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Proline-rich proteins are associated with the formation of an acquired protein layer overlying the tooth enamel surface. Previous studies have described the antioxidant activity of salivary histatin against the hydroxyl radical from Fenton's reaction, acting as the critical reactive oxygen species. However, the role of proline-rich proteins in mitigating the oxidative stress caused by reactive oxygen species in the oral cavity remains unclear. In this study, we investigated the antioxidant effects of proline-rich proteins 2 on direct reactive oxygen species using electron spin resonance spectroscopy. For the first time, we demonstrated that proline-rich proteins 2 exhibits antioxidant activity directly against the hydroxyl radical produced by hydrogen peroxide with ultraviolet. Considering that identical results were obtained when assaying 30 residues of proline-rich proteins 2, the direct antioxidant effects against the hydroxyl radical by proline-rich proteins 2 may be related to these specific 30 residues.

There are no studies on Candida colonization and micropeptides of saliva in any patient. Therefore, we studied the effects of the salivary antimicrobial peptide histatin 5 on oral fungal colonization, subjects were subdivided into Down syndrome (D) and normal (N) groups by age: N-1 and D-1, age &lt, 20 years, N-2 and D-2, age &gt, 40 years. Histatin 5 concentration in saliva was measured by enzyme-linked immunosorbent assay. Oral Candida species were identified using CHROMagar Candida. Candida colonization was significantly enhanced in the D-1 and D-2 groups compared to the N-1 and N-2 groups. There was no predominant difference in salivary histatin 5 concentration between the D-1 and N-1 groups, but it was significantly lower in the D-2 group than in the N-2 group. Only in the N-2 group was there a correlation between the concentration of histatin 5 and total protein, while no correlation was found in the other groups. In elderly patients with Down syndrome, the decrease in histatin 5 shown in this study may lead to oral Candida colony formation. Therefore, the results of this study suggest that a deficiency of the antimicrobial peptide histatin 5 could possibly induce oral Candida infection in DS.

Histatin, a salivary protein, affects oral homeostasis through preservation of tooth integrity and protection against caries and fungal infections. However, the effects of histatin in the generation of oxidative stress induced by reactive oxygen species and in the oral cavity remain unclear. In this study, the effects of histatin on direct reactive oxygen species scavenging activity were examined using electron spin resonance. We demonstrated, for the first time, that histatin exhibits antioxidant activity against hydroxyl radicals generated by Fenton's reaction by metal chelation or binding. The direct antioxidant effects of histatin, along with its antimicrobial activity, may be important in the oral protection of salivary proteins.

The acquired enamel pellicle is an oral, fluid-derived protein layer that forms on the tooth surface. It is a biologically and clinically important integument that protects teeth against enamel demineralization, and abrasion. Tooth surfaces are exposed to different proteinaceous microenvironments depending on the enamel location. For instance, tooth surfaces close to the gingival sulcus contact serum proteins that emanate via this sulcus, which may impact pellicle composition locally. The aims of this study were to define the major salivary and serum components that adsorb to hydroxyapatite, to study competition among them, and to obtain preliminary evidence in an in vivo saliva/serum pellicle model. Hydroxyapatite powder was incubated with saliva and serum, and the proteins that adsorbed were identified by mass spectrometry. To study competition, saliva and serum proteins were labeled with CyDyes, mixed in various proportions, and incubated with hydroxyapatite. In vivo competition was assessed using a split-mouth design, with half the buccal tooth surfaces coated with serum and the other half with saliva. After exposure to the oral environment for 0 min, 30 min and 2 h, the pellicles were analyzed by SDS-PAGE. In pure saliva- or serum-derived pellicles, 82 and 84 proteins were identified, respectively. When present concomitantly, salivary protein adsorbers effectively competed with serum protein adsorbers for the hydroxyapatite surface. Specifically, acidic proline-rich protein, cystatin, statherin and protein S100-A9 proteins competed off apolipoproteins, complement C4-A, haptoglobin, transthyretin and serotransferrin. In vivo evidence further supported the replacement of serum proteins by salivary proteins. In conclusion, although significant numbers of serum proteins emanate from the gingival sulcus, their ability to participate in dental pellicle formation is likely reduced in the presence of strong salivary protein adsorbers. The functional properties of the acquired enamel pellicle will therefore be mostly dictated by the salivary component.

Detoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2 mg Sub-A/g chow) (n = 9) compared to 31.9% in mice fed with chow containing unmodified Sub-A (n = 9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.

There is growing interest in the use of human whole saliva for diagnostics and disease monitoring as an alternative to blood samples. In contrast to blood, whole saliva is a non-sterile body fluid. Proper hand-ling and storage are required to preserve the integrity of potential biomarkers. We investigated salivary autoproteolytic degradation using a variety of approaches. We determined inhibition of protease activities by monitoring the endogenous proteome. In addition, the stability of highly protease-susceptible proteins—histatin 5, statherin, and PRP1—was assessed. Experimental variables included (a) protease inhibitors, (b) salivary pH, (c) incubation temperatures, and (d) sample heating. A cocktail containing AEBSF, aprotinin, pancreatic trypsin inhibitor, leupeptin, antipain, and EDTA could not prevent histatin 5, statherin, or PRP1 degradation in whole saliva. Among the other treatments evaluated, short-term storage of freshly collected samples on ice was effective without interfering with the chemistry of the proteome. In conclusion, whole saliva contains a unique mixture of enzymes as evidenced from their resilience to protease inhibition. Analytical evidence on protein stability is needed to ensure the validity of salivary biomarker study outcomes. Analysis of the data presented will provide help and guidance for the use of saliva samples for diagnostic purposes.

Objective The aim of this study was to investigate the type and the nature of peptides present in the in vivo formed human acquired enamel pellicle. Design Pellicle material was collected from 10 volunteers and subjected to sample preparations consisting of centrifugal filtration using a 10 kDa molecular weight cut-off membrane and high-resolution gel filtration chromatography. The fractions containing peptides Results 78 natural pellicle peptides with molecular weights ranging from 766.9 Da to 3981.4 Da were identified originating from 29 different proteins. Conclusions The number of peptides present in acquired enamel pellicle appears to be large and this is likely to enhance the functional spectrum of this protein film. The presence of small peptides in pellicle may be functionally important since structure/function studies of many salivary proteins have shown that specific domains within these native proteins retain or even exhibit enhanced biological activities. The data present the basis for determining the precise function of these pellicle peptides and for gaining insights into the role pellicle plays in the oral cavity.

