nearly full-length) copy of an approximately 640-bp statherin mRNA. Statherin appears to be coded by a single-copy gene that maps to chromosome 4q11-4q13 when somatic-cell hybrids are used.     

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 Sjögren’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.     

Expression,purification, phosphorylation and characterization of recombinant human statherin

This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.     

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.     

Cross‐species comparison of mammalian saliva using an LC–MALDI based proteomic approach

Despite the importance of saliva in the regulation of oral cavity homeostasis, few studies have been conducted to quantitatively compare the saliva of different mammal species. Aiming to define a proteome signature of mammals’ saliva, an in‐depth SDS‐PAGE–LC coupled to MS/MS (GeLC–MS/MS) approach was used to characterize the saliva from primates (human), carnivores (dog), glires (rat and rabbit), and ungulates (sheep, cattle, horse). Despite the high variability in the number of distinct proteins identified per species, most protein families were shared by the mammals studied with the exception of cattle and horse. Alpha‐amylase is an example that seems to reflect the natural selection related to digestion efficacy and food recognition. Casein protein family was identified in all species but human, suggesting an alternative to statherin in the protection of hard tissues. Overall, data suggest that different proteins might assure a similar role in the regulation of oral cavity homeostasis, potentially explaining the specific mammals’ salivary proteome signature. Moreover, some protein families were identified for the first time in the saliva of some species, the presence of proline‐rich proteins in rabbit's saliva being a good example.     

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.     

Thermodynamics of statherin adsorption onto hydroxyapatite

Statherin is a salivary protein that inhibits the nucleation and growth of hydroxyapatite crystals in the supersaturated environment of the oral cavity. The thermodynamics of adsorption of statherin onto hydroxyapatite crystals have been characterized here by isothermal titration calorimetry and equilibrium adsorption isotherm analysis. At 25 degrees C, statherin adsorption is characterized by an exothermic enthalpy of approximately 3 kcal/mol that diminishes to zero at approximately 25% surface coverage. The initial heat of statherin adsorption increases with temperature, displaying a positive heat capacity change of 194 +/- 7 cal K(-)(1) mol(-)(1) at 25 degrees C. The heat of adsorption during this initial phase is strongly dependent on the buffer species, and from the differential heats of buffer ionization, it can be calculated that approximately one proton is taken up by the crystal or protein upon adsorption. The free energy of adsorption is dominated at all coverages by a large positive entropy (>or=23 cal K(-)(1) mol(-)(1)), which may be partially due to the loss of organized water that hydrates the protein and the mineral surface prior to adsorption. These results are interpreted using a two-site model for adsorption of statherin onto the hydroxyapatite crystals.     

Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches

Precursor proteins of the acquired enamel pellicle derive from glandular and non-glandular secretions, which are components of whole saliva. The purpose of this investigation was to gain further insights into the characteristics of proteins in whole saliva and in vivo formed pellicle components. To maximize separation and resolution using only micro-amounts of protein, a two-dimensional gel electrophoresis system was employed. Protein samples from parotid secretion, submandibular/sublingual secretion, whole saliva, and pellicle were subjected to isoelectric focusing followed by SDS-PAGE. Selected protein spots were excised, subjected to "in-gel" trypsin digestion, and examined by mass spectrometry (MS). The data generated, including peptide maps and tandem MS spectra, were analyzed using protein data base searches. Components identified in whole saliva include cystatins (SA-III, SA, and SN), statherin, albumin, amylase, and calgranulin A. Components identified in pellicle included histatins, lysozyme, statherin, cytokeratins, and calgranulin B. The results showed that whole saliva and pellicle have more complex protein patterns than those of glandular secretions. There are some similarities and also distinct differences between the patterns of proteins present in whole saliva and pellicle. MS approaches allowed identification of not only well characterized salivary proteins but also novel proteins not previously identified in pellicle.     

Human infant saliva peptidome is modified with age and diet transition

In order to describe developmental changes in human salivary peptidome, whole saliva was obtained from 98 infants followed longitudinally at 3 and 6months of age. Data on teeth eruption and diet at the age of 6months were also recorded. Salivary peptide extracts were characterised by label-free MALDI-MS. Peptides differentially expressed between the two ages, and those significantly affected by teeth eruption or introduction of solid foods were identified by MALDI TOF-TOF and LC ESI MS-MS. Out of 81 peaks retained for statistical analysis, 26 were overexpressed at the age of 6months. Exposure to solid foods had a more pronounced effect on profiles (overexpression of nine peaks) than teeth eruption (overexpression of one peak). Differential peaks corresponded to fragments of acidic and basic PRPs, statherin and histatin. Comparison with existing knowledge on adult saliva peptidome revealed that proteolytic processing of salivary proteins is qualitatively quite comparable in infants and in adults. However, age and diet are modulators of salivary peptidome in human infants.     

