Contribution of Cationic Amino Acids toward the Inhibition of Arg-Specific Cysteine Proteinase (Arg-gingipain) by the Antimicrobial Dodecapeptide, CL(14–25), from Rice Protein Masayuki Taniguchi,1,2 Yoshiyasu Matsuhashi,1 Takako K. Abe,1 Yohei Ishiyama,3 Eiichi Saitoh,4 Tetsuo Kato,5 Akihito Ochiai,1 Takaaki Tanaka1 1
Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan 2
Center for Transdisciplinary Research, Niigata University, Niigata 950-2181, Japan
3
Center for Fostering Innovative Leadership, Niigata University, Niigata 950-2181, Japan
4
Graduate School of Technology, Niigata Institute of Technology, Niigata 945-1195, Japan
5
Department of Chemistry, Tokyo Dental College, Tokyo 101-0062, Japan
Received 18 February 2014; revised 11 June 2014; accepted 28 June 2014 Published online 21 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22525
ABSTRACT: CL(14–25), a dodecapeptide, exhibits antimicrobial activity against Porphyromonas gingivalis with the 50% growth-inhibitory concentration (IC50) value of 145 mM, and arginine-specific gingipain (Rgp)-inhibitory activity. Kinetic analysis revealed that CL(14–25) is a mixed-type inhibitor, with inhibition constants (Ki and Ki0 values) of 1.4 3 1026 M and 4.3 3 1026 M, respectively. To elucidate the contributions of four cationic amino acid resi-
almost as high as that of CL(14–25). Rgp-inhibitory activities of alanine-substituted analogs, CL(R14A) and CL(R14A, R15A) also significantly decreased, whereas those of CL(K25A) and CL(R24A, K25A) were higher than that of CL(14–25). These results suggest that the arginine residue at position 15 substantially contributes to the Rgp-inhibitory activity and that the arginine residue at position 14 plays important roles in exerting Rgpinhibitory activity. In this study, we demonstrated that CL(K25A) was a potent, dual function, peptide inhibitor
dues at the N- and C-termini of CL(14–25) toward Rgp-
candidate, exhibiting Rgp-inhibitory activity with Ki and
inhibitory activity, we investigated the Rgp-inhibitory
Ki0 of 9.6 3 1027 M and 1.9 3 1026 M, respectively, and
activities of truncated and alanine-substituted analogs of
antimicrobial activity against P. gingivalis with an IC50
CL(14–25). Rgp-inhibitory activities significantly
C 2014 Wiley Periodicals, Inc. value of 51 mM. V
decreased by truncated analogs, CL(15–25) and CL(16–
Biopolymers (Pept Sci) 102: 379–389, 2014.
25), whereas those of CL(14–24) and CL(14–23) were
Keywords: Arg-gingipain inhibitor; antimicrobial peptide; inhibition in a mixed manner; bifunctional peptide;
Additional Supporting Information may be found in the online version of this article. Correspondence to: Masayuki Taniguchi, Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan; e-mail: [emailprotected] Contract grant sponsor: KAKENHI, the Ministry of Education, Culture, Sports, Science and Technology of Japan Contract grant number: 23560939 Contract grant sponsor: Development of Fundamental Technology for Analysis and Evaluation of Functional Agricultural Products and Functional Foods C 2014 Wiley Periodicals, Inc. V
PeptideScience Volume 102 / Number 5
cationic peptide
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INTRODUCTION
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hronic periodontitis is one of the most common infectious diseases that results in the destruction of periodontal tissues. Proliferation of specific Gramnegative bacteria in subgingival dental plaque causes a chronic inflammatory response, leading to tooth 1,2 loss. In addition, chronic periodontitis has recently been associated with an increased risk of systemic diseases such as atherosclerosis,3,4 diabetes,5,6 respiratory diseases,7 and pancreatic cancer.8 Porphyromonas gingivalis, a Gram-negative, obligate anaerobic, and asaccharolytic black pigmented bacterium, is a major etiological agent in the onset and progression of chronic periodontitis.9,10 The main virulence factors of P. gingivalis are two-types of cysteine proteinases with specificity for arginine–Xaa or lysine–Xaa peptide bonds, commonly referred to as arginine-(Rgp) and lysine-(Kgp) gingipains, respectively. These enzymes are essential for the proliferation and survival of the bacterium in vitro and in vivo11,12 and play crucial roles in the acquisition of nutrients and evasion of host defenses.13,14 Gingipains have also been implicated in adhesion to host tissue,15 hemagglutination,16 processing of bacterial cell-surface and secretory proteins,17 induction of apoptosis,18 disruption of polymorphonulcear leukocytes,19 and modulation of the proinflammatory response in host cells.20 Rgp and Kgp, which have low sequence similarity to other cysteine proteinases, contribute