Identification of Porphyromonas gingivalis lipopolysaccharide-binding proteins in human saliva

Molecular Immunology 48 (2011) 2207–2213

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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm

Short communication

Identification of Porphyromonas gingivalis lipopolysaccharide-binding proteins in human saliva Seulggie Choi a,1 , Jung Eun Baik a,1 , Jun Ho Jeon b , Kun Cho c , Deog-Gyu Seo d , Kee-Yeon Kum d , Cheol-Heui Yun b , Seung Hyun Han a,∗ a

Department of Oral Microbiology and Immunology, Dental Research Institute, and BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea c Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang 863-883, Republic of Korea d Department of Conservative Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 4 May 2011 Received in revised form 9 June 2011 Accepted 10 June 2011 Available online 13 July 2011 Keywords: Porphyromonas gingivalis Lipopolysaccharide Lipopolysaccharide-binding proteins Saliva Mass spectrometry

a b s t r a c t Porphyromonas gingivalis causes periodontal diseases and its lipopolysaccharide (LPS) is considered as a major virulence factor responsible for pathogenesis. Since initial recognition of P. gingivalis LPS (Pg.LPS) in the oral cavity might be crucial for the host response, we identified Pg.LPS-binding proteins (Pg.LPSBPs) using Pg.LPS-immobilized beads and a high-resolution mass spectrometry. LPS purified from P. gingivalis was conjugated onto N-hydroxysuccinimidyl-Sepharose® 4 Fast Flow beads. Notably, Pg.LPSconjugated beads could stimulate Toll-like receptor 2 (TLR2) as determined by a TLR2-depdendent reporter expression system using CHO/CD14/TLR2. In addition, the Pg.LPS-conjugated beads induced the production of inflammatory mediators such as nitric oxide and interferon-gamma-inducible protein-10 in the macrophage cell-line, RAW 264.7. These results imply that Pg.LPS retained its immunological properties during the conjugation process. Then, the Pg.LPS-conjugated beads were mixed with a pool of saliva obtained from nine human subjects to capture Pg.LPS-BPs and molecular identities were determined by LTQ-Orbitrap hybrid fourier transform mass spectrometry. Pg.LPS-BPs captured at high frequencies included alpha-amylase, cystatin, prolactin-inducible protein, lysozyme C, immunoglobulin components, serum albumin, lipocalin-1, and submaxillary gland androgen-regulated protein 3B. These proteins are known to be involved in bacterial adhesion and colonization, anti-microbial functions or modulation of immune responses. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Porphyromonas gingivalis is a Gram-negative anaerobe which is closely associated with periodontitis, a chronic inflammatory disease of the tissue surrounding tooth root surface (Socransky et al., 1998). P. gingivalis is found in greater frequency in the oral cavities of periodontal patients than those of healthy subjects (Griffen et al., 1998). In addition to periodontitis, P. gingivalis has also been reported to have positive correlation with systemic diseases such as cardiovascular diseases, rheumatoid arthritis, and atherosclerosis (Bartold et al., 2010; Southerland et al., 2006). P. gingivalis expresses various virulence factors such as lipopolysaccharide (LPS), cytotoxins, and leukotoxins causing the pathogenesis (Holt et al., 1999). Among them, LPS, a cell-wall component, is considered as a major virulence factor contributing to bacterial adherence, colonization,

∗ Corresponding author. Tel.: +82 2 740 8641; fax: +82 2 743 0311. E-mail address: [email protected] (S.H. Han). 1 These authors contributed equally to this article. 0161-5890/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2011.06.434

and invasion to the host cells and the induction of inflammatory responses and alveolar bone loss (Erridge et al., 2002; Miyata et al., 1997). During infections, the host immune responses are initiated upon the recognition of pathogen-associated molecular patterns (PAMPs) exposed on the surface of and/or released from the pathogen. The recognition of PAMPs is mediated through specific interaction with the proteins, called pattern recognition receptors (PRRs) in either soluble or membrane-anchored forms (Janeway and Medzhitov, 2002). For example, LPS is first recognized by LPS-binding protein (LBP) in the blood and the LPS–LBP complex is further transported to CD14 and Toll-like receptors (TLRs) to activate dendritic cells, macrophages, neutrophils, and endothelial cells leading to the production of various inflammatory molecules (Schumann et al., 1990; Wright et al., 1990). Otherwise, the LPS–LBP complex can be deposited to high density lipoproteins for neutralization (Kitchens et al., 1999). Notably, LBP is present at low levels (ca. 100–200 ng/ml in the blood) under normal conditions whereas it is highly increased during infections (ca. 10–20 ␮g/ml) to neutralize LPS for the prevention of

