HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 51, 39860-39866, December 22, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/51/39860    most recent M006928200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Prakobphol, A. Articles by Fisher, S. J. Search for Related Content PubMed PubMed Citation Articles by Prakobphol, A. Articles by Fisher, S. J. Social Bookmarking                      What's this?

Salivary Agglutinin, Which Binds Streptococcus mutans and Helicobacter pylori, Is the Lung Scavenger Receptor Cysteine-rich Protein gp-340^*

From the Departments of ^a Stomatology, ⁱ Anatomy, ^d Pharmaceutical Chemistry, and ^j Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, California 94143, the ^c Department of Odontology/Cariology and Oral Microbiology, Umeå University, SE-901 87 Umeå, Sweden, the ^f Department of Medical Microbiology, Institute of Medical Biology, University of Odense, DK-5000 Odense C, Denmark, the ^e Department of Medical Biochemistry, University of Göteborg, SE-413 90 Göteborg, Sweden, the ^g Department of Molecular Medicine, Lund University, SE-221 00 Lund, Sweden, and the ^h Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, August 1, 2000, and in revised form, September 22, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le^x carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human saliva has several critical functions, including lubrication (1-3), digestion (4), formation of a bioactive semipermeable barrier (pellicle) that coats oral surfaces (5-8), and regulation of the composition of the oral flora. Saliva fulfills the latter function by virtue of its antimicrobial activity (9, 10) and by promoting selective microbial clearance or adherence (11-14). The diverse functions attributed to saliva are allocated among its many components, which include amylases, cystatins, proline-rich proteins, proline-rich glycoproteins, carbonic anhydrases, peroxidases, statherins, histatins, lactoferrin, lysozyme, sIgA, mucins, and salivary agglutinin. Protein sequences have now been deduced for all the major salivary components except the agglutinin (15-18).

Salivary agglutinin was identified as a protein fraction that mediates specific adhesion and aggregation of Streptococcus mutans (19-22). Monoclonal antibodies (mAb)¹ to agglutinin block adherence of S. mutans to experimental pellicles and aggregation of the bacterial cells by parotid saliva (23, 24). Several studies have related the levels of agglutinin in saliva to the numbers of S. mutans in dental plaque (19, 25), the rate of plaque formation (26), and the susceptibility to dental caries (27). Other studies have not found these associations (28, 29).

Despite a potentially important role of agglutinin in regulating the composition of the oral flora, very little is known about the chemical nature of the molecule. When isolated from parotid saliva, it behaves as a 5 × 10⁶-Da oligomeric complex that contains a major 440-kDa glycoprotein (20). Immunoblotting experiments show that this protein is recognized by the same mAb that blocks S. mutans adherence and its parotid saliva-induced aggregation in vitro (20, 23). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) shows that the complex contains other proteins, including secretory IgA, and minor, as yet unidentified, components (20, 21, 30). In the absence of additional structural information about the salivary agglutinin, we isolated the glycoprotein and analyzed its peptide portion by using mass spectrometry techniques. The results showed identity with gp-340 discovered in airway secretions by virtue of its affinity for surfactant protein D (31, 32). We also obtained additional interesting information about its glycosylation and bacterial binding properties.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All chemicals, unless otherwise noted, were obtained from Sigma. Nitrocellulose membranes (0.45 µm) were obtained from Schleicher & Schuell. Polyvinylidene difluoride membranes (Immobilon-P, 0.45 µm) were obtained from Millipore (Bedford, MA). Human lung gp-340 was purified from a lavage pellet as described (33). Rabbit polyclonal antibodies that recognized a synthetic peptide of gp-340 (anti-gp-340-1) and a purified human lung gp-340 (anti-gp-340-3) were prepared as described (31-33). A mAb that recognized human purified lung gp-340 (anti-gp-340-2 (Hyb 213-6)) was prepared as described (31, 32). We used two mAbs that specifically reacted with salivary agglutinin: mAb 143 and mAb 303 (34). The latter two antibodies were from Dr. Daniel Malamud (University of Pennsylvania, Philadelphia, PA). The anti-sLe^x mAb was prepared by culturing the hybridoma cell line CSLEX-1, obtained from the American Type Culture Collection (Manassas, VA), in RPMI 1640 medium supplemented with 15% fetal bovine serum. The culture supernatant containing the desired mouse IgM was collected after 48 h. Horseradish peroxidase-conjugated goat anti-rabbit IgG (heavy and light chains) was purchased from Bio-Rad and DAKO (Glostrup, Denmark). Goat anti-mouse IgG (heavy and light chains) and goat anti-mouse IgM, both conjugated to horseradish peroxidase, were purchased from Jackson Immuno Research Laboratories Inc. (West Grove, PA). High purity trypsin was from Promega (Madison, WI). Fast-stain^® (Coomassie Blue) was from Zoion Biotech (Shrewsbury, MA). Brucella agar and yeast extract were from Difco. IsoVitalex, trypticase soy broth, and Columbia-II-agar were from Becton Dickinson Microbiology Systems (Cockeysville, MD). Chemiluminescence detection reagents (SuperSignal® substrate) were purchased from Pierce.