The present study was undertaken to investigate the rate and mode of degradation of individual histatin proteins in whole saliva to establish the impact on its functional domains. Pure synthetic histatins 1, 3, and 5 were incubated with whole saliva supernatant as the enzyme source, and peptides in the resultant digests were separated by reverse-phase-HPLC and structurally characterized by electrospray ionization-tandem mass spectrometry. The overall Vmax/Km ratios, a measure of proteolytic efficiency, were on the order of histatin-5 > histatin-3 > histatin-1. Mathematical models predict that histatins 1, 3, and 5 levels in whole saliva stabilize at 5.1, 1.9, and 1.2 μM, representing 59, 27, and 11% of glandular histatins 1, 3, and 5 levels, respectively. Monitoring of the appearance and disappearance of histatin fragments yielded the identification of the first targeted enzymatic cleavage sites as K13 and K17 in histatin 1, R22, Y24, and R25 in histatin 3, and Y10, K11, R12, K13, H15, E16, K17, and H18 in histatin 5. The data indicate that metal-binding, antifungal, and wound-healing domains are largely unaffected by the primary cleavage events in whole saliva, suggesting a sustained functional activity of these proteins in the proteolytic environment of the oral cavity.—Sun, X., Salih, E., Oppenheim, F. G., Helmerhorst, E. J. Kinetics of histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties.

The nonsterile environment of the oral cavity facilitates substantial proteolytic processing, not only of resident salivary proteins but also of dietary proteins. To gain insight into whole saliva enzymatic processes, the in vivo generated peptides in this oral fluid were subjected to nano-flow liquid chromatography electrospray ionization tandem mass spectrometry. The 182 peptides identified were predominantly derived from acidic and basic proline-rich proteins, statherin, and histatins. The proteolytic cleavages in the basic proline-rich proteins occurred preferentially after a Gln residue with predominant specificity for the tripeptide Xaa-Pro-Gln, where Xaa in the P3 position was mostly represented by Lys. Using the synthetic substrates Lys-Pro-Gln-pNA and Gly-Gly-Gln-pNA, the overall Km values were determined to be 97 ± 7.7 and 611 ± 28 μm, respectively, confirming glutamine endoprotease activity in whole saliva and the influence of the amino acids in positions P2 and P3 on protease recognition. The pH optimum of Lys-Pro-Gln-pNA hydrolysis was 7.0, and the activity was most effectively inhibited by antipain and 4-(2-aminoethyl) benzenesulfonyl fluoride, was metal ion-dependent, and not inhibited by cysteine protease inhibitors. A systematic evaluation of enzyme activities in various exocrine and nonexocrine contributors to whole saliva revealed that the glutamine endoprotease is derived from dental plaque and likely microbial in origin. The P1 site being occupied by a Gln residue is a nonarchetype with respect to known proteases and indicates the presence of novel glutamine-specific endoprotease(s) in oral fluid.

The proteome of whole saliva, in contrast to that of serum, is highly susceptible to a variety of physiological and biochemical processes. First, salivary protein secretion is under neurologic control, with protein output being dependent on the stimulus. Second, extensive salivary protein modifications occur in the oral environment, where a plethora of host- and bacteria-derived enzymes act on proteins emanating from the glandular ducts. Salivary protein biosynthesis starts with the transcription and translation of salivary protein genes in the glands, followed by post-translational processing involving protein glycosylation, phosphorylation, and proteolysis. This gives rise to salivary proteins occurring in families, consisting of structurally closely related family members. Once glandular secretions enter the non-sterile oral environment, proteins are subjected to additional and continuous protein modifications, leading to extensive proteolytic cleavage, partial deglycosylation, and protein-protein complex formation. All these protein modifications occur in a dynamic environment dictated by the continuous supply of newly synthesized proteins and removal by swallowing. Understanding the proteome of whole saliva in an environment of continuous turnover will be a prerequisite to gain insight into the physiological and pathological processes relevant to oral health, and be crucial for the identification of meaningful biomarkers for oral disease.

The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva. The structural analysis of this integument relies on efficient pellicle harvesting and protein identification procedures. Material from three individual subjects and two pooled samples yielded the identification by LC−ESI−MS/MS of 130 pellicle proteins of which 89 were found in three or more experiments. A high intersubject consistency in pellicle composition was observed. Keywords: acquired enamel pellicle • proteomics • oral • mass spectrometry • LC−ESI−MS/MS • linear ion trap • proteins • saliva

MUC7 is a low molecular weight monomeric mucin secreted by submandibular, sublingual and minor salivary glands. This mucin has been implicated in the non-immune host defense system in the oral cavity since it binds and agglutinates a variety of oral microbes. To investigate interactions between this mucin and other secretory salivary proteins, a submandibular gland prey library was screened with baits encoding the N- and C-terminal regions of MUC7 in the yeast two-hybrid system. The N-terminal region interacted with several secretory salivary proteins, whereas the C-terminal region did not. Interacting proteins included amylase, acidic proline-rich protein 2, basic proline-rich protein 3, lacrimal proline-rich protein 4, statherin and histatin 1. Formation of complexes between these proteins and the N-terminal region of MUC7 was confirmed in Far Western blotting experiments. Interactions between mucin and non-mucin proteins in saliva could protect complex partners from proteolysis, modulate the biological activity of complexed proteins or serve as a delivery system for distribution of secretory salivary proteins throughout the oral cavity.

Naturally occurring salivary antifungal proteins have been of major interest, due to their potential to provide the basis for peptide antimycotics effective in combating fungal infections in the oral cavity or elsewhere. We tested the fungistatic activity of a number of cationic antifungal proteins, with major emphasis on histatin 5, a basic protein secreted by the human parotid and submandibular glands. Histatin 5 inhibited the growth of Candida albicans and that of other medically important Candida species, such as C. kefyr, C. krusei, and C. parapsilosis, with IC50 values in the range of 10-20 microg/ml. Two Cryptococcus neoformans strains were also sensitive (IC50 5.2 and 5.6 microg/ml). On the other hand, three C. glabrata strains (ATCC 90030, 2001 and 64677) were entirely insensitive to histatin 5 (IC50>225 microg/ml). Four genetically very similar species to C. glabrata, Candida castelli (CBS 4332), Saccharomyces cerevisiae (S288C, BY4741 and CBS 1171), Kluyveromyces delphensis (CBS 2170) and Kluyveromyces bacillisporus (CBS 7720) were all sensitive to histatin 5 (IC50 2.6-64.6 microg/ml). C. glabrata was also insensitive to other members of the histatin family; histatin 1, 3 and P-113 (IC50 values in all cases >225 microg/ml). In addition, two entirely different cationic antifungal proteins originating from frog skin, PGLa and magainin 2, also showed a strong reduced activity toward this fungus. Besides the well-described inherent resistance of C. glabrata to azole-derived antifungal agents, our studies indicate that this species is also able to withstand the otherwise detrimental activities of cationic antifungal proteins.

