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Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors...
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Identification of Proteinaceous Inhibitors of a Cysteine Proteinase (an Arg-Specific Gingipain) from Porphyromonas gingivalis in Rice Grain, Using Targeted-Proteomics Approaches Mayumi Taiyoji,†,‡ Yasuyuki Shitomi,¶,# Masayuki Taniguchi,‡,⊥ Eiichi Saitoh,§ and Sadami Ohtsubo*,† Food Research Center, Niigata Agricultural Research Institute, Kamo, Niigata 959-1381, Japan, Graduate School of Science and Technology, Niigata University, Niigata, Niigata 950-2181, Japan, Department of Materials Science and Technology, Niigata University, Niigata, Niigata 950-2181, Japan, Venture Business Laboratory, Niigata University, Niigata, Niigata 950-2181, Japan, and Graduate School of Technology, Niigata Institute of Technology, Kashiwazaki, Niigata 945-1195, Japan Received June 13, 2009

Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors that this bacterium produces, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major determinants of the pathogenicity of P. gingivalis. Here, we report on our finding that there are inhibitors of these cysteine proteinases in a rice protein fraction. Comprehensive affinity chromatography and MS analyses resulted in the identification of 17 Arg-gingipain (Rgp)-interacting proteins in the rice endosperm. Of these, four proteins (i.e., a 26 kDa globulin, a plant lipid transfer/trypsin-R amylase inhibitor, the RA17 seed allergen, and an R amylase/trypsin inhibitor) were estimated to account for 90% of the Rgp inhibitory activity in the rice protein fraction, using a two-dimensional gel system of double-layer reverse zymography. In addition, a synthetic peptide derived from an Rgp-interacting protein, cyanate hydratase, could inhibit the growth of P. gingivalis and showed inhibitory activity against both the Arg- and Lys-gingipains. These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases. Keywords: Porphyromonas gingivalis • Oryza sativa • cysteine proteinase inhibitor • Arg-gingipain

Introduction Periodontal diseases are highly prevalent chronic infections caused by a specific group of Gram-negative anaerobic bacteria, leading to inflammation of the gingiva and destruction of periodontal tissues. Control of periodontal conditions is important for the maintenance of good oral hygiene, but perhaps more importantly, emerging studies have demonstrated a strong association between periodontal diseases and systemic diseases such as atherosclerosis and coronary heart diseases,1-3 diabetes,4,5 respiratory diseases,6 and preterm delivery along with low birth weight.7,8 These findings suggest that the control of periodontal diseases is also critical in the prevention and management of these systemic conditions. Porphyromonas gingivalis, which is an asaccharolytic blackpigmented bacterium, is known to be a major etiologic agent * To whom correspondence should be addressed. Phone, +81-256-523267; fax, +81-256-52-6634; e-mail, [email protected]. † Food Research Center, Niigata Agricultural Research Institute ‡ Graduate School of Science and Technology, Niigata University ¶ Venture Business Laboratory, Niigata University. # Present address: Kennedy Institute of Rheumatology, Imperial College London, 65 Aspenlea Road, Hammersmith, London W6 8LH, United Kingdom. ⊥ Department of Materials Science and Technology, Niigata University. § Niigata Institute of Technology. 10.1021/pr900519z CCC: $40.75

 2009 American Chemical Society

in the onset and progression of chronic periodontitis.9-11 This bacterium produces various virulence factors,12,13 and among these, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major pathogenicity determinants.14-17 These enzymes are essential for the growth and survival of the bacterium in vitro and in vivo,18-20 and they play critical roles in the degradation of host proteins,19,21-23 the acquisition of iron from the host environment,21,24-26 and in the processing of bacterial cell-surface and secretory proteins.27,28 Arg-specific and Lys-specific gingipains (Rgp and Kgp, respectively) are also known to contribute to cell invasion by the bacterium,29,30 induction of apoptosis,31 disruption of polymorphonuclear leukocytes,15,32 and modulation of the proinflammatory response in host cells.33,34 These discoveries suggest that the gingipains are promising targets for the prevention and treatment of periodontal diseases. Indeed, gingipain inhibitors35-37 and antibodies against gingipains38-41 are reported to be effective in suppressing both the colonization by P. gingivalis and its pathogenicity in host environments. “Nutraceuticals” or “functional foods” are defined as foods or dietary components that provide a health benefit beyond the basic function of supplying nutrients. A growing understanding of how diet affects disease, increasing healthcare costs, an aging population, and a rising interest in attaining wellness Journal of Proteome Research 2009, 8, 5165–5174 5165 Published on Web 08/19/2009

research articles through diet have prompted an increasing interest in nutraceuticals. These social and scientific trends have led to investigations of the use of bioactive food constituents in the management of various diseases such as hypertension and cancer. In this study, we set out to discover food ingredients that inhibit Rgp and/or Kgp, which might be applicable to the prevention and management of periodontal diseases through nutraceuticals. We used a targeted proteomics approach and identified Rgp inhibitors in the protein fraction of rice (Oryza sativa L. japonica), which is a staple food in eastern Asia.

