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sublingual salivary adhesion-promoting protein
Archives of Oral Biology 47 (2002) 337–345
Partial characterization of a human submandibular/sublingual salivary adhesion-promoting protein S.O. Akintoye a,b,1 , M. Dasso a,b , D.I. Hay b,c , N. Ganeshkumar b,c , A.I. Spielman a,b,∗ a
Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, 345 E 24th Street, New York, NY 10010-4086, USA b Northeast Minority Oral Health Research Center, New York, NY, USA c Forsyth Dental Center, Boston, MA, USA Accepted 9 January 2002
Abstract Human submandibular/sublingual saliva contains a protein that promotes adhesion of Streptococcus mutans JBP serotype-c to spheroidal hydroxyapatite in vitro. A high molecular-weight (250,000–300,000 Da) adhesion-promoting protein (APP) was purified by Trisacryl 2000 M gel-filtration chromatography and gel electroelution before it was partially characterized. Lectin blotting identified that the terminal carbohydrates include N-acetyl glucosamine-1-4-N-acetylglucosamine, galactose and galactose-1-3-N-acetyl galactosamine. Antibodies to APP demonstrated no difference in the immunoreactive pattern of APP from saliva of caries-active or caries-resistant individuals belonging to four different ethnic groups: Asian, African–American, Hispanic or Caucasian. No immunological similarities to salivary mucins or parotid agglutinins were detected by Western blotting using immuno-cross-reactivity as a criterion. APP appears to be a unique protein found in submandibular/sublingual saliva. Understanding such a protein could help prevent S. mutans attachment to the enamel surface. © 2002 Published by Elsevier Science Ltd. Keywords: Antibodies; Bacterial adhesion; Chromatography; Dental caries; Hydroxyapatite; Immunoglobulins; Lectins; Saliva; Streptococcus mutans
1. Introduction Oral bacteria bind to soft and hard tissues/surfaces. Although specific bacterial adhesion to oral surfaces does not itself cause disease, adhesion appears to be the essential first step (Gibbons, 1984). Adherent organisms in the presence of dietary factors contribute to the growth of dental biofilms, which subsequently lead to the development of dental caries and periodontal diseases. Bacterial colonization can be simplified to two basic mechanisms: (1) direct: bacterial–oral surface interaction; (2) indirect: Abbreviations: SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis ∗ Corresponding author. Tel.: +1-212-998-9850; fax: +1-212-995-4087. E-mail address: [email protected] (A.I. Spielman). 1 Present address: Clinical Research Core/Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research/National Institutes of Health, Bethesda, MD, USA.
bacterial–bacterial interaction, i.e. attachment through other bacteria already on the oral surfaces. Bacterial interaction with oral surfaces is highly specific, as these organisms do not attach to the oral soft and hard tissues directly but to the constituents of the acquired enamel pellicle, some of which act as specific receptor sites. Similarly, the receptor sites on the bacterial surface are specific, typically a cell surface molecule called an adhesin found on the bacterial frimbriae or pilli (Gibbons, 1984, 1989; Gong et al., 2000). Saliva plays a major part in the interaction between bacteria and tooth surfaces; most of the salivary proteins have a function in host defence mechanisms. The interactions of oral microorganisms with salivary proteins such as secretory-IgA, lysozyme, lactoferrin, salivary peroxidase and agglutinin are thought to benefit the host. This advantage has been attributed either to their bactericidal and bacteriostatic actions or to their role in facilitating the clearance of these microorganisms from the oral environment (Rudney et al., 1995, 1999; Gibbons and Spinell, 1970; Hay et al., 1971; Tabak et al., 1982; Vats and Lee, 2000).
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Other bacteria–saliva interactions may be detrimental to the host because they promote adhesion of bacteria to tooth surfaces (Gibbons and Hay, 1988). Though saliva-mediated bacterial attachment may be influenced by the specific site in the mouth (Carlen et al., 1998), we have chosen to study the influence of submandibular/sublingual salivary proteins on Streptococcus mutans attachment to spheroidal hydroxyapatite in vitro. Human submandibular/sublingual saliva contains several adhesion-promoting proteins (APPs) that specifically facilitate adhesion of S. mutans JBP serotype-c to hydroxyapatite in vitro (Kishimoto et al., 1989). These proteins were identified in fractions following gel-filtration chromatography. Adhesion assays of individual fractions showed two regions that promoted adhesion of S. mutans to hydroxyapatite. The first adhesion-promoting activity was associated with the high molecular-weight mucin peak; the second fraction was associated with several salivary proteins, including the acidic proline-rich proteins (Gibbons and Hay, 1989). Subsequent studies demonstrated that the first adhesion-promoting activity in the mucin fraction is in fact associated with a number of minor but highly active proteins termed APPs (Kishimoto et al., 1989, 1990). These proteins have Mw ranging from 250,000 to 350,000 Da and demonstrate individual variability in number and relative mobility on SDSPAGE. Because S. mutans is considered a primary agent in the aetiology of dental caries (van Houte, 1994), and because the interaction of APP with S. mutans is highly specific in an in vitro assay, it is likely that APP is important in vivo in the initial colonization of the enamel surfaces. Our purpose now was to purify and further characterize a human submandibular salivary APP and identify its immunological pattern in caries-prone and caries-free individuals. Understanding the different factors that influence bacterial interaction with the oral surfaces, including the adhesion-promoting regions in APP, might enhance development of methods to modulate such interactions, with the ultimate aim of preventing pathological outcomes such as dental caries and periodontitis.
2. Materials and methods
applying a 3% citric acid solution to the dorsum and lateral borders of the tongue every 30 s. Saliva was collected in a tube placed on ice; to avoid protein degradation the samples were mixed immediately with enzyme inhibitors: phenylmethylsulphonyl fluoride (0.001%; 2 l/ml saliva) and 10 mM EDTA, pH 7.2. The saliva samples were mixed thoroughly by passing them 20 times through two 50 ml syringes connected by a two-way Luer lock valve. 2.2. Salivary sample preparation The saliva samples were dialyzed for 24 h at 4 ◦ C, (12,000–14,000 Mw cut-off) against 10 volume (2 l) of 0.1 M ammonium bicarbonate/0.5% chloroform, pH 7.8, using two changes. The dialysate was concentrated with a Centriprep® -10 concentrator, 10,000 Mw cut-off (Amicon Inc., Beverly, MA) at 3000 × g and 4 ◦ C. The protein content of the concentrate was quantified (Bradford, 1976), with bovine gamma globulin as a standard. Samples were stored at −20 ◦ C until further use. 2.3. Gel-filtration chromatography Using a 450 ml bed volume (100 cm height × 2 cm diameter) Trisacryl GF 2000 M gel-filtration column (IBF Biotechnics Inc., Savage, MD), and an automated liquid-chromatographic system (Econo; Bio-Rad, Hercules, CA), 20 mg of salivary protein concentrate from each participant was purified as described by Kishimoto et al. (1989). In preparatory runs, submandibular/sublingual saliva was collected over 3 days from one individual and 100 mg of salivary protein was fractionated. The column was developed with 0.1 M ammonium bicarbonate/0.5% chloroform at a flow rate of 0.3 ml/min, controlled by the automated Econo system. The eluted proteins were monitored by measuring absorbance at A230 and 8 ml fractions were collected. During these preparatory runs, due to the larger amount of protein and reduced resolution, rechromatography of adhesion-promoting fractions was needed and was performed under conditions similar to the original gel-filtration chromatography. All fractions, including those containing proteins of 250,000–350,000 Da, were tested for in vitro S. mutans adhesion-promoting activity (see further).