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overwhelming inflammation (Zweigner et al., 2001). In addition to LBP, other LPS-binding proteins (LPS-BPs) have been reported such as albumin, lysozyme, complements, and deleted in malignant brain tumors 1 (DMBT1) (End et al., 2009; Freudenberg and Galanos, 1978; Gioannini et al., 2002; Ohno and Morrison, 1989). LPS of P. gingivalis is distinct from LPSs of other Gram-negative bacteria in that (i) LPSs of most Gram-negative bacteria stimulate TLR4, while LPS of P. gingivalis stimulates TLR2 (Hirschfeld et al., 2001) and (ii) P. gingivalis LPS (Pg.LPS) is weak in endotoxic activity compared to those of other Gram-negative bacteria (Liu et al., 2008). Hence, the host is supposed to recognize Pg.LPS differently from those of other Gram-negative bacteria. Although the host uses specific LPS-BPs, most studies on the LPS-BPs have been conducted using LPS of enteric bacteria such as Escherichia coli and Salmonella spp. (Couturier et al., 1991) and therefore previously known LPSBPs may not be applicable to Pg.LPS. Moreover, although the fact that P. gingivalis is present in the oral cavity and the initial recognition of Pg.LPS should occur in the oral cavity rather than in the blood, little is known about the Pg.LPS-BPs in saliva. In the present study, we developed a high-throughput system with sensitivity, accuracy and efficiency using LTQ-Orbitrap hybrid fourier transform (FT) mass spectrometry (MS) to screen for Pg.LPS-BPs present in human saliva. 2. Materials and methods

2.4. Culture of RAW 264.7 cells RAW 264.7 (TIB-71), a mouse macrophage cell line, was purchased from the American Type Culture Collection. The cells were cultured for 24 h in Dulbecco’s modified Eagle’s medium (Invitrogen, Grand Island, NY, USA), which is supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/ml of penicillin, and 100 ␮g/ml streptomycin at 37 ◦ C in a humidified incubator with 5% CO2 . 2.5. Measurement of the expression of inflammatory mediators RAW 264.7 cells at 1 × 106 cells/ml were incubated with varying concentrations of Pg.LPS-beads (0, 0.5, 1, 5, and 10 mg/ml), the native beads (10 mg/ml), or Pg.LPS (50 ␮g/ml) in the presence of IFN-␥ (0.1 ng/ml) for 24 h. At the end of the incubation period, nitric oxide (NO) and IFN-␥-inducible protein-10 (IP-10) expressions were determined in the culture media. To determine NO production, the culture media were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric acid) and incubated for 5 min. Then, using NaNO2 as a standard, the optical density was measured at 540 nm with a VERSAmax microtiter-plate reader (Molecular Devices, Sunnyvale, CA, USA). Levels of IP-10 were determined using a commercial ELISA kit (R&D Systems) according to the manufacturer’s instructions.

2.1. Bacteria, reagents, and chemicals 2.6. Measurement of TLR2-stimulating activity P. gingivalis ATCC49417 was obtained from the American Type Culture Collection (Manassas, VA, USA). A synthetic lipopeptide capable of stimulating TLR2, Pam2CSK4, was purchased from InVivoGen (San Diego, CA, USA). Brain heart infusion (BHI) was obtained from BD Biosciences (Franklin, NJ, USA). N-Hydroxysuccinimidyl-Sepharose® 4 Fast Flow beads, vitamin K, and hemine were purchased from Sigma–Aldrich (St. Louis, MO, USA). Mouse recombinant interferon-gamma (IFN-␥) was obtained from R&D Systems (Minneapolis, MN, USA). LPS Extraction Kit was purchased from Intron Biotechnology (Kyunggi-do, Korea). 2.2. Purification of Pg.LPS P. gingivalis was grown in BHI media supplemented with 10 ␮g/ml of vitamin K and 5 ␮g/ml of hemine at 37 ◦ C under anaerobic conditions (85% N2 , 10% CO2 , 5% H2 ) using the Anaerobic System (Thermo forma Model 1025, Marietta, OH, USA) for 2 days. Then, the cultured P. gingivalis was centrifuged at 11,068 × g at 4 ◦ C for 10 min and washed with PBS three times. LPS was purified from P. gingivalis using the LPS Extraction Kit according to the manufacturer’s instructions. The quantity of LPS was determined by weighing its mass after freeze-drying the sample.