Human acquired enamel pellicle is composed of molecules that selectively adsorb from saliva onto tooth surfaces and provides a protective interface between the tooth enamel and the oral environment. To identify the micro-amounts of components present in pellicle, we immunized mice with in vivo-formed human acquired enamel pellicle and analyzed the serum immune responses. Selective reactivities of the serum (OD > 1.0 above background) against albumin, amylase, carbonic anhydrase II, sIgA, IgG, IgM, lactoferrin, lysozyme, proline-rich proteins, statherin, histatin 1, and mucous glycoprotein 1 were observed. We further confirmed the presence of proline-rich proteins, lactoferrin, lysozyme, and carbonic anhydrase II by probing in vivo pellicle with specific polyclonal anti-sera. The polyclonal antibody approach provided a powerful method for the identification of various pellicle proteins, including some which show mineral homeostasis or antimicrobial activity.

Human acquired enamel pellicle is formed by molecules selectively adsorbed onto tooth surfaces. The present work describes the use of monoclonal antibody (mAb) technology as a novel approach to identify micro amounts of components present in pellicle. MAbs were obtained with reactivities against statherin, histatin, mucous glycoprotein 1(MGI), albumin, amylase and human immunoglobulins (Igs), indicating that these are pellicle components, which was further confirmed by immunoblotting. No mAbs against proline-rich proteins (PRPs), lysozyme, mucous glycoprotein 2 (MG2), carbonic anhydrase, lactoferrin or peroxidase were obtained, suggesting that these components are absent, present in low amounts, or exhibit low antigenicity. Further characterization of the binding epitopes of some of th e obtained anti-MGO, anti-statherin and anti-histatin mAbs were carried out and the biological relevance is discussed. The results open up the possibility that immunization with human pellicle and mAbs production can be employed to identify hitherto unknown constituents of pellicle.

Histatins 1, 3 and 5 are the major members of a histidine-rich protein family present in human salivary secretions. These proteins are distinct from many salivary proteins in their high positive charge density at neutral pH, and their antibacterial and antifungal properties. In this study, the hydroxyapatite adsorption characteristics of histatin 1, containing a single phosphoserine residue, recombinantly expressed histatin 1, native histatin 3, synthetic histatin 5 and an internal 12-residue sequence of histatin 5 were investigated. A Langmuir-type model was used to analyse the adsorption. A comparison of the affinities and binding sites of phosphorylated and recombinant histatin 1 provided an estimate of the positive influence of the single phosphoseryl group on mineral adsorption. Furthermore, an apparent correlation was shown to exist between peptide chain length and the number of binding sites. The influence of histatin 5 adsorption on its anticandidal activity was also investigated by performing Candida albicans killing assays with histatin 5 and histatin 5/hydroxyapatite suspensions. A decrease in killing activity was observed with the increase of hydroxyapatite present. The results suggest that the anticandidal properties of histatin 5 could be impaired by the conformations resulting from mineral adsorption, or that putative cellular receptors necessary for candidacidal activity are inaccessible when histatin 5 is adsorbed on hydroxyapatite.

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.

The present investigation has characterised the influence of gustatory stimulation and duration of stimulation on the secretion pattern of salivary mucins MG1 and MG2 and non-mucin glycoproteins in submandibular/sublingual secretion (SMSL). Resting SMSL was collected for three 2 min intervals and stimulated SMSL was collected for ten 1 min intervals from six healthy subjects. Flow rates and total protein were significantly different under the two conditions. The secretion patterns of these proteins under resting and stimulated conditions was examined on periodic acid-Schiff reagent (PAS)-stained polyacrylamide gels using a Kodak Digital-Science Image Station. Image analyses revealed that the level of MG1 increased and the level of MG2 remained nearly the same after stimulation. Six other major glycoproteins (designated Band 1-6) were identified on the basis of their electrophoretic mobilities and immuno-reactivity on Western blots. After stimulation the intensity of Band 1 (lactoferrin and peroxidase) and Band 2 (amylase) decreased whereas the intensity of Band 3 (carbonic anhydrase), Band 4 (proline-rich glycoprotein) and Bands 5 and 6 (basic glycosylated proline-rich proteins) increased. These patterns probably reflect secretion from preformed vesicles since de novo synthesis would be unexpected within the time frame of these experiments. The variable patterns observed suggest that mucins and non-mucin glycoproteins in SMSL derive from different subsets of secretory vesicles, some of which may originate in mucous and others in serous acini, as well as in ductal cells. Quantification of mucins was performed by image analysis technology using purified MG1 and MG2 standards. Finally, the present investigation has shown that the secretory patterns of mucins and non-mucin glycoproteins from submandibular/sublingual glands are complex and represent an important aspect of salivary gland physiology.

Candida albicans is an opportunistic oral pathogen. The flexibility of this microorganism in response to environmental changes includes the expression of a cyanide-resistant alternative respiratory pathway. In the present study, we characterized both conventional and alternative respiratory pathways and determined their ADP/O ratios, inhibitor sensitivity profiles and the impact of the utilization of either pathway on susceptibility to commonly used antimycotics. Oxygen consumption by isolated mitochondria using NADH or malate/pyruvate as respiratory substrates indicated that C. albicans cells express both cytoplasmic and matrix NADH–ubiquinone oxidoreductase activities. The ADP/O ratio was higher for malate/pyruvate (2.2±0.1), which generate NADH in the matrix, than for externally added NADH (1.4±0.2). In addition, malate/pyruvate respiration was rotenone-sensitive, and an enzyme activity assay further confirmed that C. albicans cells express Complex I activity. Cells grown in the presence of antimycin A expressed the cyanide-insensitive respiratory pathway. Determination of the respiratory control ratio (RCR) and ADP/O ratios of mitochondria from these cells indicated that electron transport from ubiquinone to oxygen via the alternative respiratory pathway was not coupled to ATP production; however, an ADP/O ratio of 0.8 was found for substrates that donate electrons at Complex I. Comparison of antifungal susceptibility of C. albicans cells respiring via the conventional or alternative respiratory pathways showed that respiration via the alternative pathway does not reduce the susceptibility of cells to a series of clinically employed antimycotics (using Fungitest®), or to the naturally occurring human salivary antifungal peptide, histatin 5.

Human salivary mucin MG2 is a 180 kDa glycoprotein secreted by submandibular/sublingual and minor salivary glands. Secreted MG2 contains a domain with the only two cysteines (Cys45 and Cys50) present in the polypeptide backbone; in native and recombinant MG2 this domain is involved in mucin binding to oral microbes. As the reduction and alkylation of MG2 has been shown to abolish binding, the present study was undertaken to determine whether the cysteine residues exist in the dithiol or disulphide form. Electrophoretic analysis under reducing and non-reducing conditions showed that intermolecular disulphide bonds do not occur between MG2 molecules. The same incorporation of radiolabelled iodoacetamide into MG2 was obtained with or without prior reduction. When radiolabelled alkylated MG2 was digested with Endoproteinase Lys-C and the derived peptides were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), radioactivity was found in two fractions. Mass spectral analyses of these fractions showed the presence of peptides Cys-Leu-His-Lys and Arg-Cys-Arg-Pro-Lys, both containing carboxymethylated cysteines. These results show that the cysteines in the structural motif associated with bacterial binding exist in the dithiol form, and suggest the potential use of cysteine-containing peptides as agents to modify interactions of MG2 with microbes and oral surfaces.