2.1. Collection of saliva 2.4. Analytical SDS-PAGE Submandibular/sublingual saliva (200 ml) was collected from four healthy adult volunteers with at least 14 natural teeth (one Asian, one African–American, one Caucasian and one Hispanic). The participants provided informed consent and the collection protocol was reviewed and approved by the human subjects committee of our institution. A custom-made, acrylic, modified Block–Brottman saliva collection device (Block and Brottman, 1962; Nederfors and Dahlöf, 1993) was fabricated on an impression of the floor of the mouth taken with polyvinyl siloxane dental impression material. Stimulated saliva was collected by
Following gel-filtration chromatography, 10 l of each fraction was tested by gel electrophoresis (Laemmli, 1970) using precast 4–15 or 4–20% gradient SDS-PAGE (Jules Inc., New Haven, CT) and stained with Silver Stain Plus (Bio-Rad). 2.5. Adhesion-promoting assay The bacterial adhesion-promoting assay was as described by Kishimoto et al. (1989). Bacterial cells were attached
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to 0.08–0.20 mm spheroidal hydroxyapatite beads (Fluka Chemical Co., USA). To maintain the consistency of the adhesion-promoting assay, 1.4 g protein of submandibular/sublingual saliva or column fractions was always tested. S. mutans JBP serotype-c (a gift from the culture collection of the Forsyth Dental Center, Boston MA) was [3 H]-labeled in culture for 14, 16, 18, 20, 22 and 24 h, and then each time-point assayed in various ways. The bacterial cells harvested at 18 h demonstrated optimum viability and density (data not shown); an 18 h culture time was standard for all our adhesion assays. We also performed preliminary adhesion assays using various solutions (50 mM of KCl, phosphate-buffered saline, Tris, NaCl, NaHCO3 ); NaHCO3 was the solution of choice because it demonstrated minimal non-specific activity. To determine the actual number of attached bacteria and to establish the labeling efficiency, 725 × 105 [3 H]-labeled S. mutans cells (125 l of 5 × 108 cells/ml bacterial suspension), the same as that exposed to spheroidal hydroxyapatite, were always transferred to a separate scintillation vial. The counts/min of this vial represent the baseline used to convert counts/min to actual number of attached bacteria. 2.6. Preparatory SDS-PAGE and electroelution of adhesion-promoting protein APP was purified from two participants. Saliva from the first had a single 300,000 Da protein band on SDS-PAGE; saliva from the second had two bands at 300,000 Da. These bands from both participants were purified by electroelution (Hunkapillar et al., 1983; Spielman and Bennick, 1989), tested for purity (silver-stained, 4–15% gradient SDS-PAGE) and adhesion-promoting activity. 2.7. Preparation of polyclonal antibodies to adhesion-promoting protein Specific polyclonal antibodies were raised in guinea pigs (twice) to the APP obtained from the two different participants, using an established protocol (Spielman and Bennick, 1989; Spielman et al., 1991, 1995). The electroeluted proteins were mixed with complete Freund adjuvant at 1:1 ratio to a total of 1 ml solution and injected intradermally into the back of the neck of guinea pigs (six females, each weighing 400 g). The same amount of antigen was injected on days 28, 35, 42, 49, and 56; each injection contained a total of 0.4–0.5 g of pure APP. The guinea pigs were bled on day 56 and the serum tested for immunoreactivity by Western blotting as described in the following sections. On day 63 a final booster injection of 1 g APP was given with incomplete Freund adjuvant. On day 70, the guinea pigs were bled by cardiac puncture under halothane or ketamine/acepromazine anaesthesia. After overnight storage at 4 ◦ C, the antiserum was separated from the coagulum and cellular components by a 15 min centrifugation at 1000 × g.
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2.8. Purification and fragmentation of polyclonal antibodies Antiserum was further purified by removing the albumin and lipid components with the EZ-SEP® method (Pharmacia Biotech Inc., Piscataway, NJ), leaving only the immunoglobulin fraction. The IgG fraction was further separated from the rest of the immunoglobulins on an Acti-DiscTM protein G affinity column (FMC Corporations Bioproducts, Pine Brook, NJ). The F(ab )2 fragments needed to study inhibition of adhesion were generated by pepsin digestion, and were separated from the Fc fragments with the ImmunoPure® F(ab )2 preparation kit, following the manufacturer’s instructions (Pierce, Rockford, IL). The purity and functional integrity of the F(ab )2 fragments were tested by SDS-PAGE and Western blotting. Controls were prepared from custom-made F(ab )2 fragments raised to unrelated or purchased, control pure F(ab )2 fragments of normal guinea pig IgG (Jackson Immuno Research Laboratories Inc., West Grove, PA). 2.9. Testing of polyclonal antiserum and F(ab )2 fragment by Western blotting The antisera were tested by Western blotting at dilutions ranging from 1:50 to 1:1000 (Towbin et al., 1979), or 1:100 for F(ab )2 fragments, and visualized using the avidin–biotin procedure (Vector, Burlingame, CA). Non-specific binding to the nitrocellulose paper was blocked by incubating with milk buffer (10% milk buffer (w/v), 100 mM Tris, 0.9% NaCl and 0.1% Tween 20). Various control experiments included omission of anti-APP antiserum; substitution of anti-APP antiserum with either normal guinea pig serum, normal guinea pig F(ab )2 fragment or antiserum against an unrelated protein, the human apocrine secretion odorant-binding protein-1 (ASOB1) (Spielman et al., 1995). All samples and antisera were prepared in the same way. 2.10. Testing of adhesion-promoting protein with antibodies to mucins and agglutinin Western blotting experiments were done to determine any relation between APP and mucins, or APP and parotid agglutinins. APP was tested for immunoreactivity with polyclonal antibodies to MG1 (high molecular-weight mucin) and MG2 (low molecular-weight mucin) (anti-MG1 and anti-MG2 were provided courtesy of Dr. M. Levine, SUNNY, Buffalo) and with monoclonal antibodies to parotid agglutinins 143D4 B1 and 303D5 A1 (provided courtesy of Dr. D. Malamud, University of Pennsylvania, Philadelphia). Polyclonal antiserum to APP was tested at a dilution of 1:100; other antisera were tested at a dilution of 1:50.
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2.11. Lectin blotting of adhesion-promoting protein and inhibition of lectin reactivity Partial glycosylation patterns of samples from three of four volunteers were tested by lectin blotting: APP-enriched, gel-filtration chromatographic fractions from two participants and fractions from the analytical chromatography run of our third volunteer. The following seven lectins were used to identify the glycosylation pattern of the APP: Bandeiraea (Griffonia) simplicifolia II (BSL II), Datura stramonium (DSL), Erythrina cristagalli (ECL), Lycopersicon esculentum (LEL), Artocarpus integrifolia (Jacalin), Solanum tuberosum (potato) (STL), and Vicia villosa agglutinin (VVA), (Biotinylated Lectin Kit III; Vector). Lectin-blotting experiments followed the Western blotting procedure described above, except that 0.5% gelatin buffer (in 100 mM Tris–buffered saline/Tween 20, pH 7.2) was used instead of milk buffer to block non-specific binding. APP-enriched fractions (after gel-filtration chromatography) were loaded on a 4–15% gradient SDS-PAGE gel. The different lectins were incubated at 1:50 dilution; an E-Z SEP® purified guinea pig antiserum used as a control was incubated at 1:100 dilution, and all were visualized using the avidin–biotin procedure as described above. After identifying Jacalin and LEL as immunoreactive lectins, inhibitory Western blotting experiments were done to block lectin reactivity. Jacalin and LEL were preincubated for 30 min at 4 ◦ C with their competing sugars: thus, Jacalin was preincubated with a final concentration of 800 mM ␣-d-galactose, while LEL was preincubated with chitin hydrolysate (Vector) at a 1:50 final concentration. At a dilution of 1:50, lectins conjugated to their respective blockers were exposed to APP. Changes in the binding activity of the lectin–blocker complex were visualized by the avidin–biotin procedure. 2.12. Individual variability of adhesion-promoting protein immunoreactivity To identify individual variability and to attempt to correlate the presence of APP with caries incidence, we screened and Western blotted submandibular/sublingual saliva from 40 age-, sex- and caries history-matched individuals. These saliva samples were selected from those collected during another study conducted at the Research Center for Minority Oral Health, New York University (Cruz et al., 2001). The participants signed informed-consent documents approved by the University’s institutional review board. Using a cotton-tip applicator, 3% citric acid was applied briefly to the dorsum of the tongue. Immediately, 200–300 l of saliva was collected with a plastic Pasteur pipette from the pool around the orifices of submandibular salivary glands. The samples were transferred to microfuge tubes containing 2 l of 0.001% phenylmethylsulphonyl fluoride. All samples were kept on ice until stored at −80 ◦ C. Saliva samples from four broad ethnic groups were tested: Asian, African–American, Caucasian and Hispanic.