to the degradation of host defense peptides.21–23 These observations suggest the importance of both P. gingivalis and gingipains as targets for the management of periodontal diseases. A number of human proteins, peptides, and cysteine proteinase inhibitors exhibit, to some extent, antimicrobial activity against P. gingivalis and/or inhibit cysteine proteinase activity. They play important roles in innate immunity of the oral cavity. Human b-defensin 3 is widely expressed in oral tissues such as the oral epithelium and salivary glands and is found in saliva and gingival crevicular fluids.24,25 LL-37 is a 37 amino acid peptide derived from the C-terminal end of an 18 kDa human cationic antimicrobial protein.26 It has been previously reported that a deficiency of LL-37 in patients with morbus Kostmann syndrome is correlated with severe periodontitis.27 Histatins comprise a family of small, cationic, histidine-rich peptides of 3–4 kDa present in human saliva. Histatins are constitutively produced and secreted by the submandibular, sublingual, and parotid glands.21,22 Salivary histatins inhibit cysteine proteinase activity and inhibit the proliferation of P. gingivalis.28,29 The cystatins S, SA, and SN, all originally isolated from saliva, are cysteine proteinase inhibitors.30,31 In addition to these endogenous bioactive compounds, there have been many molecules, both synthetic and naturally derived,
that inhibit the proliferation of P. gingivalis and/or the activity of gingipains. Kappacin from bovine milk is an anionic antimicrobial peptide that exhibits bactericidal activity against P. gingivalis.32 Because kappacin does not contain the basic amino acid residues (arginine and lysine) it is resistant to proteolytic activity of gingipains which limits the effectiveness of cationic antimicrobial peptides.33 Recently, a peptide that corresponds to the amino acid residues 109–137 of j-casein, a partial amino acid sequence of kappacin, has been found to inhibit cysteine proteinases of P. gingivalis.34 Several synthetic gingipain inhibitors have already been reported, including tetracycline analogs,35 chlorhexidine,36 leupeptin,18 N-a-p-tosyl-Llysine-chloromethyl ketone,37 and the small peptide analogs, KYT-1 [carbobenzoxy-Lys-Arg-CO-Lys-N-(CH3)2] and KYT-36 [carbobenzoxy-Glu(NHN(CH3)Ph)-Lys-CO-NHCH2Ph] designed on the basis of cleavage specificity of histatin.38 Natural gingipain inhibitors discovered to date include melabaricone C, isolated from nutmeg,39 and polyphenolic compounds derived from plants, such as cranberry,40 green tea,41 apple,42 and hops.42 FA-70C1, isolated from the culture supernatant of Streptomyces species strain FA-70, is a potent Rgp inhibitor and also exhibits growth-inhibitory activity against P. gingivalis.43 Antimicrobial activity of major gingipain inhibitors against P. gingivalis depends on their ability to repress acquisition of peptides and amino acids by degradation of exogenous proteins in the medium, rather than that on the direct killing activity. Therefore, gingipain inhibitors exhibit little or no antimicrobial activity in media, and probably in saliva, that contain peptides and amino acids. However, recent studies have demonstrated that bovine lactoferrin exhibits inhibitory activities against Rgp and Kgp, slight inhibitory effect on the planktonic growth of P. gingivalis, and a substantial inhibitory effect on biofilm formation.44,45 An ideal approach for the management of periodontal diseases is to develop inhibitors that target both P. gingivalis and gingipains. We recently reported that a potent dodecapeptide [CL(14–25), RRLMAAKAESRK; Table I], derived from a region (residues 14–25) near the N-terminus of cyanate lyase (CL, EC 4.3.99.1, GenBank ID: Os10g0471300) from rice (Oryza sativa L. japonica), inhibits both Rgp and Kgp activities and the growth of P. gingivalis.46 In previous studies,47,48 we compared the bactericidal activity of truncated and alanine-substituted CL peptide analogs against P. gingivalis and investigated the contribution of cationic amino acids, including arginine and lysine, to the antimicrobial activity of CL(14–25). In the present study, to elucidate the contribution of two arginine residues at the N-terminus and lysine and arginine residues at the C-terminus of CL(14–25) toward proteinaseinhibitory activity, we synthesized four types of truncated CL peptide analogs: [CL(15–25), CL(16–25), CL(14–24), and Biopolymers (Peptide Science)
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Table I The Amino Acid Sequences and Properties of Peptides Used in This Study Molecular Mass (Da) Peptide Parent Truncated Analogs
AlanineSubstituted Analogs a
Peptide Name
Sequence
Size (a.a.)