Materials and Methods Bacterial Strain, Culture Conditions, and Preparation of Cell Extract. P. gingivalis ATCC 33277 and JCM 8525 were cultivated at 37 °C in brain heart infusion broth (Oxoid Ltd., Hampshire, England) supplemented with yeast extract (5 g/L), hemin (5 mg/L), vitamin K1 (1 mg/L) and cysteine (1 g/L) under anaerobic conditions (AnaeroPack system; Mitsubishi Gas Chemical Co., Tokyo, Japan).42 A cell extract of P. gingivalis ATCC 33277 was used as a Kgp source in an enzyme inhibition assay (see below). Briefly, bacterial cells were collected from an overnight culture by centrifugation (15 000g for 10 min). After washing the cells with a buffer containing 50 mM Tris-HCl (pH 7.4) and 1 mM CaCl2, the cells were suspended in the same buffer. The suspended cells were disrupted with a sonicator (INSONATOR, model 201M, Kubota Co., Tokyo, Japan) and centrifuged at 20 000g for 15 min. The clarified cell extract was stored at -80 °C until use. Purification of Rgp. The 50 kDa Rgp protein was purified from 15 L of P. gingivalis ATCC 33277 culture supernatant using the method of Pike et al.43 Briefly, proteins were precipitated in 75% saturation of ammonium sulfate and then dissolved in 50 mM Tris-HCl (pH 7.4)/150 mM NaCl/5 mM CaCl2. The protein solution was separated by size-exclusion chromatography (Sephadex G-100; GE Healthcare Bio-Science Corp., Piscataway, NJ), and the fractions containing Rgp activity but not Kgp activity were pooled. The pooled fraction was separated by anion-exchange chromatography (TOYOPEARL DEAE-650M; Tosoh Corp., Tokyo, Japan) followed by affinity chromatography (Arginine-Sepharose 4B; GE Healthcare Bio-Science Corp.). The resultant solution contained homogeneous Rgp and no contaminants were detected by denaturing PAGE (polyacrylamide gel electrophoresis). This Rgp solution was concentrated by cation-exchange chromatography (POROS HS/M; Applied Biosystems, Foster City, CA) and stored at -20 °C in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, and 50% glycerol. Enzyme and Inhibitor Assays. The proteolytic activities of Rgp and Kgp were measured using fluorogenic substrates purchased from Peptide Institute, Inc., Osaka, Japan: carbobenzoxy (Z)-Phe-Arg-4-methyl-coumaryl-7-amide (MCA) for Rgp and t-butyloxycarbonyl (Boc)-Val-Leu-Lys-MCA for Kgp. For each assay, an appropriate amount of enzyme preparation was preincubated for 5 min at 40 °C in an assay buffer containing 150 mM NaCl, 5 mM CaCl2, 4 mM dithiothreitol (DTT), 0.05% Brij35, and 100 mM 2-[4-(2-hydroxyethyl)-1piperazinyl]ethanesulfonic acid (HEPES) pH 7.5, with a total volume of 2.0 mL. Then, the fluorogenic substrate was added to the reaction mixture to a final concentration of 50 µM, and the mixture was incubated for 1-5 min at 40 °C. The release of 7-amino-4-methylcoumarin (AMC) was monitored with a 5166

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Taiyoji et al. thermostatted RF-5300PC spectrofluorophotometer (excitation, 380 nm; emission, 440 nm; Shimadzu Co., Kyoto, Japan). The purified Rgp and the P. gingivalis cell extract were used as the enzyme source of Rgp and Kgp, respectively, in the inhibition assays. Inhibitors were preincubated with the appropriate amount of enzyme preparation for 5 min at 40 °C before the addition of the fluorogenic substrate. One unit of inhibition was defined as a reduction in the liberation of 1 µmol of AMC from the fluorogenic substrate per minute. The concentration of active purified Rgp was determined by titration with the specific inhibitor KYT-1 (Peptide Institute Inc.) using Z-Phe-Arg-MCA as the substrate, according to the method of Barrett and Kirschke.35,44 Extraction of Rice Proteins. Dehulled and polished rice (O. sativa L. japonica cv. Koshihikari) was obtained from a local market. One volume of milled rice flour was homogenized in a 10-fold higher volume of 50 mM Tris-HCl (pH 8.0)/0.5 M NaCl at 4 °C, and then centrifuged at 4000g for 20 min. The clarified extract was heated at 80 °C for 10 min to inactivate proteinases and the precipitate was removed by centrifugation at 15 000g for 20 min. Solution-Phase Isoelectric Focusing (IEF). Solution-phase IEF was performed with a ZOOM IEF Fractionator (Invitrogen Co., Carlsbad, CA) according to the manufacturer’s instructions. The fractionator was equipped with four kinds of ZOOM Disks (pH 3.0, pH 5.4, pH 6.2, and pH 10.0). Each rice protein extract was concentrated in 7 M urea/2 M thiourea using an Amicon Ultra-15 filter device (molecular cutoff ) 5000 Da; Millipore Corp., Billerica, MA). The protein solution (about 0.6 mg/mL final concentration) was prepared for focusing in a sample buffer containing 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 1% Zoom Carrier Ampholytes (pH 3-10) (Invitrogen Co.), and 20 mM DTT. The proteins were focused with the following three steps: 100 V for 20 min, 200 V for 80 min, and 600 V for 80 min. Size-Exclusion Chromatography (SEC). SEC was performed with a BioCAD/SPRINT system (Applied Biosystems) equipped with a Superdex 75 HR 10/30 column (GE Healthcare BioScience Corp.). The column was equilibrated with a buffer containing 50 mM HEPES (pH 7.0), 0.15 M NaCl, and 0.05% CHAPS. Samples were fractionated at a flow rate of 0.5 mL/ min. Blue dextran 2000, bovine serum albumin (67.0 kDa), ovalbumin (43.0 kDa), chymotrypsinogen A (25.0 kDa), and ribonuclease A (13.7 kDa) (the low molecular weight Gel Filtration Calibration Kit; GE Healthcare Bio-Science) were used for calibrating the column. Immobilization of Rgp onto Magnetic Particles and Affinity Isolation of Rgp-Binding Proteins. Rgp-binding proteins were isolated from the rice protein extract using a biomagnetic procedure. Rgp was conjugated on superparamagnetic particles (Magnabind amine derivatized beads; Pierce, Rockford, IL) as follows. Five milligrams of the magnetic particles was washed three times with 300 µL of coupling buffer (20 mM HEPES, pH 7.0). The particles were retained with a magnet and the buffer was removed. The particles were then suspended in 100 µL of a solution containing 0.8 mg of purified Rgp in the coupling buffer. After gentle agitation, cross-linker (bis[sulfosuccinimidyl] suberate; Pierce) was added at a final concentration of 1 mM, and the mixture was incubated at 4 °C for 2 h with gentle agitation. After the incubation, Tris-HCl (pH 7.5) was added to a concentration of 50 mM and the solution was incubated for 30 min to stop the cross-linking