Collection of Saliva-- Human parotid and submandibular/sublingual (SM/SL) salivas were collected as the ductal secretions as described previously (11, 35). The samples were either used immediately or mixed with an equal volume of loading buffer and stored at 20 °C before the experiment.

SDS-PAGE-- Parotid and SM/SL saliva samples, purified salivary agglutinin preparations, and purified lung gp-340 were electrophoretically separated on polyacrylamide gels (36). Three types of gels were used. One had a 3% stacking gel and 10% running gel, and the others were gradient gels, either 4-15% or 10-20% (Bio-Rad). Protein bands were visualized by staining with either Fast-stain® or Coomassie Brilliant Blue.

Biochemical Purification of Salivary Agglutinin-- Four hundred µl of fresh parotid saliva from one donor was concentrated to 25 µl by using a Centricon Plus 20 filter (Millipore, Marlborough, MA) then mixed with an equal volume of loading buffer. The concentrated sample was electrophoretically separated; the stacking gel contained 3% acrylamide, and the running gel contained 10% acrylamide. After electrophoresis, the protein bands were visualized with Fast-stain®. Under these conditions the salivary agglutinin was well separated from other proteins. This allowed excision of the ~ 350-kDa band.

Affinity Purification of Salivary Agglutinin-- Agglutinin was purified from fresh parotid or SM/SL saliva using published methods (20, 30). Briefly, S. mutans strain Ingbritt was maintained as described below. The cultures were harvested in exponential phase, washed once with phosphate-buffered saline (PBS; 10 mM K₂HPO₄/KH₂PO₄, 0.15 M NaCl), pH 6.8, and suspended at a concentration of 5 × 10⁹ cells/ml. Equal volumes (100 ml) of bacterial suspension and parotid or SM/SL saliva (diluted 1:1 in PBS, pH 6.8) were mixed and incubated at 37 °C for 60 min. The aggregated bacteria were pelleted by centrifugation (15,000 × g) for 30 min at 4 °C. After the supernatant was discarded, agglutinin was released by adding 6 ml of 20 mM EDTA in PBS, pH 6.8, at room temperature. Then the sample was centrifuged (30,000 × g) for 30 min at 4 °C. The EDTA extraction and centrifugation steps were repeated once. The two supernatants were pooled and subjected to gel filtration on a Superdex 200 26/60 column (Amersham Pharmacia Biotech) which was eluted with 10 mM PBS, pH 6.8. The agglutinin-containing void volume (40 ml) was concentrated to a final volume of 600 µl by using Centriprep-10 centrifugal concentrators (Amicon, Beverly, MA). The protein concentration was estimated by using the Bio-Rad DC Protein Assay with bovine albumin as a standard. The void volume fraction was also analyzed by gradient SDS-PAGE (4-15% acrylamide).

Mass Spectrometry of Peptides Isolated from the Biochemically Purified Agglutinin-- The excised agglutinin band was macerated in a solution of high purity trypsin (0.05 µg/µl in 25 mM ammonium bicarbonate). The proteolytic digestion was allowed to continue at 37 °C for 16 h. The resulting peptides were eluted from the gel with a solution of 50% acetonitrile and 5% trifluoroacetic acid in distilled water and concentrated in a SpeedVac. First, the eluate was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Portions of unseparated tryptic digests were cocrystallized in a matrix of -cyano-4-hydroxycinnamic acid and analyzed by using a PerSeptive Biosystems DE-STR MALDI-TOF mass spectrometer equipped with delayed extraction operated in the reflector mode.