Net Charge
Calculated
Measureda
CL(14–25) CL(15–25) CL(16–25) CL(14–24) CL(14–23) CL(R14A) CL(R14A, R15A) CL(K25A) CL(R24A, K25A)
RRLMAAKAESRK -RLMAAKAESRK –LMAAKAESRK RRLMAAKAESRRRLMAAKAES– ARLMAAKAESRK AALMAAKAESRK RRLMAAKAESRA RRLMAAKAESAA
12 11 10 11 10 12 12 12 12
14 13 12 13 12 13 12 13 12
1416.85 1260.65 1104.45 1288.66 1132.46 1331.74 1246.63 1359.75 1274.64
1416.72 1260.54 1104.35 1288.55 1132.36 1331.60 1246.51 1359.63 1274.52
The values indicate molecular mass of each purified peptide confirmed by MALDI-TOF analysis.
CL(14–23); Table I] and four types of alanine-substituted CL peptide analogs: [CL(R14A), CL(R14A, R15A), CL(K25A), and CL(R24A, K25A); Table I] and examined their Rgpinhibitory activities, Rgp being a primary pathogenic determinant of P. gingivalis. Furthermore, to identify a potent peptide inhibitor targeting both Rgp and P. gingivalis, we discussed the differences between the contributions of arginine and lysine residues of CL(14–25) to Rgp-inhibitory and antimicrobial activity against P. gingivalis.
size-exclusion chromatography on a Sephadex G-100 column (GE Healthcare Bio-Science, Piscataway, NJ). Fractions with Rgp activity were pooled and were subsequently fractionated by anion-exchange chromatography using a Toyopearl DEAE-650M column (Tosho, Tokyo, Japan), followed by affinity chromatography using arginineSepharose 4B beads (GE Healthcare Bio-Science). Homogeneity of the resulting Rgp was confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The purified Rgp solution was stored at 220 C until use in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM CaCl2, and 50% glycerol.
Rgp Activity and Peptide-Inhibition Assay
MATERIALS AND METHODS Peptides Amino acid sequences and properties of CL peptides used in this study are summarized in Table I. The amino acid sequence of CL(14– 25) is identical to that of the CH peptide reported by Taiyoji et al.46 Chemically synthesized CL peptides, including CL(14–25), CL(15– 25), CL(16–25), CL(14–24), CL(14–23), CL(R14A), CL(K25A), CL(R14A, R15A), and CL(R24A, K25A) were obtained from Hokkaido System Science, Ltd. (Sapporo, Japan). Synthetic peptides were purified to >95% by reversed-phase high-performance liquid chromatography (RP-HPLC). Molecular weights of purified CL peptides were confirmed by matrix-assisted laser/desorption ionization–timeof-flight mass spectroscopy (MALDI–TOF-MS).