Arg-Gingipain Inhibitors from Rice Grain reaction. Finally, the magnetic particles were washed in a solution containing 100 mM HEPES (pH 7.5), 500 mM NaCl, 5 mM CaCl2, 4 mM DTT, and 0.05% CHAPS. The coupling efficiency was 79% (0.13 mg of Rgp/mg of magnetic particles). The rice protein solution, after fractionation by solutionphase IEF and SEC, was dialyzed against a binding buffer consisting of 50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM CaCl2, 4 mM DTT, and 0.05% CHAPS. The protein solution (42 µg/mL in 5 mL) was reduced by incubation at 50 °C for 15 min. The Rgp-conjugated particles (200 µg), which had previously been washed in the binding buffer, were then added. After gentle agitation at 4 °C for 20 min, the magnetic particles were retained with a magnet and the rice protein solution was removed. The particles were then washed three times with the binding buffer, and finally, the proteins bound to the magnetic particles were eluted with 0.1 M glycine-HCl (pH 2.7) containing 4 mM DTT. Two-Dimensional (2D) Gel Electrophoresis and Reverse Zymography. IEF for 2D gel electrophoresis was performed using the ZOOM IPGRunner system (Invitrogen Co.) according to the manufacturer’s instructions. Briefly, the extract was prefractionated by solution-phase IEF and then adjusted to 2.5 mg/mL of protein in a buffer consisting of 7 M Urea, 2 M thiourea, 4% CHAPS, 20 mM DTT, and 0.5% ZOOM Carrier Ampholytes (pH 5-7). A ZOOM dry strip (pH 5.3-6.3; Invitrogen Co.) was rehydrated with the buffer containing 400 µg of protein, and the gel was focused under the conditions described by the manufacturer. After the completion of IEF, the strip was equilibrated and reduced in NuPAGE LDS Sample Buffer containing the NuPAGE Sample Reducing Agent (Invitrogen Co.) and then loaded on a NuPAGE Novex 4-12% BisTris ZOOM Gel (Invitrogen Co.). SDS-PAGE was carried out according to the manufacturer’s protocol using NuPAGE MES SDS running buffer (Invitrogen Co.). Reverse zymography was performed using procedures based on the methods of Katunuma et al.45,46 and Saitoh et al.47 The gel was gently shaken in 2.5% Triton X-100 at room temperature for 30 min to remove SDS, and washed four times with ultrapure water. The washed gel was incubated in 30 mL of Rgp solution containing 1.0 µg/mL of purified Rgp in the enzyme assay buffer mentioned above, at 4 °C for 60 min. A cellulose acetate membrane (pore size, 0.45 µm; dimensions, 7.0 × 8.0 cm; ADVANTEC, Tokyo, Japan) was presoaked in the enzyme assay buffer containing 100 µM Z-Phe-Arg-MCA, then placed on the gel and incubated at 37 °C for 60 min. For visualizing the fluorescent spots representing Rgp inhibitory activity, the cellulose acetate membrane was observed using an ultraviolet (UV) transilluminator (model DT-20LMP, Atto Corp., Tokyo, Japan), and digital fluorescent images were generated with an EOS Kiss DigitalX digital camera (Canon, Inc., Tokyo, Japan) with a UV filter attached (Fuji Filter SC-50; FUJIFILM Co., Tokyo, Japan). After reverse zymography, the 2D gel was stained with SYPRO Ruby protein gel stain (Invitrogen Co.), and a fluorescent image was obtained with a LAS3000 Image Analyzer (FUJIFILM Co.). The digital images from reverse zymography and SYPRO Ruby staining were quantitatively analyzed using the PDQuest version 7.1 software (BioRad Laboratories, Inc., Hercules, CA). In-Gel and In-Solution Digestions. For in-gel digestions, the 2D gel used in SYPRO Ruby staining was next stained with a SilverQuest Silver Staining Kit (Invitrogen Co.), and protein spots of interest were excised manually for identification of the Rgp inhibitors. Each excised gel piece was destained using the