Each group contained 10 individuals, subdivided into five caries-active (history and presence of active caries lesions) and five caries-resistant (no presence or history of caries lesions). Thus, 40 submandibular/sublingual saliva samples from 20 caries-active and 20 caries-resistant individuals were tested for variability in APP immunoreactivity. Equal amounts of salivary proteins (50 g) were electrophoresed on a 5–20% SDS gradient gel, Western blotted and tested with antiserum to APP. In summary, we collected saliva samples from four individuals (nos. 1–4). From each, 20 mg submandibular/ sublingual salivary protein was loaded for analytical gel-filtration chromatography. In addition, from no.1, a 100 mg sample was loaded on a column for preparatory gel-filtration chromatography. Enriched APP from two individuals (nos. 1 and 2; 20 mg runs) were further purified by electroelution and injected into guinea pigs to raise polyclonal antibodies. Gel-chromatographic fractions from three individuals (nos. 1–3) were tested by lectin blotting. Adhesion assay results reported are those of samples from no. 1. Separately, submandibular/sublingual salivas of 40 individuals were screened for APP immunoreactivity.
3. Results 3.1. Salivary protein purification There was individual variability in volume (15–150 ml) and protein concentration (1.6–2.3 mg/ml (mean 1.8 mg/ml)) of saliva collected from the four participants. Loading 100 mg of submandibular/sublingual salivary proteins on to the column during the preparatory run resulted in poor protein separation (Fig. 1; first chromatographic run). However, using the adhesion assay we identified APP in fractions 12–17 and by SDS-PAGE identified fractions containing proteins of 250,000–350,000 Da (data not shown); these fractions were pooled and rechromatographed to obtain better resolution, as shown in the elution profile in Fig. 1 (second run). 3.2. Adhesion-promoting assay and activity The results of a typical adhesion assay are shown in Fig. 1, in which the gel-chromatographic elution profile (second run) is overlaid with a bar graph identifying fractions with adhesion-promoting activity. Fractions 11–16 demonstrated marked bacterial adhesion; other fractions were not significantly different from blanks included in the experiments (results not shown). 3.3. Immunological assays Fig. 2 demonstrates the purity of APP when equal protein amounts of unfractionated saliva and APP were loaded on a 4–20% gradient gel. After raising polyclonal antibodies in
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Fig. 1. Elution profiles of salivary proteins following gel-filtration chromatography and bacterial adhesion assay. Two elution profiles are presented. The first chromatographic elution profile shows gel-filtration chromatography of 100 mg protein of submandibular/sublingual saliva. Fractions 12–17 from this run demonstrated highest adhesion activity (results not shown); the same fractions 12–17 were pooled and reloaded on the same column to generate the second elution profile. The second chromatographic fractions were tested for adhesion-promoting activity. Superimposed bar graph represents adhesion assay; and shows that fractions 11–16 had the highest adhesion-promoting activity (expressed as mean number of adherent S. mutans cells ± S.E.M., n = 12). These same fractions had the highest immunoreactivity (see Fig. 3).
guinea pigs with the purified APP, we used Western blotting at a 1:100 antibody dilution to test salivary fractions obtained by gel-filtration chromatography. Antiserum (results not shown) and E-Z SEP® purified Ig fraction of the antiserum
to APP (Fig. 3) demonstrated a single immunoreactive band to APP-containing fractions. Note the immunoreactivity of fractions 12–16 (Fig. 3), and the similarity to the same fractions that have the highest adhesion-promoting activity
Table 1 Results of lectin blotting of salivary proteins. APP-enriched or unfractionated SM/SL saliva from three of four subjects were tested for reactivity with antiserum O APP (1:100 dilution) and seven different lectins (1:50 dilution). The three subjects showed reactivity to both JAC and LEL in the same molecular weight range as APP. Co-migration of immunoreactive and lectin-reactive proteins was considered as an indication of similarity. All three subjects reacted to APP and showed reactivity to JAC and LEL in the same molecular weight range as APP. Subject 3 showed mild reactivity to both JAC and LEL. The other five lectins were negative in all subjects. Negative reactivity was obtained when JAC and LEL blotting experiments were repeated in the presence of their respective competitive inhibitor. (+ = reactive; ++ = Moderately reactive; +++ = strongly reactive; − = non-reactive; NT = not tested) Lectin
Degree of lectin reactivity in three subjects Subject 1
Subject 2
Subject 3
Polyclonal antibodies to APP Lycopersicon esculentum (LEL) Artocarpus integrifolia (Jacalin) Bandeiraea (Griffonia) simplicifolia II (BSL II) Datura stramonium (DSL) Erythrina cristagalli (ECL) Solanum tuberosum (potato) (STL) Vicia villosa agglutinin (VVA)
+++ ++ + − − − − −
+++ + ++ − − − −
+++ + + − − − − −
Lectin and competitor Lycopersicon esculentum (LEL) and chitin hydrolysate Artocarpus integrifolia (Jacalin) and ␣-d-galactose
− −
NT NT
NT NT
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in Fig. 1. Similar Western blotting with monoclonal antibodies to mucins (MG1 and MG2) or agglutinin, even at dilutions of 1:50, produced negative results. 3.4. Lectin blotting Because APP coelutes with mucin when submandibular/ sublingual saliva is separated by gel-filtration chromatography, and because both mucin and APP are glycosylated, seven lectins were tested by lectin blotting to identify a potential purification strategy. While such a strategy was eventually not used in purification, it nevertheless identified specific terminal glycosylation for APP. The glycosylation pattern of APP was tested by lectin blotting using adhesion-promoting, protein-enriched, gel-filtration chromatographic fractions (similar to those in Figs. 1 and 2) from the two volunteers and a third individual whose submandibular/sublingual salivary APP had been partially purified (Table 1). Two lectins, Lycopersicon esculentum (LEL) and Artocarpus integrifolia (Jacalin), demonstrated reactivity in the three individuals tested. The degree of reactivity varied slightly among the three. The antiserum to APP used in control experiments showed reactivity in all three participants, but the degree of reactivity for LEL was highest in no. 1 and Jacalin reactivity was highest in no. 2, while no. 3 displayed minimal reactivity to both LEL and Jacalin. Five other lectins tested (listed in Table 1) did not show reactivity. Inhibitory lectin-blotting experiments showed successful blockage of Jacalin immunoreactivity by ␣-d-galactose, and LEL reactivity by chitin hydrolysate (Table 1). 3.5. Individual adhesion-promoting protein variability Fig. 2. Testing the purity of one of the electroeluted proteins (APP) by SDS-PAGE. The gel profile shows APP before electroelution (as part of unfractionated saliva, left lane) and after (right lane). The original submandibular/sublingual saliva (50 l sample) and the purified APP were loaded in separate lanes. The arrows show the migration level of APP. Note that the relative abundance of APP in original submandibular/sublingual saliva is very low (left arrow) staining much more faintly than in the enriched and overloaded APP sample (right arrow).
There was no correlation between caries experience and presence or absence of APP. Similarly, no correlation was found between ethnicity and presence or absence of APP immunoreactivity. As we anticipated, some (approximately half) of those tested did not demonstrate immunoreactivity (Fig. 4), probably due to the nature of the antibodies raised (part of its antigenic determinants are probably carbohydrates that vary among these individuals). The antibody we
Fig. 3. Western blotting of elution fractions from the gel-filtration chromatography. Note the heavy reactivity to E-Z SEP® purified immunoglobulin fraction of the antiserum to APP of fractions 12–16, corresponding to the high adhesion-promoting fractions in Fig. 1. Other fractions did not demonstrate similar reactivity.