The proteolytic activity of purified Rgp was measured using the fluorogenic substrate, carboxybenzoxy (Z)-Phe-Arg-4-methyl-coumaryl-7amide (Z-Phe-Arg-MCA; Peptide Institute, Osaka, Japan). In brief, the purified Rgp solution was diluted to 0.6 mg protein/ml in the assay buffer (100 mM HEPES at pH 7.5, 150 mM NaCl, 5 mM CaCl2, 4 mM dithiothreitol, and 0.05% Brig 35) and preincubated for 15 min at 37 C. The peptide solution was added to the assay buffer at various concentrations and preincubated for 5 min at 37 C. Rgp solution (10 ml) was added to the peptide solution (1980 ml) or the assay buffer (1980 ml) as a control without inhibitor and the reaction mixture was further preincubated for 1 min at 37 C. Then, the fluorogenic substrate (10 ml) was added to the reaction mixture at different concentrations. Release of 7amino-4-methylcoumarin (AMC) was monitored for 1–5 min by measuring the fluorescence intensity at an excitation wavelength of 380 nm and emission wavelength of 440 nm using a thermostated spectrofluorometer (PF-5300PC, Shimadzu, Kyoto, Japan). Reaction velocities were determined from the amount of AMC produced per minute.
Preparation and Purification of Rgp P. gingivalis ATCC 33277 was cultivated at 37 C in brain heart infusion broth (Oxoid, Hampshire, England) supplemented with 5 g/l yeast extract, 5 mg/l hemin, 1 mg/l vitamin K, and 1 g/l cysteine under anaerobic conditions in the presence of a deoxygenating reagent (AnaeroPack A03; Mitsubishi Gas Chemical, Tokyo, Japan). Rgp (50 kDa) was purified from 15 l of P. gingivalis culture supernatant as reported previously.46 In brief, proteins in the supernatant were precipitated by 75% saturation of ammonium sulfate and were then dissolved in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 5 mM CaCl2. This protein solution was fractionated by
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Determination of Kinetic Parameters To determine kinetic parameters, reaction velocities (V) were measured as described above using the substrate Z-Phe-Arg-MCA at various concentrations. The velocities obtained were analyzed using a Lineweaver– Burk plot (double-reciprocal plot), in which the kinetic data were plotted as 1/velocity (1/V) versus 1/substrate concentration (1/S). The Michaelis constant (Km) and the maximum velocity (Vmax) were determined by varying substrate concentrations in the reaction mixture without peptide as an inhibitor. The inhibitory activity of each peptide
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Taniguchi et al. was examined by measuring V in the presence of different concentrations of peptides. Kinetic data were analyzed using double-reciprocal plots, and the mode of inhibition of each peptide was evaluated on the basis of the intrinsic property of a family of lines with different slopes and intercepts. When Rgp activity was competitively inhibited by the peptide, the inhibition constant (Ki), which is the dissociation constant between inhibitor and enzyme, was obtained using a Dixon plot [1/V versus inhibitor (peptide) concentration (I)]. In the Dixon plot, V in the reaction mixture containing peptide at different concentrations was determined using six substrate concentrations. When a peptide inhibited Rgp in a mixed-type manner, Ki was obtained using a Dixon plot. In addition, the y-intercepts of the lines in the double-reciprocal plots correspond to a reciprocal apparent reaction velocity (1/ Vapp 5 (1 1 I/Ki0 )/Vmax, where Ki0 was the dissociation constant between the inhibitor and the enzyme–substrate complex). Values of 1/Vapp were plotted as a function of peptide concentration (I) and–Ki0 values were obtained as values of x-intercepts. Results were expressed as mean 6 standard deviation (SD) of three individual experiments or as mean of two individual experiments.
Degradation of the Parent Peptide by Rgp To evaluate the degradation of the parent peptide, CL(14–25), by Rgp, 10 ml of purified Rgp solution (0.6 mg protein/ml) was added to 150 ml of assay buffer containing 200 lM CL(14–25) and the mixture was incubated at 37 C for 1–3 h. After incubation, 40 ml of 50% trichloroacetic acid was added to the mixture and the resultant suspension was incubated in ice-cold water for 15 min to fully precipitate Rgp, according to the method of O’Brien-Simpson et al.29 The suspension was centrifuged at 2000g for 5 min and then supernatant was filtered to remove small particles using a membrane filter (13CP020AN; Advantec Toyo, Tokyo, Japan). Peptides in the filtrate were analyzed by analytical RP-HPLC using a Cazenda CD-C18 column (Imtakt, Kyoto, Japan) and a ultraviolet-visible (UV–VIS) detector (SPD 10AV, Shimadzu). Peptides were eluted with a gradient of 0–50% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The eluate was monitored at 220 nm.