research articles Destainer solution provided in the SilverQuest Silver Staining Kit according to manufacturer’s instructions, and then dehydrated with 100% acetonitrile. Following drying in a centrifugetype vacuum dryer (Spin Dryer Lite VC-36R; TAITEC, Saitama, Japan), the gel piece was reduced in a reaction with 10 mM DTT in 25 mM NH4HCO3 (56 °C, 60 min) and then alkylated with 55 mM iodoacetamide in 25 mM NH4HCO3 (room temperature, 45 min). The gel piece was dehydrated again as described above, then rehydrated by adding 2 µL of trypsin solution (sequence grade modified trypsin; Promega, Madison, WI; 20 µg/mL in 50 mM NH4HCO3). A solution of 25 mM NH4HCO3 (5-20 µL) was added, and the proteins were digested overnight at 37 °C. The proteolysis was stopped by the addition of 2 µL of 5% trifluoroacetic acid (TFA). For in-solution digestions, TFA was added to each protein solution to a final concentration of 0.1% (v/v), and the proteins were captured on a C18 ZipTip (Millipore Corp.). After washing with 0.1% TFA, the proteins were eluted with 70% acetonitrile/ 0.1% TFA and dissolved in 50 mM NH4HCO3, then dried at 60 °C. Next, the proteins were reduced with 0.23 mM DTT at 50 °C for 15 min, alkylated with 0.27 mM iodoacetamide at room temperature for 15 min, and digested overnight with 0.5 µg/ mL trypsin at 37 °C. Mass Spectrometry (MS). Each peptide solution prepared by in-solution digestion was loaded onto an Agilent 1100 capillary LC system (Agilent Technologies, Santa Clara, CA) equipped with an Inertsil Peptides C18 column (0.2 mm i.d. × 15 cm; GL science, Tokyo, Japan). The peptides were separated with a flow rate of 2 µL/min at 45 °C, using a elution pattern consisting of 95% solution A (0.1% TFA) and 5% solution B (80% acetonitrile in 0.1% TFA) for 5 min; a linear gradient from 5 to 50% of solution B over 30 min; and 100% of solution B for 5 min. At 30 s intervals, eluents at the linear stage of the gradient were spotted directly onto prespotted AnchorChip target plates, PAC384 HCCA or PAC96 HCCA (Bruker Daltonik GmbH, Bremen, Germany), using a PROTEINEER fc robot (Bruker Daltonik GmbH). Peptides from in-gel digestions were prepared for MS analysis as follows. An aliquot of the supernatant from each digestion (1 µL) was loaded onto an AnchorChip 600/384 target plate (Bruker Daltonik GmbH) and allowed to dry completely. Then, 0.8 µL of matrix solution (R-cyano-4-hydroxycinnamic acid; 0.5 mg/mL in 90% acetonitrile/0.1% TFA) was applied to the sample spot and left to dry. Subsequently, 2 µL of 0.5% TFA was loaded on the matrix deposit. After 30 s, the remaining solution was removed with a micropipet.48 MS and MS/MS spectra were obtained automatically using an Autoflex III TOF/TOF mass spectrometer controlled by the FlexControl 3.0 and WarpLC (version 1.1.63.1) software (Bruker Daltonik GmbH). MS analysis was initially performed in reflection mode, measuring from 700 to 4000 Da, with each set having a signal-to-noise ratio greater than 6 and a resolution greater than 5000. MS/MS analysis was carried out in LIFT mode with the following settings: ion source 1, 6.00 kV; ion source 2, 5.30 kV; lens, 3.00 kV; reflector 1, 27.00 kV; reflector 2, 11.70 kV; lift 1, 19.00 kV; lift 2, 4.40 kV; reflector detector, 1689 V. Data Analysis and Informatics. Peak lists were generated using the FlexAnalysis 3.0 software (Bruker Daltonik GmbH) with a signal-to-noise threshold of 7. The MS scan was smoothed with the SavitzkyGolay algorithm using a width of 0.15 m/z, and the baseline subtraction was achieved with the TopHat algorithm. Journal of Proteome Research • Vol. 8, No. 11, 2009 5167

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Figure 1. Size-exclusion chromatography of the pI 5.4-6.2 fraction of the rice protein extract, which was obtained by solution-phase IEF. The fraction was applied to a Superdex 75 HR10/30 column equilibrated with a buffer containing 50 mM HEPES (pH7.5), 150 mM NaCl, and 0.05% CHAPS. The flow rate was 0.5 mL/min and the fraction size was 0.5 mL/tube. The fractions that were pooled for biomagnetic separation are indicated with a boldface line. The retention times for the calibration standards are indicated: Al, bovine serum albumin (67.0 kDa); Ov, ovalbumin (43.0 kDa); Ch, chymotrypsinogen A (25.0 kDa); Ri, ribonuclease A (13.7 kDa).

Proteins were identified using MS/MS data to search the O. sativa subset of the National Center for Biotechnology Information nonredundant protein database (NCBInr), released in June 2007. The database was searched using the Biotools 3.1 software (Bruker Daltonik GmbH) and the MASCOT engine version 2.2 (Matrix Science, London, U.K.), run on an in-house server. The parameters used for MASCOT searches were as follows: charge state, 1+; enzyme, trypsin; number of missed cleavage sites, up to 2; fixed modification, carbamidomethyl; variable modifications, oxidation and deamidation; tolerance for MS peaks, 150 ppm; tolerance for MS/MS peaks, 0.8 Da. Sequence alignments of identified proteins were generated using the Parallel Protein Information Analysis (PAPIA) system (http://mbc.cbrc.jp/papia/papia.html), which was constructed by the Computational Biology Research Center, National Institute of Advance Industrial Science and Technology (Tsukuba, Japan). The following default settings were used: BLOSUM62; Opening gap, 16; Extension gap, 2; Out gap, 2.49 The signal peptides of the identified proteins were predicted by a Hidden Markov model using a SignalP program (version 3.0; http://www.cbs.dtu.dk/services/SignalP/).50 The molecular weights (MW) and isoelectric points (pI) were calculated based on the coding sequences excluding the signal peptides, using the GENETYX-Mac software (version 14.0.5; GENETYX Co., Tokyo, Japan). Peptide Synthesis and Other Analysis. Two peptides, RA17_a7 and CH_a12, were synthesized by Sigma-Genosys (Ishikari, Japan). Protein concentrations were determined using a BCA protein assay kit (Pierce) with bovine serum albumin as the standard. Growth Inhibition Assay. Growth inhibition assays were performed in 96-well, flat-bottomed tissue culture plates. Each well was inoculated with 1.2 × 106 viable cells of P. gingivalis JCM 8525 suspended in modified GAM broth (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan), with or without the synthetic peptides. The plates were incubated at 37 °C under anaerobic conditions. Bacterial growth was monitored by measuring the optical density at 655 nm.