An NF-␬B reporter cell line CHO/CD14/TLR2, co-expressing TLR2 and CD14, was used to analyze the ability of Pg.LPS-beads (0, 0.5, 1, 5, and 10 mg/ml) to activate TLR2 as previously described (Medvedev et al., 2001). The cell line expresses the gene which encodes membrane CD25 with the NF-␬B binding site-containing E-selectin promoter. Expression of CD25 was examined by flow cytometry using FACSCalibur with CellQuest software (BD Biosciences). 2.7. Collection of human saliva samples This study was conducted under the approval of the Institutional Review Board of the Seoul National University Dental Hospital (IRB No. CRI11008). Nine healthy subjects fasted for at least 2 h before brushing their teeth without toothpaste for 2 min. Afterward, the subjects rinsed their mouths with water for 10 min. Then, the subjects were each asked to spit about 10 ml of saliva into a 50 ml conical tube, in which each contained the Complete Mini Protease Inhibitor Cocktail EDTA-free tablets (Roche, Mannheim, Germany). To remove cells, debris, and insoluble materials, the saliva samples were centrifuged for 15 min at 7000 × g at 4 ◦ C. The supernatant was stored at −80 ◦ C until further use.

2.3. Conjugation of Pg.LPS to N-hydroxysuccinimidyl-Sepharose beads

2.8. Capturing Pg.LPS-BPs

After washing N-hydroxysuccinimidyl-Sepharose® 4 Fast Flow beads with non-pyrogenic water, 1000 mg of the beads were incubated with 6 mg of P. gingivalis LPS for 4 h at 4 ◦ C with gentle agitation. Then, the beads were incubated with 0.5 M ethanolamine (pH 8.0) for 1 h at 4 ◦ C with gentle agitation to block remaining conjugation sites. Finally, unconjugated LPS was removed by washing with non-pyrogenic water five times. Conjugation of P. gingivalis LPS onto N-hydroxysuccinimidyl-Sepharose beads was confirmed using a Limulus Amebocyte Lysate (LAL) test kit (QCL-1000; Cambrex Bio Science, Walkersville, MD).

The LPS-free and ethanolamine-treated beads (100 mg) were incubated with 200 ␮l of saliva for 1 h at 4 ◦ C with gentle agitation to eliminate bead-only binding proteins. The saliva was then incubated with Pg.LPS-conjugated beads (Pg.LPS-beads, 100 mg) for 3 h at 4 ◦ C with gentle agitation. Then, the beads were washed with PBS three times. Two hundred microliters of elution buffer (0.2 M glycine–HCl pH 2.2) were added and incubated for 10 min at 4 ◦ C, after which 30 ␮l of neutralizing buffer comprising 1 M Tris(hydroxymethyl)aminomethane-HCl, pH 9.1, was added and adjusted to pH 7.0. Half of the eluent from nine each saliva