RESULTS Rgp-Inhibitory Activity of the Parent Peptide FIGURE 1 Inhibition of arginine-specific gingipain (Rgp) activity by CL(14225). Enzyme activity was measured using carboxybenzoxy (Z)Phe-Arg-4-methyl-coumaryl-7-amide (Z-Phe-Arg-MCA) as a substrate, as described in Materials and Methods section. Each plot (A and B) shows the mean value obtained from two independent experiments. (A) Lineweaver2Burk plots for Rgp-inhibitory activity at different concentrations of CL(14225). The inserted small figure shows the periphery of the origin of the coordinate axes. Symbols: Inhibitor [(CL(14–25)] concentration: 0 lM, open circles; 2.5 lM, closed squares; 5 lM, closed triangles; 10 lM, closed circles; 15 lM closed diamonds. (B) Dixon plots for determining Ki of CL(14225) against Rgp. Symbols: Substrate concentration: 1 lM, closed diamonds; 1.5 lM, closed circles; 2 lM, closed triangles; 2.5 lM, closed squares; 5 lM, open diamonds; 10 lM, open circles. (C) Secondary plots for determining Ki0 of CL(14–25) against Rgp. Reciprocal apparent reaction velocity (1/Vapp 5 (1 1 [I]/ Ki0 )/Vmax) was plotted against the CL(14225) concentration.
As reported previously,46 the parent peptide, CL(14–25), inhibited the activity of purified Rgp, which was measured using ZPhe-Arg-MCA as the substrate. Figure 1 shows the kinetic analysis of Rgp-mediated Z-Phe-Arg-MCA hydrolysis in the absence and presence of CL(14–25). In the absence of CL(14– 25), Vmax and Km were determined to be 2.4 3 1027 M/min and 4.4 3 1026 M, respectively. CL(14–25) decreased V in a concentration-dependent manner. An increase in CL(14–25) concentration led to a decrease in Vmax and an increase in Km. The results suggested that CL(14–25) exhibited mixed-type inhibition when using a strict definition, because the Lineweaver–Burk plot yielded a family of lines with different slopes and intercepts, and they intersected with one another in the second quadrant (Figure 1A). However, because the point of Biopolymers (Peptide Science)
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Rgp-Inhibitory Activity of Truncated CL Peptide Analogs
FIGURE 2 Inhibition of arginine-specific gingipain (Rgp) activity by CL(15225). Each plot (A and B) shows the mean value obtained from two independent experiments. (A) Lineweaver2Burk plots for Rgp-inhibitory activity at different concentrations of CL(15225). (B) Dixon plots for determining Ki of CL(15225) against Rgp. Symbols are the same as those shown in Figure 1.
intersection was situated in the immediate vicinity of the horizontal axis, the inhibition type of CL(14–25) appeared close to being noncompetitive, in which Ki and Ki0 values were identical. In mixed-type inhibition, the inhibitor binds to the free enzymes and to the enzyme–substrate complex. From the Dixon plots (Figure 1B), Ki for Rgp-inhibitory activity by CL(14–25) was calculated to be 1.4 3 1026 M. In addition, to determine Ki0 for Rgp-inhibitory activity by CL(14–25), 1/Vapp values that corresponded to the y-intercepts of the lines described by the double-reciprocal plots were plotted against the CL(14–25) concentration (secondary plots, Figure 1C). Ki0 value was evaluated to be 4.3 3 1026 M, almost as high as Ki (1.4 3 1026 M). The results showed that CL(14–25) exhibits mixed-type inhibition that is close to being noncompetitive, suggesting that there is a single binding site on the enzyme that is distinct from the active site at which the peptide binds and inhibits activity. Biopolymers (Peptide Science)