Results Rgp/Kgp Inhibitory Activities of Rice Proteins. We searched for constituents of foods with inhibitory activities against P. 5168

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gingivalis cysteine proteinases, and found that rice protein extracts inhibited both Rgp and Kgp. The specific inhibitory activities (units per mg of protein) of the rice grain extract were 6.2 mU/mg against Rgp and 0.3 mU/mg against Kgp. Because the specific activity against Rgp was 20-fold higher than that against Kgp, we attempted to purify and identify the Rgp inhibitors from rice grain in this study. The Rgp inhibitory activity showed broad elution patterns after conventional chromatographic separations such as anion- and cationexchange chromatography and hydrophobic chromatography, and enrichment of the inhibitory activity could only be achieved using SEC (data not shown). These results suggested that the inhibitory activity did not originate from a single molecular species. Enrichment of Rgp Inhibitory Activity. The failure to isolate Rgp inhibitors by chromatographic methods prompted us to apply a targeted proteomics strategy. For the identification of Rgp inhibitors by mass spectrometry, we enriched the inhibitors using a combination of solution-phase IEF, SEC, and biomagnetic separation. First, the rice protein extract was separated by solution-phase IEF into fractions with pI values of 3.0-5.4, 5.4-6.2, and 6.2-10.0. The Rgp inhibitory activities of the fractions were 0.9 mU (0.3%) for pI 3.0-5.4; 240.3 mU (77.5%) for pI 5.4-6.2; and 68.9 mU (22.2%) for pI 6.2-10.0. Because the majority of the inhibitory activity was recovered in the pI 5.4-6.2 fraction, this fraction was further separated by SEC (Figure 1). SEC yielded three peaks of inhibitory activity, with the bulk of the activity in fractions containing proteins with molecular masses of approximately 13-42 kDa. This major peak was subjected to biomagnetic separation. Identification of Rgp Binding Proteins. Rgp-binding proteins that were present in the major active peak from the SEC were captured onto Rgp-conjugated magnetic beads and identified by LC-MALDI TOF/TOF, following in-solution trypsin digestion. A MASCOT search identified 46 proteins with scores higher than 40 (p < 0.05). Of these, 20 proteins included at least one reliable peptide match (rank 1 and individual peptide score higher than 40). Three proteins were excluded: the NCBInr accession number 1304217 whose gene was not found in rice genome sequence (Build 4) released by The International Rice Genome Sequencing Project; a hypothetical protein OsI_036991 originated from the indica cultivar group (accession no.

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Arg-Gingipain Inhibitors from Rice Grain Table 1. Arg-Gingipain Binding Proteins Identified by LC-MALDI TOF/TOF scorea

MPb

1

946

15

115471167

2 3 4 5 6 7 8 9 10

729 462 349 322 236 212 198 193 98

11 11 4 8 4 5 3 5 2

41469581 115464709 115471171 114152827 115455865 115461925 115463191 115471173 115471187

11 12 13 14 15 16

97 94 75 56 55 53

2 3 2 1 1 1

17

52

1

115470671 115482468 115471201 115457104 115483198 115452975 115454509 62733815

no.

accession no.

c

predicted structure and/or function

Plant lipid transfer/seed storage/trypsin-alpha amylase inhibitor domain containing protein Putative globulin (with alternative splicing) 26 kDa Globulin (alpha-globulin) Seed allergenic protein RA17 precursor Bowman-Birk type bran trypsin inhibitor precursor Globulin 2 (fragment) Nitrogen regulator protein P-II Manganese-superoxide dismutase precursor Seed allergenic protein RAG2 precursor Cereal seed allergen, trypsin/alpha-amylase inhibitor family protein E1 protein and Def2/Der2 allergen family protein Cyanate hydratase Alpha-amylase/trypsin inhibitor (RBI) (RATI) Lectin precursor (Agglutinin) EC protein I/II (Zinc-metallothionein class II) 40S ribosomal protein S21 40S ribosomal protein S21 Hypothetical protein, LOC_Os11g26400

MW (Da)

d

pId

13000

5.30

61109 18903 15129 25385 48871 20211 22393 15237 14069

6.10 6.47 6.54 5.11 6.29 10.30 5.80 7.43 7.38

13994 18596 13163 20156 8487 9248 13172 19950

4.94 5.54 6.80 5.65 7.15 7.29 5.75 11.79

a Mascot MS/MS ion search score. b Number of peptides that matched the predicted protein sequence. c Accession number in the NCBI nonredundant database. d The signal sequence was predicted by a Hidden Markov model using a SignalP program (version 3.0, http://www.cbs.dtu.dk/services/SignalP/). The molecular weights (MW) and isoelectric points (pI) were calculated based on the coding sequences excluding the signal sequences, using the GENETYX-Mac software (version 14.0.5).