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Fig. 4. Western blots showing the immunoreactivity of unfractionated submandibular/sublingual saliva from 40 individuals tested with polyclonal antibody to APP purified from an Asian individual. The participants belong to four different ethnic groups: Asian (A), African–American (B), Caucasian (C) and Hispanic (H); 50 g of submandibular/sublingual salivary proteins was tested in each case. These results show each group consisting of five individuals with no history of caries (no caries) and five caries-active individuals (caries). Note that saliva from individuals in the four groups demonstrated some degree of immunoreactivity with anti-APP (arrows). Minimal immunoreactivity was noticed in the Caucasian participants with no caries history.
used was raised against an APP purified from an Asian individual.
4. Discussion Our purpose was to purify and partially characterize an APP from human submandibular/sublingual saliva; our ultimate aim is to clone the genes for APP. Purification of APP proved to be more difficult than initially anticipated, because it is a very large molecule (250,000–300,000 Da), glycosylated and closely associated with, but apparently unrelated to, mucins. Collection of pure, uncontaminated submandibular saliva is easier if the gland is cannulated, but this is a more invasive procedure and not practical when collecting 300–500 ml of saliva. Therefore, we collected mixed submandibular/sublingual saliva, using a custom-made device, which prevented loss of sample and contamination from parotid saliva, gingival crevicular fluid or oral debris. We suspect that APP is present in the submandibular saliva but did not try to confirm this here. To compare different chromatographic runs for their relative adhesion-promoting activity, equal protein amounts (20 mg) were loaded and separated by gel-filtration chromatography. As previously reported, the APPs coelute with mucin (Kishimoto et al., 1989); this was confirmed when the adhesion-promoting assay was performed on each of the eluted fractions. To maintain consistency and a common base for comparison, 1.4 g of protein was tested from each fraction. Initial attempts to separate mucin from APP were unsuccessful, so we considered using lectin chromatography. Two of the seven different lectins tested with APP
samples from three participants showed positive reactivity. These were: Lycopersicon esculentum (LEL), specific for N-acetyl glucosamine-1-4-N-acetylglucosamine (GlcNAc (1,4)GlcNAc) oligomers; and Artocarpus integrifolia (Jacalin), specific for galactose and galactose-1-3-N-acetyl galactosamine (Gal (1,3)GalNac). They were noted as carbohydrates that could be targeted in the design of lectin chromatography experiments. However, after several unsuccessful attempts, probably due to individual variability and weak interaction between lectins and terminal carbohydrates, it proved to be more efficient to use electroelution of APP as a way to obtain purified protein, even though this yielded very small amounts. The concentration of APP in human submandibular/sublingual saliva is estimated to be below 1.5% of total protein; this is further illustrated in Fig. 2, showing SDS-PAGE that demonstrates the low abundance of APP in unfractionated submandibular/sublingual saliva. For analytical purifications, we used 20 mg of salivary protein as starting material. For preparative gel-filtration chromatography, the sample was scaled up to 100 mg salivary protein (Figs. 1 and 3), but poor resolution and low yield following electroelution still produced only a limited amount of pure protein. We purified approximately 25–50 pmol of APP, which was sufficient for antibody production and adhesion assays; this amount translates into 7.5 g of protein if the molecular-weight is assumed to be approximately 300,000 Da. Attempts were made to block the adhesion-promoting activity of the protein in unfractionated submandibular/sublingual saliva using anti-APP–F(ab )2 fragments. Though there was a partial decrease in the number of bacteria adhering to hydroxyapatite, it was not statistically
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significant (data not shown). This finding could be due to the use of unfractionated saliva instead of pure APP and the weak reactivity of our antibodies to adhesion-promoting epitopes of the protein. We could not establish an immunological relationship between APP, mucins and agglutinin. There was no cross-reactivity between APP and monoclonal antibodies raised to MG1 and MG2. Similarly, using monoclonal antibody 143D4 B1 , which recognizes the protein backbone of parotid agglutinin, and monoclonal antibody 303D5 A1 , which recognizes its carbohydrate portion, we could not establish any immuno-cross-reactivity between APP and agglutinin (data not shown). Based on lack of immunoreactivity alone, we cannot exclude the possibility that APP, agglutinin and mucins are related. These experiments need to be further corroborated by the sequencing of agglutinin and APP. Based on lectin blotting, the terminal carbohydrates of APP in three individuals appear to be: GlcNAc (1,4)GlcNac, galactose and Gal (1,3)GalNac, or a combination of these. The presence or absence of galactosyl residues on APP terminal oligosaccharides might influence bacterial attachment and caries susceptibility in different individuals (Seemann et al., 2001). We have also confirmed the individual variability of APP by testing its immunoreactivity with submandibular/sublingual saliva from 40 different individuals. About half of them, in all ethnic groups, demonstrated immunoreactivity to the protein antibody. Some displayed no immunoreactivity, possibly due to individual variability in the protein core or the glycosylation pattern. No correlation was found between ethnicity, immunoreactive pattern of salivary APP and caries experience, probably due to the multiple aetiological factors involved in caries incidence. In conclusion, we have purified an APP from submandibular/sublingual saliva, raised specific antibodies against it and characterized its terminal carbohydrates. This protein is apparently distinct from mucins and parotid agglutinins. No correlation could be found between ethnic group, caries incidence and APP. Further characterization of this protein is needed, including amino acid sequencing, cloning and the identification of the S. mutans-binding region to understand the specific role it may play in bacterial recognition. This site could be targeted for inhibitory activity to reduce the binding of S. mutans to enamel surfaces.
Acknowledgements This project was supported by Grant no. DE10593 (Research Center for Minority Oral Health, New York University College of Dentistry, Forsyth Dental Center) from NIH/NIDR. We would like to thank Drs. P. Bivona, P. Khurana, T. Shritavaj, J. Lee and R.Z. LeGeros for their help in the early stages of this project and for monoclonal antibodies to agglutinin (D. Malamud), and two different
antibodies to mucin (R. Troxler and M. Levine, respectively).
References Block, P.L., Brottman, B.S., 1962. A method of submaxillary saliva collection. NY State Dent. J. 28, 116–118. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Carlen, A., Borjesson, A.C., Nikdel, K., Olsson, J., 1998. Composition of pellicles formed in vivo on tooth surfaces in different parts of the dentition, and in vitro on hydroxyapatite. Caries Res. 32 (6), 447–455. Cruz, G.D., Galvis, D.L., Kim, M., LeGeros, R.Z., Barrow, S.Y., Tavares, M., Bachiman, R., 2001. Self-perceived oral health among three subgroups of Asian–Americans in New York city: a preliminary study. Community Dent. Oral Epidemiol. 29 (2), 99–106. Gibbons, R.J., 1984. Adherent interactions which may affect microbial ecology in the mouth. J. Dent. Res. 63, 378–385. Gibbons, R.J., 1989. Bacterial adhesion to oral tissues: a model for infectious diseases. J. Dent. Res. 68, 750–760. Gibbons, R.J., Hay, D.I., 1988. Human salivary acidic prolinerich protein and statherin promote the attachment of A. viscosus LY7 to apatitic surfaces. Infect. Immun. 56, 439–445. Gibbons, R.J., Hay, D.I., 1989. Adsorbed salivary acidic prolinerich proteins Contribute to the adhesion of S. mutans to apatitic surfaces. J. Dent. Res. 68, 1303–1312. Gong, K., Mailloux, L., Herzberg, M.C., 2000. Salivary film expresses a complex, macromolecular binding site for Streptococcus sanguis. J. Biol. Chem. 275, 8970–8974. Hay, D.I., Gibbons, R.J., Spinell, D.M., 1971. Characteristics of some high molecular-weight constituents with bacterial aggregating activity from whole saliva and dental plaque. Caries Res. 5, 111–123. Hunkapillar, M.W., Lujan, E., Ostrander, F., Hood, L.E., 1983. Isolation of microgram quantities of protein from polyacrylamide gels from amino acid sequence analysis. Meth. Enzymol. 91, 227–236. Kishimoto, E., Hay, D.I., Gibbons, R.J., 1989. A human salivary protein which Promotes adhesion of S. mutans serotype-c strains to hydroxyapatite. Infect. Immun. 57, 3702–3707. Kishimoto, E., Hay, D.I., Kent, R., 1990. Polymorphism of submandibular/sublingual salivary proteins which promote the adhesion of S. mutans serotype-c strains to hydroxyapatite. J. Dent. Res. 69, 1741–1745. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680–685. Nederfors, T., Dahlöf, C., 1993. A modified device for collection and flow-rate measurement of submandibular/sublingual saliva. Scand. J. Dent. Res. 101, 210–214. Rudney, J.D., Ji, Z., Larson, C.J., Liljemark, W.F., Hickey, K.L., 1995. Saliva protein binding to layers of oral streptococci in vitro and in vivo. J. Dent. Res. 74, 1280–1288. Rudney, J.D., Hickey, K.L., Ji, Z., 1999. Cumulative correlations of lysozyme, lactoferrin, peroxidase, S-IgA, amylase, and total protein concentrations with adherence of oral viridans streptococci to microplates coated with human saliva. J. Dent. Res. 78, 759–768.