To clarify the contribution of cationic amino acids (arginine and lysine) at the N- and C-termini of CL(14–25) toward Rgpinhibitory activity, we synthesized four truncated CL peptides [CL(15–25), CL(16–25), CL(14–24), and CL(14–23); Table I] and examined their Rgp-inhibitory activity. Figure 2 and Supporting Information Figure S1 show the kinetic analysis of the hydrolysis of Z-Phe-Arg-MCA by Rgp in the absence and presence of the truncated CL peptide analogs, CL(15–25) and CL(16–25), respectively. Lineweaver–Burk plots revealed that CL(15–25) exhibited a competitive-type inhibition toward Rgp (Figure 2A). Ki of CL(15–25) were calculated in a manner similar to that of CL(14–25), and were found to be 9.9 3 1026 M (Figure 2B). The results showed that Rgp-inhibitory activity of CL(15–25), which was truncated by removing one arginine residue from the N-terminus of CL(14–25), was lower than that of CL(14–25) and that the arginine residue at position 14 in CL(14–25) was most probably involved in an interaction between the peptide inhibitor and the Rgp-substrate complex. Rgp was hardly inhibited by CL(16–25) (Supporting Information Figure S1). The results show that the arginine residue at position 15 in CL(14–25) plays a determining role in Rgpinhibitory activity of CL(14–25). Furthermore, we evaluated Rgp-inhibitory activity by other truncated CL peptide analogs, CL(14–24) and CL(14–23). CL(14–24) and CL(14–23), in which either a single or two cationic amino acid residues were deleted from the C-terminus of CL(14–25). Figures 3 and 4 show the kinetic analysis of the hydrolysis of Z-Phe-Arg-MCA by Rgp in the absence and presence of the truncated CL peptide analogs, CL(14–24) and CL(14–23), respectively. Lineweaver–Burk plots showed that both CL(14–24) and CL(14–23) were mixed-type inhibitors. On the basis of Dixon plots and secondary plots, Ki and Ki0 were calculated to be 1.3 3 1026 M and 2.5 3 1026 M for CL(14–24), respectively (Figures 3B and 3C), and 1.3 3 1026 M and 5.6 3 1026 M for CL(14–23), respectively (Figures 4B and 4C). The slight difference between Ki and Ki0 for CL(14– 24) and CL(14–23) indicated that CL(14–24) and CL(14–23) were close to being noncompetitive inhibitors of Rgp. Because Ki and Ki0 for CL(14–24) and CL(14–23) were almost as high as the corresponding values for CL(14–25), contributions of the arginine and lysine residues at positions 24 and 25, respectively, toward Rgp-inhibitory activity appeared to be negligible.
Rgp-Inhibitory Activity of Alanine-Substituted CL Peptide Analogs To elucidate the importance of cationic amino acids (arginine and lysine) at the N- and C-termini of CL(14–25) for Rgp-
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FIGURE 3 Inhibition of arginine-specific gingipain (Rgp) activity by CL(14224). Each plot (A and B) shows the mean value obtained from two independent experiments. (A) Lineweaver2Burk plots for Rgp-inhibitory activity at different concentrations of CL(14224). (B) Dixon plots for determining Ki of CL(14224) against Rgp. (C) Secondary plots for determining Ki0 of CL(14224) against Rgp. Reciprocal apparent reaction velocity (1/Vapp 5 (1 1 [I]/Ki0 )/Vmax) was plotted against CL(15225) concentration. Symbols are the same as those shown in Figure 1.
FIGURE 4 Inhibition of arginine-specific gingipain (Rgp) activity by CL(14223). Each plot (A and B) shows the mean value obtained from two independent experiments. (A) Lineweaver2Burk plots for Rgpinhibitory activity at different concentrations of (14223). (B) Dixon plots for determining Ki of CL(14223) against Rgp. (C) Secondary plots for determining Ki0 of CL(14223) against Rgp. Reciprocal apparent reaction velocity (1/Vapp 5 (1 1 [I]/Ki0 )/Vmax) was plotted against CL(14224) concentration. Symbols are the same as those shown in Figure 1.