During the last decade, saliva has emerged as a potentially ideal diagnostic biofluid for noninvasive testing. In this paper, we present an automated, integrated platform useable by minimally trained personnel in the field for the diagnosis of respiratory diseases using human saliva as a sample specimen. In this platform, a saliva sample is loaded onto a disposable microfluidic chip containing all the necessary reagents and components required for saliva analysis. The chip is then inserted into the automated analyzer, the SDReader, where multiple potential protein biomarkers for respiratory diseases are measured simultaneously using a microsphere-based array via fluorescence sandwich immunoassays. The results are read optically, and the images are analyzed by a custom-designed algorithm. The fully automated assay requires as little as 10 μL of saliva sample, and the results are reported in 70 min. The performance of the platform was characterized by testing protein standard solutions, and the results were comparable to those from the 3.5-h lab bench assay that we have previously reported. The device was also deployed in two clinical environments where 273 human saliva samples collected from different subjects were successfully tested, demonstrating the device’s potential to assist clinicians with the diagnosis of respiratory diseases by providing timely protein biomarker profiling information. This platform, which combines non-invasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics.

Through the analysis of a series of 25 peptides composed of various portions of the histatin 5 sequence, we have identified P-113, a 12-amino-acid fragment of histatin 5, as the smallest fragment that retains anticandidal activity comparable to that of the parent compound. Amidation of the P-113 C terminus increased the anticandidal activity of P-113 approximately twofold. The three histidine residues could be exchanged for three hydrophobic residues, with the fragment retaining anticandidal activity. However, the change of two or more of the five basic (lysine and arginine) residues to uncharged residues resulted in a substantial loss of anticandidal activity. A synthetic d -amino-acid analogue, P-113D, was as active against Candida albicans as the l -amino-acid form. In vitro MIC tests in low-ionic-strength medium showed that P-113 has potent activity against Candida albicans, Candida glabrata, Candida parapsilosis , and Candida tropicalis . These results identify P-113 as a potential antimicrobial agent in the treatment of oral candidiasis.

OBJECTIVE: The objective of this study was to investigate the molecular nature, spectrum of activity and mechanism(s) of action of those human parotid basic proline-rich proteins that exhibit anti-HIV-1 activity. DESIGN: Fractions containing the basic proline-rich proteins were obtained from human parotid saliva of presumed HIV-1 non-infected human subjects and characterized with respect to their purity, apparent molecular size and their ability to inhibit the infectivity of T-tropic and M-tropic strains of HIV-1. SUBJECTS, MATERIALS AND METHODS: Stimulated parotid saliva samples were collected from human subjects who denied having any risk factors for HIV-1 infection and whose parotid salivas inhibited HIV-1 infectivity. Such samples were subjected to affinity, molecular sieve and ion exchange chromatography to isolate individual salivary components. Those fractions demonstrating anti-HIV-1 activity were analyzed by SDS-PAGE in order to assess their purity and determine their apparent molecular weights. HIV-1 inhibitory activity was determined using HIV-1 strains LAI and BaL in a Hela cell-derived multinuclear activation of a galactosidase indicator (MAGI) assay. Amino acid analyses were performed on some fractions. RESULTS: Recombinant gp120-CH-Sepharose chromatography of one subject's parotid saliva revealed specific binding of human parotid basic proline-rich proteins, most prominently one with an apparent molecular weight of 37 kDa. Molecular sieve and cation exchange chromatography yielded a fraction greatly enriched in this protein which amino acid analysis confirmed was proline-rich. A similar fraction from two other subjects also contained basic proline-rich proteins of similar molecular size. These fractions inhibited both T-tropic and M-tropic strains of HIV-1 when assayed in the MAGI system. Since SLPI activity is not observable in the MAGI assay, this inhibition was not due to SLPI. The presence of thrombospondin-1 (TSP-1) in the active fractions was precluded on the basis of SDS-PAGE examination. CONCLUSIONS: Specific basic proline-rich proteins in human parotid saliva possess significant anti-HIV-1 activity independent of that attributable to SLPI or TSP-1. Since the inhibition is detectable with the MAGI assay, its mechanism of action involves virus-host cell interaction prior to the introduction of the tat gene product into the host cell and may be through the binding of the basic proline-rich proteins to the HIV-1 gp120 coat of the virus.

Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side-chains of histatin 5 in aqueous solutions. Three C(epsilon1)-H histidyl and the C(gamma)-H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of C(beta)-H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His-15 present within the -H-E-X-X-H- zinc binding motif, and copper binding occurs within the N-terminal D-S-H-, ATCUN motif.

Proline-rich proteins (PRPs), histatins, and statherin are salivary proteins that exhibit high affinities for hydroxyapatite surfaces. In vitro experiments with parotid submandibular/sublingual or whole saliva have shown these proteins to adsorb selectively to tooth surfaces. This investigation focuses on the histo-morphological identification of PRPs, histatins, and statherin in acquired enamel pellicles. Synthetic hydroxyapatite or bovine enamel were exposed to glandular secretions, and whole saliva and pellicle precursor proteins were identified immunohistologically by electron microscopy. Results obtained by back-scattered scanning electron microscopy showed these proteins to be present in pellicles. Pellicles displayed a distinct structure consisting of a sponge-like meshwork of microglobules. Interconnections between structural elements were identified in submandibular/sublingual and whole saliva pellicles only. Transmission electron microscopy of pellicles formed on bovine enamel surfaces revealed a tendency for preferential localization of precursor proteins within the protein film. Since the data showed the presence of pellicle precursors in pellicles derived both from glandular secretions and from whole saliva, it is likely that PRPs, histatins, and statherin are integral components of acquired enamel pellicles in vivo.