125536544); and one protein was allocated to a putative transposable element (accession no. 21671931). The remaining 17 proteins were judged to be Rgp-binding proteins derived from the grain of the japonica rice cultivar (Table 1). Protein 16 in Table 1 was assigned to NCBInr accession no. 115452975 and/or 115454509, both of which are annotated as 40S ribosomal protein S21. The same single matched peptide was found in both of these proteins. The detailed list of the identified proteins is shown in Supplementary Table A in Supporting Information. Inhibition of Rgp/Kgp Activity and P. gingivalis Growth by Peptides Derived from the Rgp-Binding Proteins. We identified the 17 Rgp-binding proteins in the protein fraction that was concentrated for Rgp inhibitory activity, in the pI range 5.4-6.2 and the size range of approximately 13-42 kDa. Ten proteins whose pI and molecular weight values were close to or within these ranges were selected for a comparison of their predicted mature protein sequences. Although the predicted functions of these 10 proteins were varied (Table 1), several regions of the sequences showed similarities (Figure 2), suggesting that these regions may be involved in interactions with Rgp. In particular, a region near the N-terminus (amino acids 30-59 in the alignment) is likely to be exposed at the molecular surface and therefore has the potential to interact with Rgp. Evidence for this comes from a report that rabbit antiserum against the allergenic protein RA17 (NCBInr accession no. 115471171) recognized this N-terminal region,51 indicating that it is exposed at the molecular surface. To examine whether the sequences within this N-terminal region interact with Rgp, we selected RA17 and cyanate hydratase (NCBInr accession no. 115482468) from among the Rgp-binding proteins and synthesized two peptides based on the sequences of their N-terminal regions. The RA17_a7 heptapeptide (RTLVRRQ) and the CH_a12 dodecapeptide (RRLMAAKAESRK) correspond to residues 50-56 of RA17 and residues 33-48 of cyanate hydratase, respectively (Figure 2). These peptides contain Lys and/or Arg residues, which are important for recognition by Rgp.35 Indeed, the

RA17_a7 and CH_a12 peptides inhibited Rgp activity, with specific inhibitory activities of 3.83 and 11.68 U/mmol, respectively (Figure 3). These results imply that the N-terminal regions of the Rgp-binding proteins are involved in interacting with Rgp and inhibiting its activity. CH_a12 also showed inhibitory action against Kgp (4.13 U/mmol), indicating that this peptide acts as a dual inhibitor. It was reported that both Rgp and Kgp are essential for the extracellular degradation of proteins to produce peptides and amino acids needed for growth by P. gingivalis,20 and that Kgp is particularly important for its growth.35 Therefore, we examined the effect of CH_a12 on the growth of P. gingivalis and found that the peptide inhibited bacterial growth in a dosedependent manner (Figure 4). This suggests that the Rgp/Kgp inhibitory proteins of rice may exhibit antimicrobial functions against P. gingivalis. Identification of Major Rgp-Inhibitory Proteins Using 2D Gel System of Double-Layer Reverse Zymography. To examine whether the Rgp-binding proteins could inhibit Rgp activity, and to estimate the relative contribution of each Rgpbinding protein to the total Rgp inhibitory activity in the rice protein extract, we used a 2D gel system of double-layer reverse zymography. We analyzed rice proteins in the pI range 5.4-6.2 that had been fractionated by solution-phase IEF. The analysis by 2D gel electrophoresis and reverse zymography was repeated over five times, and we obtained the consistent patterns with minor variations in relative spot intensities. The representative separation pattern of proteins after 2D gel electrophoresis is shown in Figure 5A, and the reverse zymogram is shown in Figure 5B. On the reverse zymogram, enzyme inhibition could be visualized as dark spots on the bright blue background produced by hydrolysis of the peptide-MCA substrate. Eleven Rgp-inhibitory spots were detected. Spots numbered 1, 7, and 11 in Figure 5B had no corresponding protein spots in Figure 5A, suggesting that these Rgp-inhibitory activities were due to particularly minor but highly active components of the rice protein fraction. Spots numbered 8 and 9 on the reverse Journal of Proteome Research • Vol. 8, No. 11, 2009 5169

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Figure 2. Comparison of amino acid sequences of selected Rgp-binding proteins. Each protein is indicated using its number shown in Table 1. The alignment was generated using the PAPIA system (http://mbc.cbrc.jp/papia/papia.html), and the figure was prepared using the BOXSHADE program (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form)boxshade). Black and gray shading indicate identical and similar residues, respectively. The sequences corresponding to the peptides synthesized for assessment of inhibitory activities against Arg- and Lys-gingipains and the growth of P. gingivalis are underlined. The predicted function of each protein is as follows: no. 1, Plant lipid transfer/seed storage/trypsin-R amylase inhibitor domain containing protein; no. 3, 26 kDa Globulin; no. 4, Seed allergenic protein RA17; no. 5, Bowman-Birk type bran trypsin inhibitor; no. 8, Manganese-superoxide dismutase; no. 11, E1 protein and Def2/Der2 allergen family protein; no. 12, Cyanate hydratase; no. 13, Alpha-amylase/trypsin inhibitor; no. 14, Lectin (Agglutinin); no. 16, 40S ribosomal protein S21.

zymogram corresponded with two and four protein spots, respectively, and the others matched single spots in Figure 5A. The spot intensities in the 2D gel and reverse zymogram were quantified, and the results are shown in Table 2. The spot quantities shown for the reverse zymogram represent the relative contribution of each Rgp-inhibitory protein to the total 5170

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inhibitory activity in the rice protein preparation. The relative specific activity of each Rgp-inhibitory protein was expressed as the ratio of its inhibitory spot quantity to the quantity of its counterpart in the stained protein gel (i.e., the Rz/Ps value in Table 2). On the reverse zymogram, spots 9 and 10 accounted for 51.1% and 20.5% of the total inhibitory activity, respectively.