inhibitory activity, we synthesized four alanine-substituted CL peptide analogs [CL(R14A), CL(R14A, R15A), CL(R24A), and CL(R24A, K25A); Table I] and investigated their inhibitory
activities against Rgp. Lineweaver–Burk plots revealed that CL(R14A) was a mixed-type inhibitor of Rgp activity (Supporting Information Figure S2), whereas CL(R14A, R15A) Biopolymers (Peptide Science)
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FIGURE 5 Inhibition of arginine-specific gingipain (Rgp) activity by CL(K25A). Each plot (A and B) shows the mean value obtained from three independent experiments. Error bars indicate the standard deviation (SD), but little or no standard deviation (SD) was observed in these experiments. (A) Lineweaver2Burk plots for Rgp-inhibitory activity at different concentrations of CL(K25A). Symbols: Inhibitor [(CL(K25A)] concentration: 0 mM, open circles; 0.5 mM, closed squares; 1 mM, closed triangles; 1.5 mM, closed circles; 2.0 mM closed diamonds. (B) Dixon plots for determining Ki of CL(K25A) against Rgp. Symbols are the same as those shown in Figure 1. (C) Secondary plots for determining Ki0 of CL(K25A) against Rgp. Reciprocal apparent reaction velocity (1/Vapp 5 (1 1 [I]/Ki0 )/Vmax) was plotted against CL(K25A) concentration.
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FIGURE 6 Inhibition of arginine-specific gingipain (Rgp) activity by CL(R24A, K25A). Each plot (A and B) shows the mean value obtained from three independent experiments. Error bars indicate the standard deviation (SD), but little or no standard deviation (SD) was observed in these experiments. (A) Lineweaver2Burk plots for Rgp-inhibitory activity at different concentrations of CL(R24A, K25A). (B) Dixon plots for determining Ki of CL(R24A, K25A) against Rgp. (C) Secondary plots for determining Ki0 of CL(R24A, K25A) against Rgp. Reciprocal apparent reaction velocity (1/Vapp 5 (1 1 [I]/Ki0 )/Vmax) was plotted against CL(R24A, K25A) concentration. Symbols are the same as those shown in Figure 5.
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Table II The Inhibition Constants (Ki and Ki ) and 50% Growth-Inhibitory Concentrations (IC50) of Peptides Used in This Study Peptide Name
Inhibition Type
CL(14–25) CL(15–25) CL(16–25) CL(14–24) CL(14–23) CL(R14A) CL(R14A,R15A) CL(K25A) CL(R24A,K25A)
Mixed Competitive – Mixed Mixed Mixed Competitive Mixed Mixed
a b
Ki (lM)
Ki (lM)
IC50 (lM)a
Remarks
1.4 9.9 – 1.3 1.3 10 21 0.96 0.50
4.3 – – 2.5 5.6 22 – 1.9 2.1
145 6 30 248 6 50 NDb 242 6 40 NDb 104 6 11 89 6 11 51 6 16 >525
Figure 1 Figure 2 Figure S1 Figure 3 Figure 4 Figure S2 Figure S3 Figure 5 Figure 6
The values of IC50 indicate 50% growth-inhibitory concentration against P. gingivalis JCM 8525.47,48 ND, not determined. The antimicrobial activities of CL(16–25) and CL(14–23) were very low.