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was lyophilized, dissolved in 10 ␮l of non-pyrogenic water to visualize the eluted Pg.LPS-BPs on SDS–PAGE gel after silver staining, and stored at −80 ◦ C until further use. The remaining half of nine each eluent was pooled in order to increase the significance and reliability. Then the pooled eluents were dialyzed using a semi-permeable dialysis membrane (Spectra/Por6; Spectrum laboratories, Inc, Ranch Dominquez, CA, USA) against 50 mM ammonium bicarbonate (pH 7.8) to remove the salt for optimal peptide sequencing. The dialyzed eluent was lyophilized and kept at −80 ◦ C until the MS analysis. 2.9. SDS–PAGE and silver staining The saliva samples (20 ␮l) or the eluents (10 ␮l) were mixed with 4× sample buffer (8% SDS, 10% 2-mercaptoethanol, 30% glycerol, 0.02% bromophenol blue in 0.25 M Tris–HCl, pH 6.8) and incubated at 100 ◦ C for 15 min. The solution was then loaded into each well and run at 120 V for 3 h in 12% SDS–PAGE gels. The SDS–PAGE gel was treated with fixation solution (50% methanol/12% acetic acid/0.0185% CH2 O) for 1 h. Then, the gel was washed twice with 50% ethanol for 20 min each, after which the gel was treated with sensitizing solution [0.02% (w/v) Na2 S2 O3 ·5H2 O] for 1 min. After washing three times with distilled water for 20 s each, the gel was incubated with silver reaction solution (0.2% AgNO3 /0.027% CH2 O) for 1 h. After washing twice with non-pyrogenic water for 20 s each, the gel was incubated with developing solution [6% Na2 CO3 /0.0004% (w/v) Na2 S2 O3 ·5H2 O/0.0185% CH2 O] for 5–10 min. After obtaining the desired band intensity, the gel was washed and incubated with stopping solution (50% methanol/12% acetic acid) to halt the silver reaction. 2.10. Identification of Pg.LPS-BPs The lyophilized eluents were resuspended and incubated in 50 mM NH4 HCO3 containing 10 mM dithiothreitol (pH 7.8) for 30 min at 55 ◦ C. At the end of the incubation, free thiol groups were alkylated with 40 mM of iodoacetamide in the dark at room temperature for 25 min. Then, tryptic digestion was performed by treating the samples in the buffer containing 50 mM ammonium bicarbonate, 5 mM CaCl2 , and 10 ␮g/ml trypsin at 37 ◦ C for 12–16 h followed by lyophilization. The lyophilized peptide mixtures were solubilized in 0.1% formic acid and loaded onto a microcapillary column packed with C18 RP resin in 75 ␮m silica tubing (8 ␮m inner diameter of the orifice, 10 cm in length). For the elution of peptides, buffers A (0.1% formic acid) and B (80% acetonitrile containing 0.1% formic acid) were prepared and eluted with 5% of the buffer B for 25 min, 20% for 5 min, 60% for 50 min, and 100% for 5 min at a flow rate of 300 nl/min. Then, the column was equilibrated with 5% of the buffer B for 15 min prior to the next running. For the identification of peptide, the eluted peptides were subjected to MS using 7-tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nano-ESI source in positive ion mode at the spray voltage of 2.5 kV. MS and MS/MS spectra were obtained with the capillary heated to 220 ◦ C, an ESI voltage of 2.5 kV, 35% of collision energy, and 1 Da of isolation width. The full scan was performed at a resolution of 100,000 FWHM (the full width at half maximum) intensity, and then data-dependent MS/MS analysis was performed from the three most abundant MS ions. The spectra were analyzed with Mascot Daemon (Matrix Science, London, UK) using the IPI human database (IPI.HUMAN.v.3.72). Peptides were considered to be identified at the peptide tolerance of ±50 ppm, fragment mass tolerance of ±0.8 Da, two missed trypsin cleavage, oxidation of Met, and fixed modification of carbamidomethyl cysteine. Peptide score is −10 × Log(P), in which P signifies the