Histatins, a group of histidine-rich proteins in human saliva, exhibit antimicrobial activity and are therefore considered to be important in the prevention of infections in the oral cavity. Although killing of C. albicans by histatins has been extensively studied, little is known about the processes responsible for this antifungal activity. Recent studies show the requirement of metabolic activity and ATP production for histatin 5 killing activity. Therefore, the goal of this study was to investigate the kinetics of histatin 5 interaction at different temperatures with C. albicanswild type cells and with respiratory deficient mutants of C. albicans. Synthetic histatin 5 was labeled with fluorescein-5-isothiocyanate (FITC) and its association with C. albicans cells was followed by epi-fluorescence microscopy and fluorescence confocal microscopy. At 37 °C, histatin 5 accumulates intracellularly, and both killing activity and uptake of unlabeled and FITC-labeled histatin 5 are time- and concentration-dependent. At 4 °C, no killing is observed and FITC-histatin 5 is only associated with the cytoplasmic membrane. Internalization and killing activity only occurs after cells are transferred to 37 °C. In addition, cellular accumulation of histatin 5 is concomitant with a moderate alteration of membrane integrity leading to the release of UV-absorbing cell components into the medium. The uptake of histatin 5, the release of UV-absorbing materials and killing of C. albicans are markedly decreased by the respiratory inhibitor sodium azide. Concomitantly, respiratory deficient mutants of C. albicans are also less susceptible to histatin 5. These results indicated that histatin 5 killing activity could be directly correlated to histatin 5 internalization. Both of these processes are prevented by modulators of cellular metabolic activity.

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

The acquired enamel pellicle is an organic film covering the surfaces of teeth. When this film was first discovered, it was thought to be of embryologic origin. Only in the middle of this century did it become clear that it was acquired after tooth eruption. Initially, the small amounts of material that could be obtained have virtually limited the investigation of pellicle proteins to amino acid analysis. Nevertheless, this technique revealed that the pellicle is mainly proteinaceous and is formed by selective adsorption of salivary proteins on tooth enamel. Later, immunologic techniques allowed for the identification of many salivary and fewer non-salivary proteins as constituents of pellicle. However, to this date, isolation and direct biochemical characterization of in vivo pellicle protein have not been possible, because only a few micrograms can be obtained from a single donor. Therefore, the composition and structure of the acquired enamel pellicle are still essentially unknown. Information on the functions of pellicle has been obtained mainly from in vitro experiments carried out with saliva-coated hydroxyapatite and enamel discs. It was found that pellicle protects enamel by reducing demineralization upon acid challenge. Improved pellicle harvesting procedures and analysis by state-of-the-art proteomics with mass spectroscopy approaches promise to make major inroads into the characterization of enamel pellicle.

Histatins are a group of small cationic peptides in human saliva which are well known for their antibacterial and antifungal activities. In a previous study we demonstrated that histatin 5 kills both blastoconidia and germ tubes ofCandida albicansin a time- and concentration-dependent manner at 37°C, whereas no killing was detected at 4°C. This indicated that killing activity depends on cellular energy. To test histatin 5 killing activity at lower cellular ATP levels at 37°C, respiratory mutants, or so-called petite mutants, ofC. albicanswere prepared. These mutants are deficient in respiration due to mutations in mitochondrial DNA. Mutants were initially identified by their small colony size and were further characterized with respect to colony morphology, growth characteristics, respiratory activity, and cytochrome spectra. The killing activity of histatin 5 at the highest concentration was only 28 to 30% against respiratory mutants, whereas 98% of the wild-type cells were killed. Furthermore, histatin 5 killing activity was also tested on wild-type cells in the presence of the respiratory inhibitor sodium azide or, alternatively, the uncoupler carbonyl cyanidem-chlorophenylhydrazone. In both cases histatin 5 killing activity was significantly reduced. Additionally, supernatants and pellets of cells incubated with histatin 5 in the presence or absence of inhibitors of mitochondrial ATP synthesis were analyzed by sodium dodecyl sulfate gel electrophoresis. It was observed that wild-type cells accumulated large amounts of histatin 5, while wild-type cells treated with inhibitors or petite mutants did not accumulate significant amounts of the peptide. These data showed first that cellular accumulation of histatin 5 is necessary for killing activity and second that accumulation of histatin 5 depends on the availability of cellular energy. Therefore, mitochondrial ATP synthesis is required for effective killing activity of histatin 5.

MG2 (the MUC7 gene product) is a low-molecular-mass mucin found in human submandibular/sublingual secretions. This mucin is believed to agglutinate a variety of microbes and thus is considered an important component of the non-immune host defence system in the oral cavity. We have shown that MUC7 can bind to cariogenic strains of Streptococcus mutans and that this binding requires a structural determinant in the N-terminal region. In the present study an expression construct, pNMuc7, encoding the N-terminal 144 amino acids of MUC7 was generated, and the recombinant protein rNMUC7 was expressed in Escherichia coli. Purified rNMUC7 was characterized and the binding of this protein to oral bacteria was investigated in an established assay. The results showed that the recombinant protein bound to S. mutans ATCC 25175 and ATCC 33402, and that alkylation of the two cysteine residues (Cys45 and Cys50) resulted in the complete loss of bacterial binding. This suggests that binding of MUC7 to S. mutans occurs between the N-terminal region of the mucin molecule and the bacterial surface, and that this interaction is dependent on a cysteine-containing domain within this region of MUC7. In addition, the killing activity of rNMUC7 was compared with that of the candidacidal salivary protein histatin 5 in an established Candida albicans (ATCC 44505) blastoconidia killing assay. It was found that the LD50 values of rNMUC7 and histatin 5 were comparable, and that the recombinant protein displayed significant killing activity at the physiological concentration range of MUC7 in whole saliva. This study is the first to show that the N-terminal region of MUC7 contains a structural determinant for bacterial binding and that this region exhibits candidacidal activity.

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

Human salivary mucin MG2 was purified from submandibular/sublingual gland secretion by ultrafiltration and sequential gel filtration chromatography on Sephadex G-200, Superose 6 (prepgrade), and Superose 6. This method differs from earlier procedures in that all steps are performed in the presence of 4 M guanidine hydrochloride and do not involve covalent modification of the mucin molecule. Electrophoretic analyses and Western blotting showed that purified MG2 did not contain detectable levels of other salivary proteins. Amino acid analysis showed that the composition of purified MG2 was in excellent agreement with the deduced sequence of MG2 apomucin encoded in the MUC7 gene. The yield of purified MG2 was 10-15 mg from 750 ml of salivary secretion. Binding of purified MG2 to Streptococcus mutans in vitro was not significantly affected by reductive methylation, but was nearly abolished by reduction and alkylation. These data identified a functional determinant for mucin-bacterial interactions in the N-terminal region where the only two cysteines (Cys45 and Cys50) in the MG2 apomucin occur. Additionally, purified MG2 bound to four strains of oral Streptococci, indicating that the binding is not dependent on complexing with other salivary proteins, such as secretory immunoglobulin A. The purification procedure described in this work will facilitate investigation of the role of MG2 in the oral environment.