Alternatively, peptide samples were subjected to high performance liquid chromatography (HPLC) separation before mass spectrometry. The apparatus was fitted with a Michrom Bioresources MagicMS C18 column (0.2 × 50 mm; 5-µm particle size; 200 Å pore size) that was equilibrated with 7% acetonitrile, 0.1% trifluoroacetic acid in H₂O. A flow rate of 1 µl/min was established by using an Eldex Micropro pump. Peptides were eluted isocratically for 10 min followed by a linear gradient (0.875%/min) to a final mobile phase composition of 63% acetonitrile, 0.082% trifluoroacetic acid in H₂O. 1-2-µl HPLC fractions were spotted directly onto a MALDI target with 1.5 µl of -cyano-4-hydroxycinnamic acid. Post-source decay (PSD) sequencing was done as described previously (37). MS/MS sequencing was by quadrapole orthogonal time-of-flight mass spectrometry (Micromass, Manchester, UK). The peptide mass and peptide fragment-ion data were used with the MS-Fit and MS-Tag programs, which are available on the Internet to search data bases to determine peptide identity.

Mass Spectrometry of Peptides Isolated from the Affinity-purified Agglutinin-- The analyses were performed essentially as described previously (38). First, the sample was subjected to SDS-PAGE on a 10-20% gradient gel (Bio-Rad) and stained with Coomassie Brilliant Blue. A single band was visible near the origin of the gel. This band was excised and digested with trypsin. The peptides were analyzed by MALDI-TOF mass spectrometry on a TofSpec E instrument (Micromass) with delayed extraction and in reflectron mode using a matrix of -cyano-4-hydroxycinnamic acid. For MS/MS, the sample was analyzed on a quadrapole orthogonal time-of-flight mass spectrometry instrument (Micromass), and the m/z 1459 ion was selected for fragmentation analysis. The results were used to search sequence data bases using the MS-Fit and MS-Tag programs as described above.

Immunoblotting-- After SDS-PAGE, samples were transferred to nitrocellulose or Immobilon-P membranes as described previously (39). Briefly, nonspecific binding was blocked by incubating blots for 1 h in PBS containing 0.05% Tween 20 and 5% Carnation nonfat dried milk (T-blotto). The blot was then incubated for 2 h with 1 of 5 antibodies that specifically recognized either gp-340 or salivary agglutinin. Anti-gp-340-1 was raised against a synthetic peptide that corresponds to a region of gp-340 (33). Anti-gp-340-2 and anti-gp-340-3 were raised against a gp-340 preparation purified from lung (31, 32). mAbs 143 and 303 were raised against a salivary agglutinin preparation purified from saliva. An earlier study showed that mAb 143 recognizes the agglutinin protein core, whereas mAb 303 recognizes the Le^y epitope in the context of the agglutinin protein core (34). All the antibodies were diluted in T-blotto (v:v) as indicated: anti-gp-340-1, 1:1,000; anti-gp 340-2, 1:10,000; anti-gp-340-3, 1:2,000; mAb143, 1:60,000; and mAb303, 1:60,000. The blots were washed three times in PBS containing 0.05% Tween 20 for 5 min each time. Binding of primary polyclonal antibodies was detected after incubation for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG diluted 1:2,000 (v:v) in T-blotto. Binding of mAbs was detected after incubation for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG diluted 1:2,000 (v:v) in T-blotto. After washing, bands were detected either by staining the blots with 3,3'-aminobenzidine (6 mg in 100 ml of PBS containing 0.03% hydrogen peroxide) or chemiluminescence detection reagents (SuperSignal^®Substrate). Proteins that carried the sLe^x determinant were identified by staining nitrocellulose transfers with mouse monoclonal anti-sLe^x IgM (diluted 1:5 (v:v) in T-blotto) essentially as described above. The horseradish peroxidase-conjugated, isotype-specific secondary antibody used for detection of the IgM was diluted 1:2,000 (v:v) in T-blotto.

Bacteria and Culture Conditions-- The origin of S. mutans strain Ingbritt was as described previously (24). All other streptococcal strains (see Table II) were from Dr. Mogens Kilian (Aarhus University, Denmark). The streptococcal cells were grown as described previously (24). The cultures were harvested at exponential phase, washed two times in PBS, and suspended at a concentration of either 2 × 10⁹ or 5 × 10⁹ cells/ml in PBS, pH 6.8. Helicobacter pylori strain CCUG17875 was grown as described previously (40). Briefly, the cells were maintained and grown on Brucella agar supplemented with 10% bovine blood, 1% IsoVitalex, amphotericin B (4 mg/liter), vancomycin (10 mg/liter), and trimetropin (5 mg/liter). After incubation for 48 h at 37 °C in microaerophilic conditions (10% CO₂, 5% O₂, 85% N₂), the cells were harvested, washed two times in PBS, and suspended at a concentration of 2 × 10⁹ cells/ml in PBS.