Tyrosine sulfation of statherin

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.     

The effect of statherin and its shortened analogues on anaerobic bacteria isolated from the oral cavity

The susceptibility (MIC) of 44 strains of anaerobic bacteria isolated from the oral cavity and 3 standard strains to statherin and its C-terminal fragments with sequences QYQQYTF, YQQYTF, QQYTF, QYTF and YTF was determined by means of plate dilution technique in Brucella agar with 5% content of defibrinated sheep's blood, menadione and hemin. The culture was anaerobic. As shown, at concentrations from 12.5 to 100 microg/ml statherin and its C-terminal fragments inhibited the growth of anaerobic bacteria isolated from the oral cavity. Peptostreptococcus strains were the most susceptible to statherin and YTF (MIC < or = 12.5 mg/ml), whereas the most susceptible to the peptides investigated were Fusobacterium necrogenes and Fusobacterium necrophorum strains: QYQQYTF, YQQYTF, QQYTF, QYTF (MIC < or = 12.5 microg/ml). Prevotella oralis, Bacteroides forsythus and Bacteroides ureolyticus strains exhibited the lowest susceptibility (MIC > 100 microg/ml). When analysing the bacteriostatic activity of statherin it should be pointed out that the concentrations of this peptide used in microbiological investigations are within the range of physiological concentrations determined for whole saliva when at rest and stimulated in healthy donors of 19-25 years of age. Since the anaerobes investigated may be involved in the diseases of periodontum, the results presented seem to have also a practical aspect, i.e. a possibility to apply the C-terminal fragments of statherin as a novel therapeutic agent, affecting favourably the oral cavity.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Treatment of an encephalitogenic peptide from guinea pig myelin basic protein with alpha-protease and thermolysin. Isolation of fragments and determination of cleavage sites.

Two peptic fragments (residues 37-88 and 43-88) of guinea pig myelin basic protein which are capable of inducing experimental allergic encephalomyelitis in Lewis rats were cleaved to shorter fragments with alpha-protease (Crotalus atrox proteinase, EC 3.4.24.1) and thermolysin (EC 3.4.24.4). The fragments were isolated, purified, and identified by amino acid composition and NH2- and COOH-terminal residues. The time courses of the reactions, monitored by thin layer electrophoresis of the digests, showed that alpha-protease cleaves peptide (43-88) initially at the Pro(71)-Gln(72) bond, and that the product peptides are subsequently attacked at the Arg(63) -Thr(64), Ser(74)-Gln(75), Arg(78)-Ser(79), and Ser(76)-Gln(80) bonds. No significant cleavages occurred at the -Leu, -Val, and -Ala bonds. These results are in striking contrast to those obtained previously by others workers with other peptide substrates, where selective cleavage at hydrophobic residues occurred. Thermolysin was found to attack peptide (37-88) at the Phe(42)-Phe(43) bond very rapidly; the product peptides were subsequently attacked at the His(60)-Ala(61), Ser(38)-Ile(39)-Tyr(67)-Gly(68), and Pro(84)-Val(85) bonds. These cleavages are compatible with the known specificity of this enzyme. Several of the fragments prepared with these two enzymes, peptides (43-71), (61-88), (75-88), and (72-84) have been used in other studies to locate the encephalitogenic site in the parent peptic peptide.     

Identification of salivary components that induce transition of hyphae to yeast in Candida albicans

Candida albicans , the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphae and hyphae filaments. The hyphae form is considered the most invasive form of the fungus. The purpose of this study is to investigate the effect of saliva on hyphae growth of C. albicans. Candida albicans hyphae were inoculated in Roswell Park Memorial Institute medium with whole saliva, parotid saliva or buffer mimicking the saliva ion composition, and cultured for 18 h at 37 °C under aerobic conditions with 5% CO₂. Whole saliva and parotid saliva induced transition to yeast growth, whereas the culture with buffer remained in the hyphae form. Parotid saliva was fractionated on a reverse-phase C8 column and each fraction was tested for inducing transition to yeast growth. By immunoblotting, the salivary component in the active fraction was identified as statherin, a phosphoprotein of 43 amino acids that has been implicated in remineralization of the teeth. Synthetically made statherin induced transition of hyphae to yeast. By deletion of five amino acids at the negatively charged N-terminal site (DpSpSEE), yeast-inducing activity and binding to C. albicans were increased. In conclusion, statherin induces transition to yeast of C. albicans hyphae and may thus contribute to the oral defense against candidiasis.