S.O. Akintoye et al. / Archives of Oral Biology 47 (2002) 337–345 Seemann, R., Zimmer, S., Bizhang, M., Kage, A., 2001. Differences in the salivary glycan pattern between children with high and low caries susceptibility. Caries Res. 35, 156–161. Spielman, A.I., Bennick, A., 1989. Isolation and characterization of six proteins from rabbit parotid saliva belonging to a unique family of protein-rich proteins. Arch. Oral Biol. 34, 117–130. Spielman, A.I., Bernstein, A., Hay, D.I., Blum, M., Bennick, A., 1991. Purification and characterization of a rabbit salivary protein, a potent inhibitor of crystal growth of calcium phosphate salts. Arch. Oral Biol. 36, 55–63. Spielman, A.I., Zeng, X.N., Leyden, J., Preti, G., 1995. Proteinaceous precursors of human axillary odor: isolation of two novel odor-binding proteins. Experientia 51, 42–48.
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Tabak, L.A., Levine, M.J., Mandel, I.D., Ellison, S.A., 1982. Role of salivary mucins in the protection of the oral cavity. J. Oral Pathol. 11, 1–17. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4354. van Houte, J., 1994. Role of micro-organisms in caries etiology. J. Dent. Res. 73, 672–681. Vats, N., Lee, S.F., 2000. Active detachment of Streptococcus mutans cells adhered to epon-hydroxylapatite surfaces coated with salivary proteins in vitro. Arch. Oral Biol. 45, 305– 314.
Partial characterization of a human submandibular/sublingual salivary adhesion-promoting protein S.O. Akintoye a,b,1 , M. Dasso a,b , D.I. Hay b,c , N. Ganeshkumar b,c , A.I. Spielman a,b,∗ a
Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, 345 E 24th Street, New York, NY 10010-4086, USA b Northeast Minority Oral Health Research Center, New York, NY, USA c Forsyth Dental Center, Boston, MA, USA Accepted 9 January 2002
Abstract Human submandibular/sublingual saliva contains a protein that promotes adhesion of Streptococcus mutans JBP serotype-c to spheroidal hydroxyapatite in vitro. A high molecular-weight (250,000–300,000 Da) adhesion-promoting protein (APP) was purified by Trisacryl 2000 M gel-filtration chromatography and gel electroelution before it was partially characterized. Lectin blotting identified that the terminal carbohydrates include N-acetyl glucosamine-1-4-N-acetylglucosamine, galactose and galactose-1-3-N-acetyl galactosamine. Antibodies to APP demonstrated no difference in the immunoreactive pattern of APP from saliva of caries-active or caries-resistant individuals belonging to four different ethnic groups: Asian, African–American, Hispanic or Caucasian. No immunological similarities to salivary mucins or parotid agglutinins were detected by Western blotting using immuno-cross-reactivity as a criterion. APP appears to be a unique protein found in submandibular/sublingual saliva. Understanding such a protein could help prevent S. mutans attachment to the enamel surface. © 2002 Published by Elsevier Science Ltd. Keywords: Antibodies; Bacterial adhesion; Chromatography; Dental caries; Hydroxyapatite; Immunoglobulins; Lectins; Saliva; Streptococcus mutans
1. Introduction Oral bacteria bind to soft and hard tissues/surfaces. Although specific bacterial adhesion to oral surfaces does not itself cause disease, adhesion appears to be the essential first step (Gibbons, 1984). Adherent organisms in the presence of dietary factors contribute to the growth of dental biofilms, which subsequently lead to the development of dental caries and periodontal diseases. Bacterial colonization can be simplified to two basic mechanisms: (1) direct: bacterial–oral surface interaction; (2) indirect: Abbreviations: SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis ∗ Corresponding author. Tel.: +1-212-998-9850; fax: +1-212-995-4087. E-mail address: [email protected] (A.I. Spielman). 1 Present address: Clinical Research Core/Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research/National Institutes of Health, Bethesda, MD, USA.
bacterial–bacterial interaction, i.e. attachment through other bacteria already on the oral surfaces. Bacterial interaction with oral surfaces is highly specific, as these organisms do not attach to the oral soft and hard tissues directly but to the constituents of the acquired enamel pellicle, some of which act as specific receptor sites. Similarly, the receptor sites on the bacterial surface are specific, typically a cell surface molecule called an adhesin found on the bacterial frimbriae or pilli (Gibbons, 1984, 1989; Gong et al., 2000). Saliva plays a major part in the interaction between bacteria and tooth surfaces; most of the salivary proteins have a function in host defence mechanisms. The interactions of oral microorganisms with salivary proteins such as secretory-IgA, lysozyme, lactoferrin, salivary peroxidase and agglutinin are thought to benefit the host. This advantage has been attributed either to their bactericidal and bacteriostatic actions or to their role in facilitating the clearance of these microorganisms from the oral environment (Rudney et al., 1995, 1999; Gibbons and Spinell, 1970; Hay et al., 1971; Tabak et al., 1982; Vats and Lee, 2000).
0003-9969/02/$ – see front matter © 2002 Published by Elsevier Science Ltd. PII: S 0 0 0 3 - 9 9 6 9 ( 0 2 ) 0 0 0 2 0 - 1
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Other bacteria–saliva interactions may be detrimental to the host because they promote adhesion of bacteria to tooth surfaces (Gibbons and Hay, 1988). Though saliva-mediated bacterial attachment may be influenced by the specific site in the mouth (Carlen et al., 1998), we have chosen to study the influence of submandibular/sublingual salivary proteins on Streptococcus mutans attachment to spheroidal hydroxyapatite in vitro. Human submandibular/sublingual saliva contains several adhesion-promoting proteins (APPs) that specifically facilitate adhesion of S. mutans JBP serotype-c to hydroxyapatite in vitro (Kishimoto et al., 1989). These proteins were identified in fractions following gel-filtration chromatography. Adhesion assays of individual fractions showed two regions that promoted adhesion of S. mutans to hydroxyapatite. The first adhesion-promoting activity was associated with the high molecular-weight mucin peak; the second fraction was associated with several salivary proteins, including the acidic proline-rich proteins (Gibbons and Hay, 1989). Subsequent studies demonstrated that the first adhesion-promoting activity in the mucin fraction is in fact associated with a number of minor but highly active proteins termed APPs (Kishimoto et al., 1989, 1990). These proteins have Mw ranging from 250,000 to 350,000 Da and demonstrate individual variability in number and relative mobility on SDSPAGE. Because S. mutans is considered a primary agent in the aetiology of dental caries (van Houte, 1994), and because the interaction of APP with S. mutans is highly specific in an in vitro assay, it is likely that APP is important in vivo in the initial colonization of the enamel surfaces. Our purpose now was to purify and further characterize a human submandibular salivary APP and identify its immunological pattern in caries-prone and caries-free individuals. Understanding the different factors that influence bacterial interaction with the oral surfaces, including the adhesion-promoting regions in APP, might enhance development of methods to modulate such interactions, with the ultimate aim of preventing pathological outcomes such as dental caries and periodontitis.