Aggregation of Bacterial Cells by Purified Agglutinin-- The ability of affinity-purified parotid agglutinin to agglutinate a panel of streptococci (Table II) and H. pylori strain CCUG17875 was assessed as described earlier (20). Briefly, 1 ml of purified agglutinin (~2 µg/ml) in PBS, pH 6.8, was mixed with 1 ml of bacteria suspended at a concentration of 5 × 10⁹ cells/ml in PBS, pH 6.8. Aggregation was recorded by measuring, at 1-min intervals for 1 h, the A₇₀₀ using a Beckman^® DU-50 series spectrophotometer (Beckman Instruments).

Binding of Agglutinin to Bacterial Cells-- 4 ml of either an H. pylori strain CCUG17875 or an S. mutans Ingbritt cell suspension (2 × 10⁹ cells/ml PBS) was incubated with an equal volume of fresh parotid saliva diluted 1:1 in PBS (experimental) or PBS alone (control). Binding took place at room temperature for 1 h. The cells were then pelleted by centrifugation, washed twice with PBS, and suspended in 150 µl of SDS-PAGE loading buffer. After 30 min, the cells were pelleted. An aliquot of the supernatant (~25 µl) was subjected to SDS-PAGE and immunoblotting with anti-gp-340-1 as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of Agglutinin for Mass Spectrometry Analyses-- The salivary agglutinin was isolated by two methods. The first was biochemical. Whole parotid saliva was electrophoretically separated on a SDS-polyacrylamide gel, and the band corresponding to the molecular mass of the salivary agglutinin was excised (Fig. 1A, arrowhead). The second method exploited the known biological properties of the molecule. Specifically, the glycoprotein was purified by selective adsorption from whole parotid saliva to S. mutans bacteria followed by release of bound proteins with EDTA-containing buffer and then fractionation of the released proteins on a Superdex 200 column. SDS-PAGE of the void volume fraction showed only a single band of ~350 kDa under nonreducing conditions (Fig. 1B, lane 1, arrowhead), which had a slightly lower molecular mass under reducing conditions (Fig. 1B, lane 2). Finally, we also purified agglutinin from SM/SL saliva by selective adsorption to S. mutans cells. SDS-PAGE of the SM/SL agglutinin showed a single band with the same electrophoretic properties as the parotid agglutinin under both nonreducing (Fig. 1B, lane 3) and reducing conditions (Fig. 1B, lane 4).

View larger version (73K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE of a parotid saliva sample containing the salivary agglutinin and purified agglutinin samples isolated from parotid and SM/SL saliva by adsorption to S. mutans. A, a sample of whole parotid saliva was separated on a 10% polyacrylamide gel as described under "Experimental Procedures." The band of 350 kDa, denoted by the arrowhead, was excised and subjected to an in-gel trypsin digest before analysis by mass spectrometry (see Fig. 2 and Table I). B, salivary agglutinin (single band denoted with an arrowhead) was also isolated from saliva by adsorption to S. mutans. SDS-PAGE (4-15% gradient gel) of the glycoproteins that were purified from parotid (lanes 1 and 2) and SM/SL saliva (lanes 3 and 4). Under both non-reducing (B, lanes 1 and 3) and reducing conditions (B, lanes 2 and 4), the agglutinin appeared as a single band, with a slightly lower molecular mass under reducing conditions. Proteins were visualized by staining with either Fast-stain® (A) or Coomassie Brilliant Blue (B). The top and bottom of the stacking gels are marked with arrows.

Analysis of the Peptide Portion of the Salivary Agglutinin by Mass Spectrometry-- Initially, both agglutinin preparations were subjected to in-gel trypsin digestion. An aliquot of each unseparated digest was analyzed by MALDI-TOF MS, which yielded a peptide mass fingerprint. A total of 15 peptides and 14 unique sequences was obtained from the biochemically purified sample (Table I). The sequence of five of these peptides was determined by MALDI-TOF/PSD (bold italic typeface in Table I). As an example, the MALDI-TOF spectrum of one of the digests is shown in Fig. 2A. A PSD spectrum and fragmentation of the peptide m/z 1459.8 is shown in Fig. 2B together with the interpretation and deduced amino acid sequence. The peptide mass fingerprint and PSD fragment ions (Table I) were used for data base searching. The results showed that the peptides were identical to regions found within both gp-340 and its splice variant DMBT1, both members of the scavenger receptor cysteine-rich protein family.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   The trypsin digest of the biochemically purified salivary agglutinin included a peptide with a molecular mass and sequence identical to an area within the cysteine-rich region of the scavenger receptor gp-340. A, a band corresponding to the estimated molecular mass of the salivary agglutinin was excised from an SDS-PAGE gel (see Fig. 1A) and digested with trypsin. MALDI-TOF MS analysis of the digest revealed a peptide that gave a particularly strong signal at m/z 1459.8. B, this peptide was sequenced by PSD. The fragment ions were used to search data bases, which allowed assignment of the amino acid sequence shown above the spectrum. This sequence is identical to a region found within the scavenger receptor cysteine-rich region of gp-340. Two classes of ions are labeled in the spectrum, immonium and related ions, which are found in the low mass region, and b- and y-ions as indicated. The b6 18 and b8 18 ions were observed due to loss of H2O. C, peptides also present in a negative control, a portion of the polyacrylamide gel that did not stain for protein. T, trypsin autoproteolysis peptides, which were used to internally calibrate the spectrum.