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probability that the observed match is a random event. Individual peptide scores over 35 are considered as identity or extensive homology (P < 0.05) and proteins comprising more than two peptides with over 35 peptide scores were identified as Pg.LPS-BPs. 2.11. Statistical analysis The mean values ± standard deviations were determined from triplicate samples. Statistical significance was measured using a two-tailed t-test. Differences were considered significant when P < 0.05 by comparing the experimental groups with the nontreatment control group. All experiments, except the mass spectrometric analysis, were performed at least three times. The mass spectrometric analysis was independently performed twice under the similar conditions. 3. Results 3.1. Conjugation of Pg.LPS onto N-hydroxysuccinimidyl-Sepharose beads To capture and identify Pg.LPS-BPs, Pg.LPS was first conjugated onto amine-reactive N-hydroxysuccinimidyl-Sepharose® 4 Fast Flow beads by the formation of stable amide bonds between the primary amine (–NH2 ) in the LPS and the bead, replacing the N-hydroxysuccinimidyl group in the process (Fig. 1). Then, the remaining reactive N-hydroxysuccinimidyl sites were replaced with ethanolamine. The LAL test conducted on the Pg.LPS-beads confirmed the binding of Pg.LPS (data not shown). 3.2. Pg.LPS-conjugated beads are biologically active As a result of the conjugation reaction, the Pg.LPS may have lost its immunological properties. To confirm that the Pg.LPS that is conjugated on the Sepharose beads is biologically active and its properties are unhindered, the ability of Pg.LPS-beads to induce the production of inflammatory mediators NO and IP-10 was examined in the murine macrophage cell line, RAW 264.7. When the cells were treated with Pg.LPS-beads (0, 0.5, 1, 5, or 10 mg/ml), the native beads (10 mg/ml), or Pg.LPS (50 ␮g/ml) in the presence of IFN-␥ (0.1 ng/ml), there was an induction of NO (Fig. 2A) and IP-10 (Fig. 2B) production by the Pg.LPS-beads and Pg.LPS but not by the native beads. These results suggest that the Pg.LPS on Pg.LPS-beads retains its inflammatory potential. 3.3. Pg.LPS-beads stimulate TLR2 The innate immune response to Pg.LPS has been shown to be mediated primarily through TLR2 (Hirschfeld et al., 2001). To confirm that Pg.LPS-beads retained the ability to stimulate TLR2, CHO/CD14/TLR2 cells were treated with Pg.LPS-beads at 0.5, 1, 5, or 10 mg/ml or with the native beads at 10 mg/ml, and the expression of CD25 was examined using flow cytometry. An increase in CD25 expression was found only on the cells stimulated with Pg.LPS-beads in a concentration-dependent manner as Pg.LPS and Pam2CSK4, which are known as TLR2-specific ligands (Fig. 3). However, such induction was not observed on the cells treated with the native beads (Fig. 3). These results suggest that the Pg.LPS conjugated onto the beads retain its TLR2-activating ability. 3.4. Capturing and identification of Pg.LPS-BPs in the human saliva Saliva is constantly present in the oral cavity, and therefore contains a number of proteins involved in the host immune response against bacterial infection (Van Nieuw Amerongen et al., 2004).

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LPS-binding proteins NHS

NHS

+ LPS

LTQ-Orbitrap mass spectrometry

+ Saliva

NHS

Fig. 1. Experimental scheme for the identification of Pg.LPS-BPs in human saliva. NHS and LPS signify N-hydroxysuccinimidyl and Pg.LPS, respectively.

To characterize the protein profiles from the saliva samples, proteins in the human saliva were visualized by silver staining on the SDS–PAGE gel. Fig. 4A demonstrates heterogeneous protein profiles with various molecular mass in the saliva samples from all individuals. When the saliva samples were incubated with Pg.LPSbeads to isolate Pg.LPS-BPs, several distinct bands were observed on the SDS–PAGE gel (Fig. 4B). High-resolution LTQ-Orbitrap FT MS spectrometry followed by peptide identification revealed a total of 35 Pg.LPS-BPs (data not shown). Among the 35 proteins, nine Pg.LPS-BPs with statistical significance (P < 0.05) were selected as shown in Table 1. Alpha-amylase and serum albumin were detected with remarkably high scores. The proteins with medium scores were cystatin, lysozyme C, submaxillary gland androgen-regulated

*

25

A

*

Nitrite (μM)

20 15

*

10 5 0

Beads (mg/ml)

-

10

-

-

-

-

-

Pg.LPS-Beads (mg/ml)

-

-

0.5

1

5

10

-

Pg.LPS (μg/ml) -

-

-

-

-

-

50

12

B

*

IP-10 (ng/ml)

*

6

*

4

*

2

Accession number

Composition of amino acids

Peptide score

Alpha-amylase (AMY1A; AMY1B; AMY1C; AMY2A)a LSGLLDLALGK WVDIALECER ALVFVDNHDNQR NWGEGWGFMPSDR IPI00300786; DVNDWVGPPNDNGVTK IPI00939512 DFPAVPYSGWDFNDGK IAEYMNHLIDIGVAGFR EVTINPDTTCGNDWVCEHR NVVDGQPFTNWYDNGSNQVAFGR

73.1 68.1 62.7 81.0 75.5 67.7 48.5 82.7 39.8

Serum albumin (ALB)a FQNALLVR QTALVELVK LVAASQAALGL LVNEVTEFAK AVMDDFAAFVEK IPI00745872 KVPQVSTPTLVEVSR QNCELFEQLGEYK RPCFSALEVDETYVPK VFDEFKPLVEEPQNLIK EFNAETFTFHADICTLSEK