2. Materials and methods
applying a 3% citric acid solution to the dorsum and lateral borders of the tongue every 30 s. Saliva was collected in a tube placed on ice; to avoid protein degradation the samples were mixed immediately with enzyme inhibitors: phenylmethylsulphonyl fluoride (0.001%; 2 l/ml saliva) and 10 mM EDTA, pH 7.2. The saliva samples were mixed thoroughly by passing them 20 times through two 50 ml syringes connected by a two-way Luer lock valve. 2.2. Salivary sample preparation The saliva samples were dialyzed for 24 h at 4 ◦ C, (12,000–14,000 Mw cut-off) against 10 volume (2 l) of 0.1 M ammonium bicarbonate/0.5% chloroform, pH 7.8, using two changes. The dialysate was concentrated with a Centriprep® -10 concentrator, 10,000 Mw cut-off (Amicon Inc., Beverly, MA) at 3000 × g and 4 ◦ C. The protein content of the concentrate was quantified (Bradford, 1976), with bovine gamma globulin as a standard. Samples were stored at −20 ◦ C until further use. 2.3. Gel-filtration chromatography Using a 450 ml bed volume (100 cm height × 2 cm diameter) Trisacryl GF 2000 M gel-filtration column (IBF Biotechnics Inc., Savage, MD), and an automated liquid-chromatographic system (Econo; Bio-Rad, Hercules, CA), 20 mg of salivary protein concentrate from each participant was purified as described by Kishimoto et al. (1989). In preparatory runs, submandibular/sublingual saliva was collected over 3 days from one individual and 100 mg of salivary protein was fractionated. The column was developed with 0.1 M ammonium bicarbonate/0.5% chloroform at a flow rate of 0.3 ml/min, controlled by the automated Econo system. The eluted proteins were monitored by measuring absorbance at A230 and 8 ml fractions were collected. During these preparatory runs, due to the larger amount of protein and reduced resolution, rechromatography of adhesion-promoting fractions was needed and was performed under conditions similar to the original gel-filtration chromatography. All fractions, including those containing proteins of 250,000–350,000 Da, were tested for in vitro S. mutans adhesion-promoting activity (see further).
2.1. Collection of saliva 2.4. Analytical SDS-PAGE Submandibular/sublingual saliva (200 ml) was collected from four healthy adult volunteers with at least 14 natural teeth (one Asian, one African–American, one Caucasian and one Hispanic). The participants provided informed consent and the collection protocol was reviewed and approved by the human subjects committee of our institution. A custom-made, acrylic, modified Block–Brottman saliva collection device (Block and Brottman, 1962; Nederfors and Dahlöf, 1993) was fabricated on an impression of the floor of the mouth taken with polyvinyl siloxane dental impression material. Stimulated saliva was collected by
Following gel-filtration chromatography, 10 l of each fraction was tested by gel electrophoresis (Laemmli, 1970) using precast 4–15 or 4–20% gradient SDS-PAGE (Jules Inc., New Haven, CT) and stained with Silver Stain Plus (Bio-Rad). 2.5. Adhesion-promoting assay The bacterial adhesion-promoting assay was as described by Kishimoto et al. (1989). Bacterial cells were attached
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to 0.08–0.20 mm spheroidal hydroxyapatite beads (Fluka Chemical Co., USA). To maintain the consistency of the adhesion-promoting assay, 1.4 g protein of submandibular/sublingual saliva or column fractions was always tested. S. mutans JBP serotype-c (a gift from the culture collection of the Forsyth Dental Center, Boston MA) was [3 H]-labeled in culture for 14, 16, 18, 20, 22 and 24 h, and then each time-point assayed in various ways. The bacterial cells harvested at 18 h demonstrated optimum viability and density (data not shown); an 18 h culture time was standard for all our adhesion assays. We also performed preliminary adhesion assays using various solutions (50 mM of KCl, phosphate-buffered saline, Tris, NaCl, NaHCO3 ); NaHCO3 was the solution of choice because it demonstrated minimal non-specific activity. To determine the actual number of attached bacteria and to establish the labeling efficiency, 725 × 105 [3 H]-labeled S. mutans cells (125 l of 5 × 108 cells/ml bacterial suspension), the same as that exposed to spheroidal hydroxyapatite, were always transferred to a separate scintillation vial. The counts/min of this vial represent the baseline used to convert counts/min to actual number of attached bacteria. 2.6. Preparatory SDS-PAGE and electroelution of adhesion-promoting protein APP was purified from two participants. Saliva from the first had a single 300,000 Da protein band on SDS-PAGE; saliva from the second had two bands at 300,000 Da. These bands from both participants were purified by electroelution (Hunkapillar et al., 1983; Spielman and Bennick, 1989), tested for purity (silver-stained, 4–15% gradient SDS-PAGE) and adhesion-promoting activity. 2.7. Preparation of polyclonal antibodies to adhesion-promoting protein Specific polyclonal antibodies were raised in guinea pigs (twice) to the APP obtained from the two different participants, using an established protocol (Spielman and Bennick, 1989; Spielman et al., 1991, 1995). The electroeluted proteins were mixed with complete Freund adjuvant at 1:1 ratio to a total of 1 ml solution and injected intradermally into the back of the neck of guinea pigs (six females, each weighing 400 g). The same amount of antigen was injected on days 28, 35, 42, 49, and 56; each injection contained a total of 0.4–0.5 g of pure APP. The guinea pigs were bled on day 56 and the serum tested for immunoreactivity by Western blotting as described in the following sections. On day 63 a final booster injection of 1 g APP was given with incomplete Freund adjuvant. On day 70, the guinea pigs were bled by cardiac puncture under halothane or ketamine/acepromazine anaesthesia. After overnight storage at 4 ◦ C, the antiserum was separated from the coagulum and cellular components by a 15 min centrifugation at 1000 × g.
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2.8. Purification and fragmentation of polyclonal antibodies Antiserum was further purified by removing the albumin and lipid components with the EZ-SEP® method (Pharmacia Biotech Inc., Piscataway, NJ), leaving only the immunoglobulin fraction. The IgG fraction was further separated from the rest of the immunoglobulins on an Acti-DiscTM protein G affinity column (FMC Corporations Bioproducts, Pine Brook, NJ). The F(ab )2 fragments needed to study inhibition of adhesion were generated by pepsin digestion, and were separated from the Fc fragments with the ImmunoPure® F(ab )2 preparation kit, following the manufacturer’s instructions (Pierce, Rockford, IL). The purity and functional integrity of the F(ab )2 fragments were tested by SDS-PAGE and Western blotting. Controls were prepared from custom-made F(ab )2 fragments raised to unrelated or purchased, control pure F(ab )2 fragments of normal guinea pig IgG (Jackson Immuno Research Laboratories Inc., West Grove, PA). 2.9. Testing of polyclonal antiserum and F(ab )2 fragment by Western blotting The antisera were tested by Western blotting at dilutions ranging from 1:50 to 1:1000 (Towbin et al., 1979), or 1:100 for F(ab )2 fragments, and visualized using the avidin–biotin procedure (Vector, Burlingame, CA). Non-specific binding to the nitrocellulose paper was blocked by incubating with milk buffer (10% milk buffer (w/v), 100 mM Tris, 0.9% NaCl and 0.1% Tween 20). Various control experiments included omission of anti-APP antiserum; substitution of anti-APP antiserum with either normal guinea pig serum, normal guinea pig F(ab )2 fragment or antiserum against an unrelated protein, the human apocrine secretion odorant-binding protein-1 (ASOB1) (Spielman et al., 1995). All samples and antisera were prepared in the same way. 2.10. Testing of adhesion-promoting protein with antibodies to mucins and agglutinin Western blotting experiments were done to determine any relation between APP and mucins, or APP and parotid agglutinins. APP was tested for immunoreactivity with polyclonal antibodies to MG1 (high molecular-weight mucin) and MG2 (low molecular-weight mucin) (anti-MG1 and anti-MG2 were provided courtesy of Dr. M. Levine, SUNNY, Buffalo) and with monoclonal antibodies to parotid agglutinins 143D4 B1 and 303D5 A1 (provided courtesy of Dr. D. Malamud, University of Pennsylvania, Philadelphia). Polyclonal antiserum to APP was tested at a dilution of 1:100; other antisera were tested at a dilution of 1:50.