The tryptic digest of the affinity-purified agglutinin sample was also analyzed by MALDI-TOF MS. The results showed a dominant peptide of m/z 1459.8. MS/MS analyses of this peak showed that the sequence was identical to that of the peptide of the same mass that was analyzed from the biochemically purified sample. Other ions corresponding to the peptides marked with an asterisk in Table I were also identified as well as two additional ions at m/z 1082.2 and 2739 that correspond to peptide regions that lie toward the C terminus of both gp-340 and DMBT1. Together, these results show that the samples isolated by the two different methods are very likely to be the same protein.

Immunoblot Analyses of Salivary Agglutinin with Antibodies Specific for either gp-340, Salivary Agglutinin, or the sLe^x Epitope-- Next, we determined whether antibodies raised against gp-340 cross-react with the salivary agglutinin. Fig. 3A shows immunoblot analyses (anti-gp-340-1) of whole parotid saliva samples from six donors as well as purified gp-340 isolated from a human lung lavage pellet as a control. The sample of lung gp-340 reacted with the antibody (lane 1). Immunoreactive bands of ~350 kDa were also observed in the parotid saliva samples obtained from all six donors (lanes 2-7). Additional immunoblot experiments were performed with anti-gp-340-2. The latter antibody recognized single bands of ~350 kDa in samples of whole parotid saliva (Fig. 3B, lane 1) and affinity-purified agglutinin (Fig. 3B, lane 2) only under non-reducing conditions, as did anti-gp-340-3. Bands of the same estimated molecular mass in both samples also reacted with mAb 143, which was raised against the agglutinin protein core (data not shown). Together, the data from the immunoblot experiments suggested that the salivary agglutinin and gp-340 contained the same immunogenic regions.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Immunoblotting showed that the salivary agglutinin reacted with anti-gp-340 and anti-sLex. Immunoblotting was carried out as described under "Experimental Procedures." The transfers shown in A and C contained the same samples: gp-340 purified from lung lavage (lane 1) and parotid saliva samples from six individuals (lanes 2-7). The transfer shown in B contained a sample of parotid saliva (lane 1) and the agglutinin glycoprotein purified from parotid saliva by adsorption to S. mutans (lane 2). A, polyclonal anti-gp-340-1 reacted with both lung lavage gp-340 and bands of ~350 kDa in all the parotid saliva samples. B, monoclonal antibody anti-gp-340-3 (Hyb 213-6) reacted with a band of ~350 kDa in parotid saliva and with the affinity-purified agglutinin from parotid saliva. C, anti-sLex did not react with lung gp-340 (lane 1), whereas all the samples of whole parotid saliva contained a single high molecular mass band that stained (lanes 2-7). The top and bottom of the stacking gels are marked with arrows.

During the course of the immunoblot analyses we noted that mAb 303, which recognizes the Le^y epitope in the context of the agglutinin protein core (34), reacted with a single band in parotid saliva and in the affinity-purified agglutinin sample (data not shown). This result suggested the presence of specialized oligosaccharide structures. To obtain additional information about glycosylation of this molecule we surveyed expression of the sLe^x epitope, which is carried by the low molecular weight salivary mucin and which interacts with L-selectin (42, 43). Interestingly, immunoblotting showed that the salivary agglutinin reacted with anti-sLe^x, whereas gp-340 purified from lung did not (Fig. 3C). This result suggested the possibility of different gp-340 glycoforms due to differences in the genotypes of the donors.