High molecular weight salivary mucin (MG1) is an important component of saliva, contributing to the lubricative and tissue-protective functions of this biological fluid. We have shown previously that the human mucin gene MUC5B is expressed at high levels in sublingual gland and is a significant constituent of MG1. Since many epithelia express multiple mucin genes, it seemed likely that MG1 in salivary secretions is also a heterogeneous mixture of mucin gene products. The aim of this study was to determine whether MUC4, a mucin shown in Northern blotting experiments to be expressed in salivary glands, was a significant protein component of MG1 in salivary secretions. Two cDNA clones containing MUC4 tandem repeats were isolated from a human submandibular gland cDNA library. In addition, recombinant MUC4 produced in a bacterial expression system cross-reacted with an antibody directed against deglycosylated MG1. This shows conclusively that human salivary mucin MG1 contains both MUC5B and MUC4 gene products suggesting that each mucin may perform distinct functions in the oral cavity.

Heterotypic complexes between the high-molecular-weight mucin MG1 and other salivary proteins in human submandibular/sublingual secretion (HSMSL) could have a significant impact on the biological properties of these proteins in oral fluids in both health and disease. We describe a mild procedure for isolation and purification of native MG1 by gel filtration chromatography on Sepharose CL-2B which does not involve dialysis, lyophilization, use of denaturing agents, or covalent modification. Western blots of native MG1 probed with antibodies against 8 different salivary proteins showed that complexing occurs between MG1 and salivary amylase, proline-rich proteins (PRPs), statherins, and histatins but not MG1, slgA, secretory component, or cystatins. When native MG1 was placed in 4 M guanidine hydrochloride and chromatographed on Sepharose CL-4B, ELISA measurement of column fractions showed that amylase, PRPs, statherins, and histatins were released. Interestingly, gel filtration resolved the material which eluted into 4 or 5 distinct peaks, suggesting that the released entities were heterotypic complexes. From these studies, the occurrence of at least three different types of complexes between MG1 and other salivary proteins has been identified. Type I complexes are dissociated by SDS-PAGE and in 4 M guanidine hydrochloride. Type II complexes are not dissociated under these conditions. Type III complexes are dissociated during SDS-PAGE and by 4 M guanidine hydrochloride, but the released proteins appear to be complexes containing amylase, PRPs, statherins, and histatins. The possible functional role of heterotypic complexes between MG1 and other salivary proteins as a physiologic delivery system, a mechanism for protection against proteolysis, a repository for precursors of the acquired enamel pellicle, and a vehicle for modulation of the viscoelastic and rheological properties of saliva is discussed.

Human submandibular/sublingual gland secretions containa multimeric high molecular weight mucin (MG1) and amonomeric low molecular weight mucin (MG2). MG2 isthe product of the MUC7 gene, whereas the gene for MG1has not been identified. Previously, we isolated a clone(pSM2-l) from a human sublingual gland cDNA expres-sion library using an antibody against deglycosylated MG1(Troxler et al, Biochem. Biophys. Res Commun., 217,1112—1119,1995). In order to identify the mucin gene from whichpPM2-l was derived, Northern blots of human subman-dibular and sublingual gland RNA were hybridized with aseries of probes for tandem repeats found in the high mo-lecular weight secreted mucins MUC2, MUC3, MUC4,MUC5AC, MUC5B, and MUC6. The only known mucinexpressed at high levels in sublingual gland was MUC5B,and no known mucin was expressed at high levels in sub-mandibular gland. A series of overlapping clones was ob-tained by rescreening the sublingual gland cDNA libraryand by reverse transcriptase-polymerase chain reaction.The resulting clones connected pSM2-l to a series ofMUC5B tandem repeats at the 3' end of the repeat domainand provided the complete nudeotide and deduced aminosequence of the carboxyl terminal region of MG1. Thisregion is enriched with respect to cysteine (-10 mol %) andcontained a D domain and a carboxyl terminal domain thatcould be aligned with the corresponding domains in humanintestinal MUC2, human tracheobronchial MUC5AC, andhuman von Willebrand factor. The limited expression ofknown mucin genes, together with the considerable mucinsynthesizing capacity of submandibular gland, suggeststhat a novel (previously not described) mucin gene is ex-pressed in this gland and constitutes a portion of MG1 insalivary secretions.Key words: human sublingual gland/mucin/MGl/cDNA se-quence/MUC5BIntroductionSalivary mucins are important contributors to the lubricativeand rheological properties of whole saliva and thus play acritical role in physiological processes such as mastication,swallowing and speech (Cohen and Levine, 1989). Mucins areconstituents of the biofilm covering both hard and soft tissuesin the oral cavity providing protection from mechanical orchemical injury and from microbial assault (Mandel, 1987;Scannapieco and Levine, 1993; Tabak, 1995). Mucins are se-creted predominantly by a pair of submandibular glands, lo-cated medial to and partially under the mandible, and a pair ofsublingual glands, located anterior to the submandibular glandsunder the tongue and beneath the mucous membrane of thefloor of the mouth. The larger submandibular gland is a"mixed" gland containing both mucous and serous aciniwhereas the smaller sublingual gland is referred to as a "mu-cous" gland comprised almost entirely of mucous acini (TenCate, 1994). Both glands originate from budding of buccalepithelium in early development and are functionally classifiedas exocrine glands. Mucins are also produced to a much lesserextent by minor salivary glands (labial, buccal, and palatine)distributed throughout the submucosa in the oral cavity. Twodistinct mucins, named mucous glycoprotein 1 (MG1) and mu-cous glycoprotein 2 (MG2), have been isolated and partiallycharacterized with respect to their biochemical and functionalproperties (Levine et al., 1987; Loomis et al., 1987; Cohen andLevine, 1989; Scannapieco and Levine, 1993). These mucinswere isolated from secretion collected at the orifice of Whar-ton's duct located on the frenulum of the tongue. Since thissecretion is derived from both submandibular and sublingualglands, there is no way a priori to determine whether thesemucins are secreted by submandibular gland, sublingual gland,or both. MG1 is a large multimeric glycoprotein with an ap-parent molecular weight greater than 40 million, comprised ofsubunit monomers of approximately 3 x 10

Histatin 5 (Hst5) is a 24-amino acid (aa) member of the Hst family that is found in human salivary secretions and exhibits candidacidal activity. Hst5 contains a 13-aa region that alone is capable of killing fungal pathogens and is referred to as the functional domain. To investigate the role of specific aa located within the functional domain, the pRSET bacterial expression system was used to produce recombinant Hst5 (re-Hst5) and several re-variants that were generated by site-directed mutagenesis. The vector pRSETC expresses genes of interest as fusion proteins attached to the carboxy end of an N-terminal His6 tag that binds to nickel (Ni2+). The re-variants were generated using the polymerase chain reaction (PCR) and had Gly substituted for either the His, Glu or Lys/Arg within the functional domain. PCR products that encoded either the wild-type or variant forms of re-Hst5 were inserted into pRSETC and produced as fusion proteins which were affinity purified from cell lysates by Ni(2+)-Sepharose chromatography. Fusion proteins were digested with CNBr and re-Hsts were purified by reversed-phase high performance liquid chromatography (RP-HPLC). Re-Hsts were tested in bioassays to measure the ability to kill both Candida albicans (C. albicans) blastoconidia and spheroplasts which were generated by removal of the cell wall. In both assays, re-Hst5 displayed dose-dependent candidacidal activity that was nearly identical to that of native Hst5 purified from human salivary secretions. Re-Hst5 variants with either Glu or Lys/Arg substitutions demonstrated significantly lower candidacidal activity in both assays, while the variant with His mutated showed essentially no activity at physiological concentrations. These results indicate that acidic and basic aa within the functional domain contribute to candidacidal activity and that the His are essential for candidacidal activity. Additionally, since C. albicans spheroplasts were also susceptible to Hsts, the cell wall is not an essential component in the Hst mechanism of candidacidal action.