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2.11. Lectin blotting of adhesion-promoting protein and inhibition of lectin reactivity Partial glycosylation patterns of samples from three of four volunteers were tested by lectin blotting: APP-enriched, gel-filtration chromatographic fractions from two participants and fractions from the analytical chromatography run of our third volunteer. The following seven lectins were used to identify the glycosylation pattern of the APP: Bandeiraea (Griffonia) simplicifolia II (BSL II), Datura stramonium (DSL), Erythrina cristagalli (ECL), Lycopersicon esculentum (LEL), Artocarpus integrifolia (Jacalin), Solanum tuberosum (potato) (STL), and Vicia villosa agglutinin (VVA), (Biotinylated Lectin Kit III; Vector). Lectin-blotting experiments followed the Western blotting procedure described above, except that 0.5% gelatin buffer (in 100 mM Tris–buffered saline/Tween 20, pH 7.2) was used instead of milk buffer to block non-specific binding. APP-enriched fractions (after gel-filtration chromatography) were loaded on a 4–15% gradient SDS-PAGE gel. The different lectins were incubated at 1:50 dilution; an E-Z SEP® purified guinea pig antiserum used as a control was incubated at 1:100 dilution, and all were visualized using the avidin–biotin procedure as described above. After identifying Jacalin and LEL as immunoreactive lectins, inhibitory Western blotting experiments were done to block lectin reactivity. Jacalin and LEL were preincubated for 30 min at 4 ◦ C with their competing sugars: thus, Jacalin was preincubated with a final concentration of 800 mM ␣-d-galactose, while LEL was preincubated with chitin hydrolysate (Vector) at a 1:50 final concentration. At a dilution of 1:50, lectins conjugated to their respective blockers were exposed to APP. Changes in the binding activity of the lectin–blocker complex were visualized by the avidin–biotin procedure. 2.12. Individual variability of adhesion-promoting protein immunoreactivity To identify individual variability and to attempt to correlate the presence of APP with caries incidence, we screened and Western blotted submandibular/sublingual saliva from 40 age-, sex- and caries history-matched individuals. These saliva samples were selected from those collected during another study conducted at the Research Center for Minority Oral Health, New York University (Cruz et al., 2001). The participants signed informed-consent documents approved by the University’s institutional review board. Using a cotton-tip applicator, 3% citric acid was applied briefly to the dorsum of the tongue. Immediately, 200–300 l of saliva was collected with a plastic Pasteur pipette from the pool around the orifices of submandibular salivary glands. The samples were transferred to microfuge tubes containing 2 l of 0.001% phenylmethylsulphonyl fluoride. All samples were kept on ice until stored at −80 ◦ C. Saliva samples from four broad ethnic groups were tested: Asian, African–American, Caucasian and Hispanic.
Each group contained 10 individuals, subdivided into five caries-active (history and presence of active caries lesions) and five caries-resistant (no presence or history of caries lesions). Thus, 40 submandibular/sublingual saliva samples from 20 caries-active and 20 caries-resistant individuals were tested for variability in APP immunoreactivity. Equal amounts of salivary proteins (50 g) were electrophoresed on a 5–20% SDS gradient gel, Western blotted and tested with antiserum to APP. In summary, we collected saliva samples from four individuals (nos. 1–4). From each, 20 mg submandibular/ sublingual salivary protein was loaded for analytical gel-filtration chromatography. In addition, from no.1, a 100 mg sample was loaded on a column for preparatory gel-filtration chromatography. Enriched APP from two individuals (nos. 1 and 2; 20 mg runs) were further purified by electroelution and injected into guinea pigs to raise polyclonal antibodies. Gel-chromatographic fractions from three individuals (nos. 1–3) were tested by lectin blotting. Adhesion assay results reported are those of samples from no. 1. Separately, submandibular/sublingual salivas of 40 individuals were screened for APP immunoreactivity.
3. Results 3.1. Salivary protein purification There was individual variability in volume (15–150 ml) and protein concentration (1.6–2.3 mg/ml (mean 1.8 mg/ml)) of saliva collected from the four participants. Loading 100 mg of submandibular/sublingual salivary proteins on to the column during the preparatory run resulted in poor protein separation (Fig. 1; first chromatographic run). However, using the adhesion assay we identified APP in fractions 12–17 and by SDS-PAGE identified fractions containing proteins of 250,000–350,000 Da (data not shown); these fractions were pooled and rechromatographed to obtain better resolution, as shown in the elution profile in Fig. 1 (second run). 3.2. Adhesion-promoting assay and activity The results of a typical adhesion assay are shown in Fig. 1, in which the gel-chromatographic elution profile (second run) is overlaid with a bar graph identifying fractions with adhesion-promoting activity. Fractions 11–16 demonstrated marked bacterial adhesion; other fractions were not significantly different from blanks included in the experiments (results not shown). 3.3. Immunological assays Fig. 2 demonstrates the purity of APP when equal protein amounts of unfractionated saliva and APP were loaded on a 4–20% gradient gel. After raising polyclonal antibodies in
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Fig. 1. Elution profiles of salivary proteins following gel-filtration chromatography and bacterial adhesion assay. Two elution profiles are presented. The first chromatographic elution profile shows gel-filtration chromatography of 100 mg protein of submandibular/sublingual saliva. Fractions 12–17 from this run demonstrated highest adhesion activity (results not shown); the same fractions 12–17 were pooled and reloaded on the same column to generate the second elution profile. The second chromatographic fractions were tested for adhesion-promoting activity. Superimposed bar graph represents adhesion assay; and shows that fractions 11–16 had the highest adhesion-promoting activity (expressed as mean number of adherent S. mutans cells ± S.E.M., n = 12). These same fractions had the highest immunoreactivity (see Fig. 3).
guinea pigs with the purified APP, we used Western blotting at a 1:100 antibody dilution to test salivary fractions obtained by gel-filtration chromatography. Antiserum (results not shown) and E-Z SEP® purified Ig fraction of the antiserum
to APP (Fig. 3) demonstrated a single immunoreactive band to APP-containing fractions. Note the immunoreactivity of fractions 12–16 (Fig. 3), and the similarity to the same fractions that have the highest adhesion-promoting activity
Table 1 Results of lectin blotting of salivary proteins. APP-enriched or unfractionated SM/SL saliva from three of four subjects were tested for reactivity with antiserum O APP (1:100 dilution) and seven different lectins (1:50 dilution). The three subjects showed reactivity to both JAC and LEL in the same molecular weight range as APP. Co-migration of immunoreactive and lectin-reactive proteins was considered as an indication of similarity. All three subjects reacted to APP and showed reactivity to JAC and LEL in the same molecular weight range as APP. Subject 3 showed mild reactivity to both JAC and LEL. The other five lectins were negative in all subjects. Negative reactivity was obtained when JAC and LEL blotting experiments were repeated in the presence of their respective competitive inhibitor. (+ = reactive; ++ = Moderately reactive; +++ = strongly reactive; − = non-reactive; NT = not tested) Lectin
Degree of lectin reactivity in three subjects Subject 1
Subject 2
Subject 3
Polyclonal antibodies to APP Lycopersicon esculentum (LEL) Artocarpus integrifolia (Jacalin) Bandeiraea (Griffonia) simplicifolia II (BSL II) Datura stramonium (DSL) Erythrina cristagalli (ECL) Solanum tuberosum (potato) (STL) Vicia villosa agglutinin (VVA)
+++ ++ + − − − − −
+++ + ++ − − − −
+++ + + − − − − −
Lectin and competitor Lycopersicon esculentum (LEL) and chitin hydrolysate Artocarpus integrifolia (Jacalin) and ␣-d-galactose
− −
NT NT
NT NT
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in Fig. 1. Similar Western blotting with monoclonal antibodies to mucins (MG1 and MG2) or agglutinin, even at dilutions of 1:50, produced negative results. 3.4. Lectin blotting Because APP coelutes with mucin when submandibular/ sublingual saliva is separated by gel-filtration chromatography, and because both mucin and APP are glycosylated, seven lectins were tested by lectin blotting to identify a potential purification strategy. While such a strategy was eventually not used in purification, it nevertheless identified specific terminal glycosylation for APP. The glycosylation pattern of APP was tested by lectin blotting using adhesion-promoting, protein-enriched, gel-filtration chromatographic fractions (similar to those in Figs. 1 and 2) from the two volunteers and a third individual whose submandibular/sublingual salivary APP had been partially purified (Table 1). Two lectins, Lycopersicon esculentum (LEL) and Artocarpus integrifolia (Jacalin), demonstrated reactivity in the three individuals tested. The degree of reactivity varied slightly among the three. The antiserum to APP used in control experiments showed reactivity in all three participants, but the degree of reactivity for LEL was highest in no. 1 and Jacalin reactivity was highest in no. 2, while no. 3 displayed minimal reactivity to both LEL and Jacalin. Five other lectins tested (listed in Table 1) did not show reactivity. Inhibitory lectin-blotting experiments showed successful blockage of Jacalin immunoreactivity by ␣-d-galactose, and LEL reactivity by chitin hydrolysate (Table 1). 3.5. Individual adhesion-promoting protein variability Fig. 2. Testing the purity of one of the electroeluted proteins (APP) by SDS-PAGE. The gel profile shows APP before electroelution (as part of unfractionated saliva, left lane) and after (right lane). The original submandibular/sublingual saliva (50 l sample) and the purified APP were loaded in separate lanes. The arrows show the migration level of APP. Note that the relative abundance of APP in original submandibular/sublingual saliva is very low (left arrow) staining much more faintly than in the enriched and overloaded APP sample (right arrow).