Salivary Agglutinin Interacts with a Variety of Bacteria-- Next, we analyzed interactions between salivary agglutinin and a panel of commensal and pathogenic bacteria (Table II). The agglutinin mediated strong aggregation of S. mutans (three out of three strains), implicated in dental caries, and S. agalactiae (two out of two strains), implicated in neonatal meningitis, but not Streptococcus pyogenes (two out of two strains), implicated in tonsillitis and invasive infections. Furthermore, although agglutinin mediated strong aggregation of some strains of Streptococcus oralis, Streptococcus mitis, and Streptococcus intermedius, several other strains of the same bacteria as well as other streptococci that colonize dental and oral mucosal surfaces (see Table II) were not subject to aggregation.

We were also interested in whether the agglutinin interacts with other medically important bacteria that are likely to pass through, rather than permanently colonize, the oral cavity. We were particularly interested in H. pylori, an organism implicated in gastritis and peptic ulcer disease (44, 45). Results of these experiments showed that the salivary agglutinin also mediated aggregation of H. pylori to nearly the same extent as whole parotid saliva (Table II).

We used a second method to confirm this potentially important observation. S. mutans and H. pylori were incubated with parotid saliva, and bound agglutinin was detected by immunoblotting with anti-gp-340-1 as described under "Experimental Procedures." The results are shown in Fig. 4. As expected, parotid saliva (lane 1) showed an ~350-kDa immunoreactive band. Components of the extract of control S. mutans cells that were incubated in PBS failed to react with the antibody (Fig. 4, lane 2), but cells incubated in parotid saliva showed an immunoreactive band of the anticipated molecular mass (Fig. 4, lane 3). When H. pylori cells were incubated with parotid saliva or PBS, an antibody reactive band of ~350 kDa was detected only in the extract prepared from cells that were incubated with parotid saliva (Fig. 4, compare lanes 4 and 5).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Salivary agglutinin binds to H. pylori. The bacterial cells were incubated with fresh parotid saliva and washed several times with PBS. Bound salivary components were eluted, separated by SDS-PAGE, transferred to nitrocellulose and visualized by immunoblotting with anti-gp-340-1. The transfer contained the following samples: lane 1, parotid saliva; lane 2, extract from control S. mutans cells that were incubated with PBS; lane 3, extract from experimental S. mutans cells that were incubated with parotid saliva; lane 4, extract from control H. pylori cells that were incubated with PBS; lane 5, extract from experimental H. pylori cells that were incubated with parotid saliva. Anti-gp 340-1 stained only the agglutinin in parotid saliva (lane 1) and a band of the same estimated molecular mass in extracts prepared from bacterial cells that were incubated with parotid saliva (lanes 3 and 5). No antibody-reactive bands were seen in the control cell extracts (lanes 2 and 4). Arrows indicate the top and the bottom of the stacking gel.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here we report that salivary agglutinin is very likely gp-340. The primary structure of gp-340, recently established by molecular cloning, shows a polypeptide chain of 2413 amino acids (32). The N terminus consists of a signal peptide and a sequence of 69 unique amino acids. Residues 95 to 1741 contain highly repetitive regions consisting of 13 scavenger receptor cysteine-rich (SRCR) domains. These domains are separated by SRCR-interspersed domain sequences, of which there are 12. The interval between residues 1742 and 2134 contains a Ser-Thr-Pro-rich region and an additional SRCR domain flanked by two CUB (C1r/C1s Uegf Bmp1) domains, protein modules initially found in complement subcomponents C1r/C1 s, Uegf, and bone morphogenetic protein-1. The remaining residues contain a hydrophobic zona pellucida domain.

The 14 unique agglutinin peptides we detected by mass spectrometry were scattered throughout the entire protein and occurred within all the major domains (see Table I). The identification of salivary agglutinin as the gp-340 protein was also suggested by the results of immunoblotting experiments. The ~350-kDa protein we isolated from parotid saliva, by using either biochemical or affinity methods, reacted with antibodies raised against salivary agglutinin (34) and antibodies raised against lung gp-340 (31-33). Moreover, the size of the salivary molecule was the same as that of gp-340 isolated from lung lavage, evidence that the agglutinin is the full-length molecule rather than the splice variant DMBT1, which lacks 628 amino acids consisting of 5 paired SRCR and SRCR-interspersed domains (32, 46). However, we have not ruled out the possibility that the salivary molecule is a differentially glycosylated form of DMBT1. Agglutinin in parotid saliva obtained from all six individuals of diverse ethnic backgrounds reacted specifically with anti-gp-340 (Fig. 3A), suggesting that expression is probably widespread in the population. However, there could be genetic variability in the expression and/or splicing of this molecule, an issue we have yet to address. In addition, differences among individuals in glycosylation seem highly likely, since the agglutinin reacted with anti-sLe^x, an oligosaccharide epitope whose expression depends on genotype. Functional heterogeneity exists as well. For example, previous studies demonstrated differences among subjects with regard to agglutinin binding of S. mutans (21, 47) that coincide with susceptibility and resistance to dental caries (47). Finally, the presence of this glycoprotein in saliva is consistent with reverse transcription-polymerase chain reaction analyses that show the main sites of gp-340 expression are lung, trachea, salivary glands, small intestine, and stomach (32).