41.2 43.4 83.3 55.2 69.2 68.2 50.0 37.4 35.1 59.9

Cystatin (CST4, CST1)a EQTFGGVNYFFDVEVGR IPI00032294; IIPGGIYDADLNDEWVQR IPI00305477 SQPNLDTCAFHEQPELQK

82.1 85.4 91.3

Lysozyme C (LYZ)a

10 8

Table 1 Pg.LPS-BPs identified with high-resolution LTQ-Orbitrap FT MS.

0

Beads (mg/ml)

-

10

-

-

-

-

-

Pg.LPS-Beads (mg/ml)

-

-

0.5

1

5

10

-

Pg.LPS (μg/ml) -

-

-

-

-

-

50

Fig. 2. Pg.LPS-beads were biologically active. RAW 264.7 cells were stimulated with Pg.LPS-beads (0, 0.5, 1, 5, and 10 mg/ml), the native beads (10 mg/ml), or Pg.LPS (50 ␮g/ml) in the presence of IFN-␥ (0.1 ng/ml) for 24 h. Then, the culture media were analyzed for (A) NO and (B) IP-10 productions as described in Section 2. Error bars indicate the standard deviations. Asterisk (*) indicates the experimental group significantly different (P < 0.05) from the non-treatment control group.

IPI00019038

GISLANWMCLAK STDYGIFQINSR TPGAVNACHLSCSALLQDNIADAVACAK

63.3 69.6 74.3

Submaxillary gland androgen-regulated protein 3B (SMR3B)a IPPPPPAPYGPGIFPPPPPQP GPYPPGPLAPPQPFGPGFVPPPPPPPYGPGR IPI00023011 SQPNLDTCAFHEQPELQK

38.6 62.4 91.3

Immunoglobulin lambda chain (IGLV2-14, IGLC3)a SYSCQVTHEGSTVEK IPI00944677; YAASSYLSLTPEQWK IPI00830047 AAPSVTLFPPSSEELQANK

63.6 91.8 36.1

Immunoglobulin heavy chain (IGHA1, IGHA2)a YLTWASR IPI00644497; IPI00386879; WLQGSQELPR IPI00383164; TFTCTAAYPESK IPI00642017; DASGVTFTWTPSSGK IPI00784830 Prolactin-inducible protein (PIP)a FYTIEILK IPI00022974 ELGICPDDAAVIPIK

36.1 71.5 50.5 65.5

55.2 84.1

Lipocalin-1 (LCN1)a IPI00009650 a

GLSTESILIPR NNLEALEDFEK

Protein name (gene symbol).

50.8 52.6

S. Choi et al. / Molecular Immunology 48 (2011) 2207–2213

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Fig. 3. Pg.LPS-beads stimulated TLR2. CHO/CD14/TLR2 cells were stimulated with Pg.LPS-beads (0, 0.5, 1, 5, and 10 mg/ml), the native beads (10 mg/ml), Pam2CSK4 (0.1 ␮g/ml), or Pg.LPS (10 ␮g/ml) for 18 h. Then, flow cytometry was conducted for the analysis of CD25 expression, a marker of TLR2-dependent NF-␬B activation.

protein 3B, immunoglobulin components, prolactin-inducible protein (PIP), and lipocalin-1 (Table 1).

4. Discussion

Fig. 4. The protein profiles of the human saliva and the Pg.LPS-BPs. (A) Saliva samples from nine human subjects (#1–9) were separated using SDS–PAGE and visualized by silver staining. (B) Pg.LPS-BPs in saliva samples from nine subjects (#1–9) were separated using SDS–PAGE and visualized by silver staining. SM; size marker.