Arg-Gingipain Inhibitors from Rice Grain

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Figure 3. Inhibitory activities of the synthetic peptides RA17_a7 and CH_a12 against Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Appropriate amounts of enzyme preparation (Rgp, 12.4 ng of purified enzyme; Kgp, 69 µg of cell-extract protein) were preincubated for 5 min at 40 °C in a total volume of 2.0 mL of assay buffer containing 100 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM CaCl2, 4 mM DTT and 0.05% Brij35. The fluorogenic substrate (Z-Phe-Arg-MCA for Rgp and Boc-Val-Leu-Lys-MCA for Kgp) was added to the reaction mixture to a final concentration of 50 µM; then, the mixture was incubated for 0-2 min at 40 °C. The release of AMC was monitored as the change in fluorescence intensity. (A) Rgp inhibition. Symbols: closed circle, enzyme only; open circle, 14.2 µM CH_a12; open square, 21.6 µM RA17_a7. (B) Kgp inhibition. Symbols: closed circle, enzyme only; open circle, 14 µM CH_a12; open triangle, 35 µM CH_a12; open square, 107 µM RA17_a7.

Figure 4. The CH_a12 peptide derived from cyanate hydratase inhibits the growth of P. gingivalis in a dose-dependent manner. P. gingivalis cells (1.2 × 106 per well) were suspended in modified GAM broth and cultured in 96-well, flat-bottomed tissue culture plates at 37 °C under anaerobic conditions, with or without the synthetic peptides. Growth was monitored by measuring the optical density at 655 nm. Symbols: closed circle, no peptide; open circle, 5 µM CH_a12; open square, 20 µM CH_a12; open triangle, 100 µM CH_a12.

A comparison of Rz/Ps values indicated that spot 10 had the highest specific activity, followed by spot 9. Furthermore, the in-gel digestion and MALDI TOF/TOF analysis enabled us to identify four of these proteins (Supplementary Table B in Supporting Information), all of which had also been identified as Rgp-binding proteins (Table 1). Spots 2, 4, and 5 on the reverse zymogram were assigned to the 26 kDa globulin, and the largest protein spot (no. 9-1) was identified as a plant lipid transfer/trypsin-R amylase inhibitor. Inhibitory spot no. 10, which showed the highest specific activity, comprised two proteins: the seed allergen RA17 and an R-amylase/trypsin inhibitor. The sum of the inhibitory values corresponding to these four proteins accounted for 90% of the total. Therefore, these four Rgp-binding proteins can actually inhibit Rgp activity, and are responsible for the majority of the Rgpinhibitory activity in the rice protein fraction.

Discussion Many previous reports have indicated that the cysteine proteinases Rgp and Kgp play critical roles in the growth and pathogenesis of the oral pathogen P. gingivalis. The physiological functions of these enzymes include acquisition of carbon sources and iron for bacterial growth via degradation of host proteins, and destruction of host tissues. Therefore,

potent inhibitors against these proteinases might be promising for the treatment and prevention of diseases caused by P. gingivalis. Several inhibitors have been reported. For example, Kadowaki et al. developed KYT-1 and KYT-36 as small, specific inhibitors of Rgp and Kgp, based on the cleavage specificities of these enzymes on histatins.35 When combined, these inhibitors strongly suppressed the virulence activities of P. gingivalis, such as the degradation of host proteins, disruption of the bactericidal activity of polymorphonuclear cells, and enhancement of vascular permeability. When used alone, KYT-1, which is specific for Rgp, suppressed the bacterial effects on the adhesiveness and viability of human fibroblasts. These findings confirm the effectiveness of gingipain inhibitors against P. gingivalis infection. To develop nutraceuticals that are applicable to the prevention and management of periodontal diseases, we searched for Rgp/Kgp inhibitory activities among food plant materials and found these activities in a protein fraction from rice grain. A combination of affinity separation and mass spectroscopic analyses suggested that the Rgp inhibitory activity was attributable to several different proteins (Table 1). Interestingly, cysteine proteinase inhibition is a previously unknown function of these rice proteins, and no known cysteine proteinase inhibitors were detected in this study. Martı´nez et al. identified 12 nonredundant rice genes (OC-I to OC-XII) that encode members of the cystatin superfamily.52 Of these, OC-I, OC-VIII and OC-XII are expressed in rice seeds based on expressed sequence tag expression data.52 However, we found that OC-I and OC-XII each have little or no inhibitory activity against Rgp and Kgp (data not shown). These results suggest that the Rgp and Kgp inhibitory activities in the rice seed are not due to these cystatin family members. It is unlikely that the failure to detect cystatins as Rgp-interacting proteins was a false negative in this study. An alignment of cystatin sequences has identified three regions that were conserved during molecular evolution: a glycine residue in the N-terminal region, a QxVxG motif in one hairpin loop, and a PW motif in a second binding loop. Of these regions, the QxVxG motif is found in almost all members of the cystatin superfamily, and contributes to the interaction between the cystatin and its target enzymes.53,54 It has been reported that proteins containing the QxVxG sequence but not the other motifs conserved in the cystatin superfamily, such as lactoferrin and lipocalin from the human Von Ebner’s gland, Journal of Proteome Research • Vol. 8, No. 11, 2009 5171