Knowing the structure of salivary agglutinin will greatly facilitate experiments to understand its various biological roles. One important general function likely arises from the ability of the glycoprotein to interact with bacteria. The agglutinin-mediated aggregation of S. mutans, implicated in dental caries, and S. agalactiae, implicated in neonatal meningitis, suggests a role in microbial clearance of potentially pathogenic microorganisms. Some strains of S. oralis, Streptococcus gordonii, and S. mitis were also aggregated by the agglutinin, suggesting that this glycoprotein interacts with commensal organisms as well. In either case, the outcome of agglutinin binding may depend on the microbial ligand and its method of interaction as well as whether the interaction occurs in solution, which probably favors clearance, or on oral surfaces, where adherence becomes possible. In this regard, it is interesting to note that the agglutinin-binding adhesin AgI/II of S. mutans possesses different domains that are involved in either agglutinin-mediated adhesion to hydroxyapatite surfaces or aggregation in solution (48). Finally, since gp-340 is an opsonin receptor for surfactant proteins A and D (32), future studies should investigate whether this interaction influences the bacteria binding properties of the salivary molecule.

Dissecting the interactions between salivary agglutinin and various bacteria at a molecular level offers an interesting opportunity to restore, in the case of relevant disease states, the normal balance found in healthy individuals. With regard to H. pylori, the results from our study, which show an affinity of this bacterium for the salivary agglutinin, support the hypothesis of oral transmission, which could include transient adherence in the mouth (49). It is interesting that salivary mucins also interact with this bacterium.² The latter observation is in accord with two previous studies. First, we showed that salivary mucins carried the fucosylated blood group antigens (i.e. the ABO and Lewis type) (39). Second, the Le^b and H-1 histo-blood group antigens mediate adherence of H. pylori to human gastric mucosa (41). Eradication of H. pylori infection with antibiotic treatment has proved to be difficult in patients who harbor H. pylori in the oral cavity/dental plaque (50). The results of this study suggest new, pharmacological strategies for inhibiting infections by this organism.

With regard to interactions with cells of the host immune system, we found that the salivary agglutinin can carry the sLe^x epitope. This suggests that this molecule, like the low molecular weight salivary mucin (43), could tether both bacteria and leukocytes, a potentially important consideration for immune interactions in the oral cavity. Also with regard to immune function, it is interesting to consider data that show gp-340 from lung stimulates random migration (chemokinesis) of alveolar macrophages (33). This activity could also enhance bacterial interactions with cellular components of the host immune system. Finally, studies by Nagashunmugam et al. (51) suggest that salivary agglutinin has human immunodeficiency virus-neutralizing properties. Thus, the agglutinin could be a component of the molecular system that prevents oral transmission of the virus. Understanding the relationship between the structure of the salivary agglutinin and the many interesting functions it may carry out is the focus of our future experiments.

	ACKNOWLEDGEMENTS

We thank Eduardo Caceres and Patricia Urso for excellent technical assistance and Evangeline Leash for excellent editorial assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DE 07244, HL 51134, and RR 01614, Swedish Medical Research Council Grants 10435, 12165, 10906, and 11218, and the Swedish Foundation for Strategic Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^b This paper is an equal contribution of two research groups with two senior authors (S. J. F. and N. S.) and three first authors (A. P., F. X., and V. M. H.).

^k To whom correspondence should be addressed: HSW 604, University of California, San Francisco, CA 94143-0512. Tel.: 415-476-5297; Fax: 415-476-4204; E-mail: sfisher@cgl.ucsf.edu.