There was no correlation between caries experience and presence or absence of APP. Similarly, no correlation was found between ethnicity and presence or absence of APP immunoreactivity. As we anticipated, some (approximately half) of those tested did not demonstrate immunoreactivity (Fig. 4), probably due to the nature of the antibodies raised (part of its antigenic determinants are probably carbohydrates that vary among these individuals). The antibody we
Fig. 3. Western blotting of elution fractions from the gel-filtration chromatography. Note the heavy reactivity to E-Z SEP® purified immunoglobulin fraction of the antiserum to APP of fractions 12–16, corresponding to the high adhesion-promoting fractions in Fig. 1. Other fractions did not demonstrate similar reactivity.
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Fig. 4. Western blots showing the immunoreactivity of unfractionated submandibular/sublingual saliva from 40 individuals tested with polyclonal antibody to APP purified from an Asian individual. The participants belong to four different ethnic groups: Asian (A), African–American (B), Caucasian (C) and Hispanic (H); 50 g of submandibular/sublingual salivary proteins was tested in each case. These results show each group consisting of five individuals with no history of caries (no caries) and five caries-active individuals (caries). Note that saliva from individuals in the four groups demonstrated some degree of immunoreactivity with anti-APP (arrows). Minimal immunoreactivity was noticed in the Caucasian participants with no caries history.
used was raised against an APP purified from an Asian individual.
4. Discussion Our purpose was to purify and partially characterize an APP from human submandibular/sublingual saliva; our ultimate aim is to clone the genes for APP. Purification of APP proved to be more difficult than initially anticipated, because it is a very large molecule (250,000–300,000 Da), glycosylated and closely associated with, but apparently unrelated to, mucins. Collection of pure, uncontaminated submandibular saliva is easier if the gland is cannulated, but this is a more invasive procedure and not practical when collecting 300–500 ml of saliva. Therefore, we collected mixed submandibular/sublingual saliva, using a custom-made device, which prevented loss of sample and contamination from parotid saliva, gingival crevicular fluid or oral debris. We suspect that APP is present in the submandibular saliva but did not try to confirm this here. To compare different chromatographic runs for their relative adhesion-promoting activity, equal protein amounts (20 mg) were loaded and separated by gel-filtration chromatography. As previously reported, the APPs coelute with mucin (Kishimoto et al., 1989); this was confirmed when the adhesion-promoting assay was performed on each of the eluted fractions. To maintain consistency and a common base for comparison, 1.4 g of protein was tested from each fraction. Initial attempts to separate mucin from APP were unsuccessful, so we considered using lectin chromatography. Two of the seven different lectins tested with APP
samples from three participants showed positive reactivity. These were: Lycopersicon esculentum (LEL), specific for N-acetyl glucosamine-1-4-N-acetylglucosamine (GlcNAc (1,4)GlcNAc) oligomers; and Artocarpus integrifolia (Jacalin), specific for galactose and galactose-1-3-N-acetyl galactosamine (Gal (1,3)GalNac). They were noted as carbohydrates that could be targeted in the design of lectin chromatography experiments. However, after several unsuccessful attempts, probably due to individual variability and weak interaction between lectins and terminal carbohydrates, it proved to be more efficient to use electroelution of APP as a way to obtain purified protein, even though this yielded very small amounts. The concentration of APP in human submandibular/sublingual saliva is estimated to be below 1.5% of total protein; this is further illustrated in Fig. 2, showing SDS-PAGE that demonstrates the low abundance of APP in unfractionated submandibular/sublingual saliva. For analytical purifications, we used 20 mg of salivary protein as starting material. For preparative gel-filtration chromatography, the sample was scaled up to 100 mg salivary protein (Figs. 1 and 3), but poor resolution and low yield following electroelution still produced only a limited amount of pure protein. We purified approximately 25–50 pmol of APP, which was sufficient for antibody production and adhesion assays; this amount translates into 7.5 g of protein if the molecular-weight is assumed to be approximately 300,000 Da. Attempts were made to block the adhesion-promoting activity of the protein in unfractionated submandibular/sublingual saliva using anti-APP–F(ab )2 fragments. Though there was a partial decrease in the number of bacteria adhering to hydroxyapatite, it was not statistically
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significant (data not shown). This finding could be due to the use of unfractionated saliva instead of pure APP and the weak reactivity of our antibodies to adhesion-promoting epitopes of the protein. We could not establish an immunological relationship between APP, mucins and agglutinin. There was no cross-reactivity between APP and monoclonal antibodies raised to MG1 and MG2. Similarly, using monoclonal antibody 143D4 B1 , which recognizes the protein backbone of parotid agglutinin, and monoclonal antibody 303D5 A1 , which recognizes its carbohydrate portion, we could not establish any immuno-cross-reactivity between APP and agglutinin (data not shown). Based on lack of immunoreactivity alone, we cannot exclude the possibility that APP, agglutinin and mucins are related. These experiments need to be further corroborated by the sequencing of agglutinin and APP. Based on lectin blotting, the terminal carbohydrates of APP in three individuals appear to be: GlcNAc (1,4)GlcNac, galactose and Gal (1,3)GalNac, or a combination of these. The presence or absence of galactosyl residues on APP terminal oligosaccharides might influence bacterial attachment and caries susceptibility in different individuals (Seemann et al., 2001). We have also confirmed the individual variability of APP by testing its immunoreactivity with submandibular/sublingual saliva from 40 different individuals. About half of them, in all ethnic groups, demonstrated immunoreactivity to the protein antibody. Some displayed no immunoreactivity, possibly due to individual variability in the protein core or the glycosylation pattern. No correlation was found between ethnicity, immunoreactive pattern of salivary APP and caries experience, probably due to the multiple aetiological factors involved in caries incidence. In conclusion, we have purified an APP from submandibular/sublingual saliva, raised specific antibodies against it and characterized its terminal carbohydrates. This protein is apparently distinct from mucins and parotid agglutinins. No correlation could be found between ethnic group, caries incidence and APP. Further characterization of this protein is needed, including amino acid sequencing, cloning and the identification of the S. mutans-binding region to understand the specific role it may play in bacterial recognition. This site could be targeted for inhibitory activity to reduce the binding of S. mutans to enamel surfaces.
Acknowledgements This project was supported by Grant no. DE10593 (Research Center for Minority Oral Health, New York University College of Dentistry, Forsyth Dental Center) from NIH/NIDR. We would like to thank Drs. P. Bivona, P. Khurana, T. Shritavaj, J. Lee and R.Z. LeGeros for their help in the early stages of this project and for monoclonal antibodies to agglutinin (D. Malamud), and two different
antibodies to mucin (R. Troxler and M. Levine, respectively).
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