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Figure 5. Separation of proteins and detection of Arg-gingipain (Rgp) inhibitors in a rice protein fraction isolated by solution-phase isoelectric focusing. (A) Image of the 2D gel after SYPRO Ruby staining. The spots found within areas corresponding to active spots on the reverse zymogram are numbered for comparison with the image obtained by 2D reverse zymography. (B) The reverse zymogram showing Rgp inhibitory proteins. The active spots are marked and numbered. Table 2. The Analysis of Gel Images Obtained by SYPRO Ruby Staining and Reverse Zymography reverse zymography [Rz]a spot

quantity

b

%

protein staining [Ps]d

c

spot

quantityb

%c

82822928 12671359 19854732 17433614 7444207 3788510 1216512 41434952 3120171 7918941 2386258 2911257

40.8 6.2 9.8 8.6 3.7 1.9 0.6 20.4 1.5 3.9 1.2 1.4

-

1 2 3 4 5 6 7 8

871 7102 1362 3166 4305 1189 495 2392

1.1 9.0 1.7 4.0 5.4 1.5 0.6 3.0

9

40502

51.1

10

16248

20.5

2 3 4 5 6 8-1 8-2 9-1 9-2 9-3 9-4 10

11

1574

2.0

-

Rz/Pse (× 10-6)

proteins identified by MALDI TOF/TOFf

85.7 107.5 159.5 246.9 159.7 477.9

26 kDa Globulin (115464709) 26 kDa Globulin (115464709)

738.3

Plant lipid transfer/ trypsin-R amylase inhibitor (115471167)

5581.1

Seed allergen RA17 (115471171) R-amylase/trypsin inhibitor (115471201)

-

26 kDa Globulin (115464709)

-

a The analysis of the two-dimensional reverse zymography image shown in Figure 5B. b Spot quantities were obtained using the PDQuest software version 7.1 (Bio-Rad Laboratories). c The percentage values represent the proportion of each spot quantity in the sum of quantities. d The analysis of the two-dimensional gel image revealed by SYPRO Ruby fluorescence staining shown in Figure 5A. e Rz/Ps values were calculated by dividing the quantity of an active spot on the reverse zymogram by the total quantity of protein spots within the corresponding area on the stained 2D gel. f Proteins were identified by in-gel digestion and MALDI TOF/TOF analysis. The NCBInr accession number is in parentheses.

have inhibitory activity against cysteine proteinases.55,56 However, none of the Rgp-binding proteins identified in this study contained the QxVxG motif, indicating that other sequences are responsible for the Rgp/Kgp inhibitory activities of these proteins. This assumption is supported by the finding that the synthetic peptides RA17_a7 and CH_a12, derived from the N-terminal regions of the Rgp-binding proteins RA17 and cyanate hydratase, showed Rgp/Kgp inhibitory activities. The precise inhibitory sequences must be identified by further investigation. In this study, a 2D gel system of double-layer reverse zymography was used to estimate the relative contribution of each Rgp-binding protein to the total Rgp inhibitory activity of the rice protein extract. We previously developed 2D gel systems for zymography and reverse zymography, and reported that these methods are useful for detecting and characterizing proteases and protease inhibitors in biological samples.47 In those studies, we employed SDS-PAGE gels that were copolymerized with gelatin as the proteinase substrate for separa5172

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tion in the second dimension. On the other hand, Katunuma et al. developed a 1D double-layer zymographic method using specific fluorescent substrates to detect specific proteinases.46 In their method, the target proteinase was separated using SDSPAGE, and the gel was covered with a cellulose acetate membrane presoaked in the fluorescent enzyme substrate solution (“double-layer”). The location of the target proteinase on the gel could be visualized as a fluorescent band on the cellulose acetate membrane after incubation under appropriate conditions. This double-layer system would be suitable for identifying the protein from the active band using a combination of in-gel digestion and MS analysis, because this system does not require the co-polymerization of the polyacrylamide gel with large amounts of protein (such as 0.1% gelatin). Therefore, we applied this double-layer system to our protocol of 2D reverse zymography in order to identify the Rgpinhibitory proteins in the rice protein fraction. Of these, the inhibitory spot due to the seed allergen RA17 and the R-amylase/trypsin inhibitor had the highest specific activities for Rgp

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Arg-Gingipain Inhibitors from Rice Grain inhibition (Table 2). These proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases. Our results also indicate that the combination of 2D reverse zymography and MS analysis is a valuable tool for comprehensive studies of proteinase inhibitors with varying degrees of activity in biological samples. In summary, we used a targeted proteomics approach to detect Rgp-interacting proteins in rice endosperm, and identified four proteins that together account for the majority of the Rgp inhibitory activity in the rice protein fraction. These proteins were difficult to identify using conventional purification-based approaches. Our results imply that the targeted proteomics approach is a powerful tool for investigating bioactive food constituents. Among the Rgp-inhibitory proteins, there were no known cysteine proteinase inhibitors, and cysteine proteinase inhibition is a novel function of these proteins. These results suggest that many more proteins than have been identified to date may play roles as proteinase inhibitors in vivo. The rice proteins that we identified may be useful as the nutraceutical ingredients for the treatment and/ or prevention of periodontal diseases. The authors are now investigating the functions of these Rgp inhibitory proteins in relation to the suppression of pathogenicity and growth of P. gingivalis.

Acknowledgment. The authors thank Professor Kenji Yamamoto (Kyushu University) for valuable discussion. Note Added after ASAP Publication. This article was published ASAP on September 1, 2009. A unit was modified in the Methods Section. The correct version was published on November 6, 2009. Supporting Information Available: Supplementary tables provide detailed lists of the proteins identified by MS/ MS analyses in this study. This material is available free of charge via the Internet at http://pubs.acs.org.

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