In healthy individuals, approximately 750–1000 ml of saliva is secreted daily from the salivary glands including the parotid gland, the submandibular gland, the sublingual gland, and the minor salivary glands (Ogra et al., 1999). Although saliva flushes microorganisms from oral mucosa and tooth surfaces, saliva also contains various effector molecules with anti-microbial activities. Xerostomia patients with lowered secretion of the saliva are prone to oral infections (Walker, 2004). Conversely, pathogens often use certain components of the saliva for their adherence, colonization, and invasion to the host (Scannapieco, 1994). Thus, it is important to identify the salivary components with high affinity to PAMPs for the understanding of pathogenesis on the infection and immunity in the oral cavity. In this study, we captured Pg.LPS-BPs with Pg.LPS-immobilized beads and identified the proteins with LTQOrbitrap FT MS spectrometry. Pg.LPS-BPs identified in this study were categorized as to be related to either, (i) bacterial adhesion and colonization, (ii) anti-microbial functions or (iii) modulation of immune responses (Table 2). Other molecules with unknown functions are yet to be characterized. Alpha-amylase, cystatin, and PIP are likely involved in bacterial adhesion and colonization to the host. Alpha-amylase is found in acquired enamel pellicle as well as dental plaque (Birkhed and Skude, 1978; Orstavik and Kraus, 1973). Alpha-amylase is known to have high affinity to bacteria such as Streptococcus sanguinis (Scannapieco et al., 1989). Remarkably, previous studies demonstrated that alpha-amylase contributes to the adhesion of hydroxyapatite to bacteria that bind to amylase (Bennick, 1982). Cystatins are known to inhibit cysteine proteases degrading immunoglobulins and type I collagen while salivary cystatins bind to hydroxyapatite, which are present in dental enamel (Shomers et al., 1982). In addition, salivary PIP binds to the surface of various oral bacteria such as Gemella haemolysans, Gemella morbillorium, Streptococcus acidominimus, Streptococcus oralis, Streptococcus salivarius, and Streptococcus parasanguinis (Schenkels et al., 1997). Lysozyme C and immunoglobulins are likely to be anti-microbial proteins. Although lysozymes are well-known to disrupt bacterial

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Table 2 Classification of identified Pg.LPS-BPs and their respective functions. Related to

Protein name

Functions

Alpha-amylase

Digestion of starch. Association with bacteria. Promotion of bacterial adhesion to hydroxyapatite. Inhibition of cysteine protease degrading immunoglobulins and type I collagen. Binding to hydroxyapatite. Binding to oral bacteria.

Bacterial adhesion and colonization Cystatin Prolactin-inducible protein Anti-microbial functions

Lysozyme C Immunoglobulins

Modulation of immune responses

Unknown functions

Hydrolysis of peptidoglycan. Binding to LPS resulting in alteration of the conformational change of LPS. Inhibition of LPS-induced immunostimulatory activity. Inhibition of bacterial adhesion, colonization, and infection. Clearance of pathogens and toxins.

Serum albumin Lipocalin-1

Binding to LPS for the modulation of immune cell activation. Transportation of lipophilic molecules including fatty acids, phospholipids, glycolipids, and arachidonic acids. Regulation of immune responses. Inhibition of bacterial growth by scavenging siderophores.

Submaxillary gland androgen-regulated protein 3B

Function yet to be identified, but probably related in promoting angiogenesis and establishing microvasculature.