The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins.

A human sublingual gland cDNA library was screened with a polyclonal antiserum against deglycosylated MG1 and a positive clone, pSM2-1, was isolated which codes for 196 amino acids in the carboxyl-terminal region of this mucin. This region is cysteine-rich and contains a C2-like domain upstream of the extreme carboxyl-terminal domain in which the arrangement of cysteines is nearly identical to that in human von Willebrand factor, human intestinal mucin MUC2, human tracheobronchial mucin MUC5 and porcine and bovine submaxillary gland mucins. Northern analyses with pSM2-1 showed MG1 transcripts are abundant in sublingual gland and barely detectable in submandibular gland. This study provides the first primary sequence data on human salivary mucin MG1 and the significance of the results is discussed with respect to the biosynthesis and differential expression of MG1 in human salivary glands.

Histatin 1 is a histidine-rich phosphoprotein present in human parotid saliva that possesses candidacidal activity and functions in mineralization by adsorbing to hydroxyapatite. The objective of the present study was to develop a system for recombinant production of histatin 1 and to examine the role of phosphorylation in the functional activities of this molecule. Native histatin 1 (containing a phosphoserine at residue 2) was purified from parotid saliva, whereas a bacterial expression system was used to produce a recombinant form of histatin 1 (re-Hstl) that lacked phosphorylated serine. Histatin 1 cDNA was inserted into the vector pGEX-3X, which expresses foreign genes as soluble fusion proteins attached to the carboxyl-terminus of glutathione S-transferase (GST). The GST/re-Hstl fusion protein was isolated from cell lysates by affinity chromatography on glutathione (GSH)-Sepharose and digested with cyanogen bromide to separate re-Hstl from the GST fusion partner. The digest was subjected to reversed-phase high-performance liquid chromatography on a C18 column, and re-Hstl was eluted as a well-defined peak. The yield of re-Hstl was 4 mg/L of bacterial culture. Amino-terminal sequencing and amino acid analysis confirmed the final product as re-Hstl. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that native histatin 1 and re-Hstl had the same apparent molecular weights, while cationic PAGE showed that re-Hstl was more basic. Phosphate analysis indicated 1 mol phosphate/mol of native histatin 1, while re-Hstl lacked any detectable phosphate. Re-Hstl demonstrated candidacidal activity comparable to that of native histatin 1, but displayed substantially lower binding to hydroxyapatite. These results show that phosphorylation of histatin 1 at residue 2 contributes significantly to its ability to bind to hydroxyapatite.

Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events that have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of Candida albicans treated with histatin 5. Cell killing was determined by plating and differential protein expression levels in the mitochondrial samples were determined by quantitative proteomics approaches employing mTRAQ and ICAT labeling and Western blotting. Relative quantitation ratios were established for 144 different proteins. Up-regulated mitochondrial proteins were predominantly involved in genome maintenance and gene expression, whereas proteins that constitute the respiratory enzyme complexes were mostly down-regulated. The differential expression of ATP synthase gamma chain and elongation factor 1-alpha were confirmed by Western blotting by comparison to levels of cytochrome c which were unchanged upon histatin treatment. The mTRAQ and ICAT proteomics results suggest that key steps in the histatin 5 antifungal mechanism involve a bioenergetic collapse of C. albicans, caused essentially by a decrease in mitochondrial ATP synthesis.

Background Celiac disease is a T cell mediated-inflammatory enteropathy caused by the ingestion of gluten in genetically predisposed individuals carrying HLA-DQ2 or HLA-DQ8. The immunogenic gliadin epitopes, containing multiple glutamine and proline residues, are largely resistant to degradation by gastric and intestinal proteases. Salivary microorganisms however exhibit glutamine endoprotease activity, discovered towards glutamine- and proline-rich salivary proteins. The aim was to explore if gliadins can serve as substrates for oral microbial enzymes. Methodology/Principal Findings Proteolytic activity in suspended dental plaque was studied towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates b) a mixture of natural gliadins and c) synthetic highly immunogenic gliadin peptides (33-mer of α2-gliadin and 26-mer of γ-gliadin). In addition, gliadin zymography was conducted to obtain the approximate molecular weights and pH activity profiles of the gliadin-degrading oral enzymes and liquid iso-electric focusing was performed to establish overall enzyme iso-electric points. Plaque bacteria efficiently hydrolyzed Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA, with Z-YPQ-pNA being most rapidly cleaved. Gliadin immunogenic domains were extensively degraded in the presence of oral bacteria. Gliadin zymography revealed that prominent enzymes exhibit molecular weights >70 kD and are active over a broad pH range from 3 to 10. Liquid iso-electric focusing indicated that most gliadin-degrading enzymes are acidic in nature with iso-electric points between 2.5 and 4.0. Conclusions/Significance This is the first reported evidence for gluten-degrading microorganisms associated with the upper gastro-intestinal tract. Such microorganisms may play a hitherto unappreciated role in the digestion of dietary gluten and thus protection from celiac disease in subjects at risk.

Human salivary statherin inhibits both primary and secondary calcium phosphate precipitation and, upon binding to hydroxyapatite, associates with a variety of oral bacteria. These functions, crucial in the maintenance of tooth enamel integrity, are located in defined regions within the statherin molecule. Proteases associated with saliva, however, cleave statherin effectively, and it is of importance to determine how statherin functional domains are affected by these events. Statherin was isolated from human parotid secretion by zinc precipitation and purified by reversed-phase high performance liquid chromatography (RP-HPLC). To characterize the proteolytic process provoked by oral proteases, statherin was incubated with whole saliva and fragmentation was monitored by RP-HPLC. The early formed peptides were structurally characterized by reversed phase liquid chromatography electrospray-ionization tandem mass spectrometry. Statherin was degraded 3.6× faster in whole saliva than in whole saliva supernatant. The main and primary cleavage sites were located in the N-terminal half of statherin, specifically after Arg(9), Arg(10), and Arg(13); after Phe(14) and Tyr(18); and after Gly(12), Gly(15), Gly(17) and Gly(19) while the C-terminal half of statherin remained intact. Whole saliva protease activities separated the charged N-terminus from the hydrophobic C-terminus, negatively impacting on full length statherin functions comprising enamel lubrication and inhibition of primary calcium phosphate precipitation. Cryptic epitopes for bacterial binding residing in the C-terminal domain were likewise affected. The full characterization of the statherin peptides generated facilitates the elucidation of their novel functional roles in the oral and gastro-intestinal environment.