Published, JBC Papers in Press, September 27, 2000, DOI 10.1074/jbc.M006928200

² A. Prakobphol, T. Borén, and S. J. Fisher, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; SM/SL, submandibular/sublingual; PBS, phosphate-buffered saline; MALDI-TOF MS, matrix-assisted laser/desorption ionization time-of-flight mass spectrometry; HPLC, high performance liquid chromatography; PSD, post-source decay; SRCR, scavenger receptor cysteine-rich.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. Cannon, Y. Yi, H. Ni, E. Stoddard, D. A. Scales, D. I. Van Ryk, I. Chaiken, D. Malamud, and D. Weissman HIV Envelope Binding by Macrophage-Expressed gp340 Promotes HIV-1 Infection J. Immunol., August 1, 2008; 181(3): 2065 - 2070. [Abstract] [Full Text] [PDF]

				E. J. Helmerhorst, X. Sun, E. Salih, and F. G. Oppenheim Identification of Lys-Pro-Gln as a Novel Cleavage Site Specificity of Saliva-associated Proteases J. Biol. Chem., July 18, 2008; 283(29): 19957 - 19966. [Abstract] [Full Text] [PDF]

				P. J. Crowley, T. B. Seifert, R. Isoda, M. van Tilburg, M. W. Oli, R. A. Robinette, W. P. McArthur, A. S. Bleiweis, and L. J. Brady Requirements for Surface Expression and Function of Adhesin P1 from Streptococcus mutans Infect. Immun., June 1, 2008; 76(6): 2456 - 2468. [Abstract] [Full Text] [PDF]

				R. C. De Lisle, W. Xu, B. A. Roe, and D. Ziemer Effects of Muclin (Dmbt1) deficiency on the gastrointestinal system Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G717 - G727. [Abstract] [Full Text] [PDF]

				S. W. Kerrigan, N. S. Jakubovics, C. Keane, P. Maguire, K. Wynne, H. F. Jenkinson, and D. Cox Role of Streptococcus gordonii Surface Proteins SspA/SspB and Hsa in Platelet Function Infect. Immun., December 1, 2007; 75(12): 5740 - 5747. [Abstract] [Full Text] [PDF]

				E. Stoddard, G. Cannon, H. Ni, K. Kariko, J. Capodici, D. Malamud, and D. Weissman gp340 Expressed on Human Genital Epithelia Binds HIV-1 Envelope Protein and Facilitates Viral Transmission J. Immunol., September 1, 2007; 179(5): 3126 - 3132. [Abstract] [Full Text] [PDF]

				E.J. Helmerhorst and F.G. Oppenheim Saliva: a Dynamic Proteome J. Dent. Res., August 1, 2007; 86(8): 680 - 693. [Abstract] [Full Text] [PDF]

				P. Rosenstiel, C. Sina, C. End, M. Renner, S. Lyer, A. Till, S. Hellmig, S. Nikolaus, U. R. Folsch, B. Helmke, et al. Regulation of DMBT1 via NOD2 and TLR4 in Intestinal Epithelial Cells Modulates Bacterial Recognition and Invasion J. Immunol., June 15, 2007; 178(12): 8203 - 8211. [Abstract] [Full Text] [PDF]

				M. Drobni, T. Li, C. Kruger, V. Loimaranta, M. Kilian, L. Hammarstrom, H. Jornvall, T. Bergman, and N. Stromberg Host-Derived Pentapeptide Affecting Adhesion, Proliferation, and Local pH in Biofilm Communities Composed of Streptococcus and Actinomyces Species Infect. Immun., November 1, 2006; 74(11): 6293 - 6299. [Abstract] [Full Text] [PDF]

				B. Grubor, D. K. Meyerholz, and M. R. Ackermann Collectins and cationic antimicrobial peptides of the respiratory epithelia. Vet. Pathol., September 1, 2006; 43(5): 595 - 612. [Abstract] [Full Text] [PDF]

				S. Zhang, N. M. Green, I. Sitkiewicz, R. B. LeFebvre, and J. M. Musser Identification and Characterization of an Antigen I/II Family Protein Produced by Group A Streptococcus Infect. Immun., July 1, 2006; 74(7): 4200 - 4213. [Abstract] [Full Text] [PDF]

				D. Takamatsu, B. A. Bensing, A. Prakobphol, S. J. Fisher, and P. M. Sullam Binding of the Streptococcal Surface Glycoproteins GspB and Hsa to Human Salivary Proteins Infect. Immun., March 1, 2006; 74(3): 1933 - 1940. [Abstract] [Full Text] [PDF]

				S. A. Shelburne III, P. Sumby, I. Sitkiewicz, C. Granville, F. R. DeLeo, and J. M. Musser Central role of a bacterial two-component gene regulatory system of previously unknown function in pathogen persistence in human saliva PNAS, November 1, 2005; 102(44): 16037 - 16042. [Abstract] [Full Text] [PDF]

N. S. Jakubovics, S. W. Kerrigan, A. H. Nobbs, N. Stromberg, C. J. van Dolleweerd, D. M. Cox, C. G. Kelly, and H. F. Jenkinson Functions of Cell Surface-