cell walls by hydrolyzing peptidoglycans (Holler et al., 1975), lysozymes have been shown to bind to LPS (Ohno and Morrison, 1989) resulting in the reduction of immunostimulatory activity to produce TNF-␣ and IL-6 (Takada et al., 1994a,b). More comprehensive studies proposed that the binding of lysozymes to LPS induced a conformational change of LPS, which could account for the reduced immunostimulatory activity (Brandenburg et al., 1998). Immunoglobulins that bind to LPS (i.e., anti-LPS-antibodies) are involved in protection against Gram-negative bacterial infection by inhibiting colonization and adhesion of bacteria to host cells while promoting bacterial clearance by immune cells such as macrophages, dendritic cells, and neutrophils (Pudifin et al., 1985; Wells et al., 1987). Furthermore, previous reports indicated that mice deficient in anti-LPS antibodies were more prone to septic shock upon bacterial infection compared to wild-type mice (Reid et al., 1997). Interestingly, BLAST search analysis demonstrated that the majority of the identified immunoglobulin sequences matched with those of anti-viral antibodies. This suggests that the Pg.LPScaptured immunoglobulins may be cross-reactive against both LPS and some viral antigens. Such cross-reactivity could be explained by natural antibodies, which are considered to be used as initial defense mechanisms against bacterial and viral invasions (Casali and Notkins, 1989). Indeed, monoclonal natural antibodies from mice could bind to multiple antigens, including those from ssDNA, beta-galactosidase, insulin, thyroglobulin, and LPS (Zhou et al., 2007). Therefore, the identified immunoglobulins may be a member of the natural antibody family with polyreactive properties that contribute to host defense against bacterial infection. Serum albumin and lipocalin-1 seem to be related with the modulation of immune responses. Serum albumin is known to bind to LPS of Gram-negative bacteria (Rietschel et al., 1973) and lipooligosaccharide (LOS) of Neisseria meningitidis (Gioannini et al., 2002). However, unlike lysozyme, serum albumin seems to facilitate the immune cell stimulation rather than inhibition. Serum albumin assists the transfer of LPS or LOS to the immunostimulatory proteins such as CD14 and TLR4/MD-2 complexes (Gioannini et al., 2002). Although serum albumin is a major constituent of plasma (ca. 35–50 mg/ml) (Anderson and Anderson, 2002), it is also a normal constituent of human saliva occupying 6% of the total salivary proteins (Scarano et al., 2010). The albumin contents in the saliva seem to be constant regardless of periodontitis because there was no significant change in the salivary albumin contents of healthy subject (123 ± 60 ␮g/ml) and periodontal patients (118 ± 64 ␮g/ml) (Henskens et al., 1996). On the other hand, lipocalin possesses diverse functions involved in transporting hydrophobic molecules, mediating prostaglandin D synthesis, and immune modulation (Flower, 1996). Lipocalin-1 is able to bind to various lipophilic ligands including fatty acids, phospholipids,

glycolipids, cholesterols, retinols, and arachidonic acids (Flower, 1996). Lipocalin-1, also known as the lingual von Ebner’s gland protein, is found in various mucosal tissues such as eyes (tears), nasal mucosa, and tracheal mucosa (Redl, 2000). Although lipocalin-1 acts as the principal lipid binding protein (Glasgow et al., 1995), it is also involved in protection against bacterial infection due to its ability to inhibit bacterial growth by scavenging microbial siderophores (Fluckinger et al., 2004). We found the heterogeneous protein profiles with various molecular mass in the saliva samples from nine individuals. Especially, the bands around 50–70 kDa were distinct in the subjects 1, 2, 3, and 4 compared to others. The bands could be for albumin (67 kDa), amylase (56 kDa), or immunoglobulin heavy chain (53 kDa) because these proteins exist in relatively high concentrations in saliva but nonetheless differ among individuals: amylase (11–178 U/ml), serum albumin (29–238 ␮g/ml), and immunoglubulin A (19–439 ␮g/ml) (Henskens et al., 1996). One may suspect the assay specificity in that abundant proteins such as serum albumin and amylase are possibly over-represented and non-specifically bound to the Pg.LPS-beads resulting in the falsepositive identification as Pg.LPS-BPs. However, this possibility is unlikely since other abundant proteins including mucins and basic proline-rich proteins, which exist 20% each of the total salivary proteins (Scarano et al., 2010), were not detected as Pg.LPS-BPs in the present study. This study demonstrated that interaction between Pg.LPS-beads and proteins followed by a high-resolution MS analysis could be applied to the screening of LPS-BPs in saliva. Furthermore, we were able to identify potential LPS-BPs from human saliva samples without arduous biological assays. Features of the method include (i) simple and easy conjugation process, (ii) irreversible and firm binding of Pg.LPS to the beads through covalent bond, (iii) no alteration of the immunostimulating property of Pg.LPS during the conjugation process, and (iv) high-throughput identification of the LPS-BPs, even in minute quantities, using highly sensitive LTQ-Orbitrap FT MS spectrometry. Therefore, this method is a potential tool to capture and identify proteins that play a central role in the infection and immunity because it is simple, convenient, sensitive, accurate, and high-throughput. While the presence of P. gingivalis is associated with periodontitis, the exact role of Pg.LPS in the pathogenesis of the disease is yet unknown. The elucidation of novel Pg.LPS-BPs may shed further light into the action mode of pathogenesis of periodontitis by P. gingivalis and the subsequent host response. With further efforts, the novel LPS-BPs may become subjects of drug development targets which could lead to a deeper understanding of bacterial infection and the advancement of improved treatment methods.

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