The protein compositions of in vitro pellicles formed from whole saliva and parotid and submandibular secretions were determined by use of synthetic hydroxyapatite as a model for dental enamel. The adsorbed and unadsorbed protein fractions were analyzed by amino acid analysis and both anionic and cationic discontinuous polyacrylamide gel electrophoresis. For further characterization of the in vitro pellicle, the adsorbed fractions were subjected to gel filtration on Sephadex G-100 and reversed-phase chromatography on C18 columns. Amylase, acidic and glycosylated proline-rich proteins, statherins, and histatins were identified in the parotid-derived pellicle. Detailed analysis of the statherin-containing fractions resulted in the observation of several statherin-like proteins. The use of cationic gel electrophoresis allowed for the identification of histatin 3 and histatin 5, which have not been previously detected in pellicle formed in vitro. The protein composition of submandibular-derived pellicle was similar to that of parotid-derived pellicle except for the presence of cystatins and the absence of glycosylated proline-rich proteins. In contrast, in vitro pellicle derived from whole saliva exhibited a vastly different composition, consisting primarily of amylase, acidic proline-rich proteins, cystatins, and proteolytically-derived peptides. The results indicate that acidic phosphoproteins as well as neutral and basic histatins from pure secretions selectively adsorb to hydroxyapatite, whereas in whole saliva some of these proteins are proteolytically degraded, dramatically changing its adsorption pattern.

We have determined transmembrane protein tyrosine phosphorylation (outside-in signaling) in cultured osteoclasts and macrophages in response to added native purified bone sialoprotein (nBSP) and its dephosphorylated form (dBSP). There were selective/differential and potent inhibitory effects by dBSP and minimal effect by nBSP on intracellular tyrosine phosphorylation in macrophages and osteoclasts. Further studies on the downstream gene expression effects led to identification of a large number of differentially expressed genes in response to nBSP relative to dBSP in both macrophages and osteoclasts. These studies were extended to a bone resorption model using live mouse neonatal calvarial bone organ cultures stimulated by parathyroid hormone (PTH) to undergo bone resorption. Inclusion of nBSP in such cultures showed no effect on type I collagen telopeptide fragment release, hence overall bone resorption, whereas addition of dBSP abolished the PTH-induced bone resorption. The inhibition of bone resorption by dBSP was shown to be unique since in complementary experiments use of integrin receptor binding ligand, GRGDS peptide, offered only partial reduction on overall bone resorption. Quantitative RANKL analysis indicated that mechanistically the PTH-induced bone resorption was inhibited by dBSP via down-regulation of the osteoblastic RANKL production. This conclusion was supported by the RANKL analysis in cultured MC3T3-E1 osteoblast cells. Overall, these studies provided direct evidence for the involvement of covalently bound phosphates on BSP in receptor mediated "outside-in" signaling via transmembrane tyrosine phosphorylation with concurrent effects on downstream gene expressions. The use of a live bone organ culture system augmented these results with further evidence that links the observed in vivo variable state of phosphorylation with bone remodeling.

Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50-75 kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer.

Saliva plays a critical role in the protection of oral hard and soft tissues and contains a multitude of constituents with well-characterized biological activities in vitro. Among these are histatins and acidic proline-rich proteins (PRPs). Nevertheless, few functional studies have recognized the structural instability of these proteins in the proteolytic environment of whole saliva. The aim of this investigation was to determine histatin and acidic PRP levels in parotid secretion (PS) and in whole saliva (WS) as well as to establish their susceptibility to proteolysis in these salivary fluids. Using cationic polyacrylamide gel electrophoresis and densitometric analysis the average total histatin concentration (histatin 1+3+5) in WS was determined to be 33.3+/-16.7 microg/ml (n=22) and the average total acidic PRP concentration (PRP1/PIF-s+PRP3/PIF-f) was 427.9+/-123.3 microg/ml (n=22). Histatin and acidic PRP concentrations in PS were 6 and 1.5 times higher than in WS (n=7), respectively. WS histatin and acidic PRP levels each correlated significantly with WS total protein concentrations (P

We have previously shown that histatins 1, 3, and 5 are homologous, histidine-rich proteins present in human parotid and submandibular secretions which contain 38, 32, and 24 amino acids, respectively. Interest in these proteins stems from the fact that histatins exhibit candidacidal and candidastatic activities. The goal of the present investigation was a detailed functional characterization of these anticandidal activities of histatins at the levels of killing of blastoconidia, killing of germinated cells, and inhibition of germination by using three bioassays. Candidacidal activities were evaluated at several ionic strengths, in the presence of different mono- and divalent ions, and at multiple pH values. In addition, the susceptibility of Candida albicans in different growth phases to histatins was investigated. While all three major human histatins demonstrated candidacidal activities, they differed in their abilities to kill blastoconidia and germinated cells, with histatin 5 being the most active, histatin 3 showing moderate activity, and histatin 1 exhibiting the lowest level of activity. For the inhibition of germination, however, histatin 3 exhibited more activity than either histatin 1 or histatin 5. The candidacidal activity of histatins was inversely proportional to both the ionic strength and the divalent cation concentration in the medium. Stepwise reduction of the pH of the assay medium enhanced the candidacidal activities of histatins 1 and 3, while the activity of histatin 5 was pH independent over the range of pHs 4 to 8. C. albicans in log-phase growth was more susceptible to histatins 1 and 3 than cells in stationary phase. Cells in either growth phase were still more vulnerable to histatin 5 than to histatins 1 and 3. The results obtained establish the functional relationship of the major histatins with respect to both their fungicidal and fungistatic activities and provide insights into their activities under ionic and pH conditions likely to be encountered in vivo in the oral cavity. Moreover, the data point towards possible mechanisms responsible for the anticandidal activities of histatins.

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

The acquired enamel pellicle (AEP) is a protein film with unique composition and properties, which is formed by the selective adsorption of a variety of oral fluid-derived proteins onto tooth enamel surfaces. Since events leading to caries and periodontal disease occur in close proximity to the tooth surface, pellicle constituents are likely to contain biomarkers valuable for diagnostic applications. Despite the importance of this oral structure, progress in understanding its formation and composition has been slow because of difficulties in efficient pellicle collection methods and limitations of biochemical techniques for the characterization of microgram amounts of proteins/peptides. Recent developments in both pellicle collection methods and nanoscale sensing technologies have brought the exploitation of pellicle analysis into the realm of point-of-care oral diagnostics.