inhibited Rgp activity in a competitive manner (Supporting Information Figure S3). Ki and Ki0 for CL(R14A) were calculated to be 1.0 3 1025 M and 2.2 3 1025 M, respectively (Supporting Information Figure S2). Rgp-inhibitory activity of CL(R14A), obtained by replacing the N-terminal arginine residue at position 14 with an alanine residue decreased in a manner similar to that of CL(15–25), obtained by deleting the Nterminal arginine residue at position 14. For CL(R14A, R15A), Ki was evaluated to be 2.1 3 1025 M by Dixon plots (Supporting Information Figure S3). The results showed that Rgpinhibitory activities of CL peptide analogs, CL(R14A) and CL(R14A, R15A), were lower than that of CL(14–25). The decrease in Rgp-inhibitory activities of alanine-substituted CL peptide analogs was almost similar to those of CL(15–25) and CL(16–25), obtained by deleting either one or two arginine residues from the N-terminus of CL(14–25), respectively. These results strongly suggest that the two arginine residues, particularly arginine at position 15 at the N-terminus of CL(14–25), play important roles in Rgp-inhibitory activity of CL(14–25). Moreover, we investigated the kinetics of inhibition for other alanine-substituted CL peptide analogs, CL(K25A) and CL(R24A, K25A), in which one or two cationic amino acid residues at the C-terminus of CL(14–25), respectively, were replaced with alanine residues. Figures 5 and 6 show the kinetic analysis of Rgp-mediated hydrolysis of the fluorogenic substrate in the absence and presence of alanine-substituted CL peptide analogs, CL(K25A) and CL(R24A, K25A), respectively. Analytical results using Lineweaver–Burk plots confirmed that CL(K25A) and CL(R24A, K25A) were mixed-type inhibitors (Figures 5A and 6A). Results of Dixon and secondary plots indicated that Ki and Ki0 were 9.6 3 1027 M and 1.9 3 1026 M for CL(K25A), respectively (Figures 5B and 5C), and 5.0 3 1027 M and 2.1 3 1026 M for CL(R24A, K25A), respectively (Figures 6B and 6C). CL(K25A) appeared close to being a non-
competitive inhibitor of Rgp because there was only a slight difference between its Ki and Ki0 values. Ki and Ki0 for CL(K25A) and CL(R24A, K25A) were significantly lower than that for CL(14–25) (Table II). Therefore, substitution of both the lysine residue at position 25 and arginine and lysine residues at positions 24 and 25, respectively, with alanine residues appeared to result in the enhancement of Rgp-inhibitory activity, although the detailed mechanisms remain unclear.
Degradation of the Parent Peptide by Rgp Rgp produced by P. gingivalis degrades human antimicrobial peptides, including human b-defensins24,25 and histatin 5,28,29 and consequently abolishes their antimicrobial activities against oral pathogenic bacteria including P. gingivalis. To examine the ability of Rgp to degrade CL(14–25), CL(14–25) was incubated with Rgp, as described in the Materials and Methods section. In this experiment, to clearly elucidate the ability of Rgp to hydrolyze CL(14–25), the Rgp concentration added was 12.5-fold higher than that used in the usual enzyme reactions using ZPhe-Arg-MCA as the substrate. Figure 7 shows analytical RPHPLC chromatograms of the peptide and its fragments. CL(14–25) was detected at the retention time of 14.6 min (Figure 7A). Two minor peaks with a retention time of 13.9 and 14.8 min were also observed, but there was little or no change in their areas or heights at 3 h. The peak area of CL(14–25) decreased by 24% after 3 h. Therefore, fragmentation of CL(14–25) by hydrolysis with Rgp appeared to be negligible during the 1–5 min reactions to measure its inhibitory activity.
DISUSSION Several synthetic inhibitors of Rgp have already been reported, including doxycycline (a tetracycline analog),35 chlorhexidine,36 leupeptin,18,28 and KYT-1 (a small peptide analog).38 Biopolymers (Peptide Science)
Inhibition of Arg-Specific Cysteine Proteinase by Antimicrobial Peptide
FIGURE 7 Analytical chromatograms of CL(14225) and its fragments after incubation with arginine-specific gingipain (Rgp). Incubation times were 0 (A), 1 (B), 2 (C), and 3 h (D). Peptides recovered were analyzed using an analytical reversed-phase highperformance liquid chromatography system (RP-HPLC) equipped with a Cazenda CD-C18 column (Imtakt, Kyoto, Japan). Peptides were eluted with a gradient of 0250% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min and the eluate was monitored at 220 nm.
Kinetic studies indicate that doxycycline is a uncompetitive inhibitor with Ki0 of 5.4 3 1025 M, whereas chlorhexidine, leupeptin, and KYT-1 are competitive inhibitors with Ki of 2.6 3 1024 M, 1.0 3 1027 M, and 1.3 3 10210 M, respectively. KYT-1 is the most potent synthetic inhibitor of Rgp, designed by Kadowaki et al., on the basis of the cleavage specificity of Rgp toward histatins.38 However, a series of KYT inhibitors appear to have substantial safety and regulatory hurdles for human use because they are synthetic peptide analogs containing components other than amino acids.34 In addition to synthetic Rgp-inhibitors, Rgp-inhibitory activities by peptide Biopolymers (Peptide Science)
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inhibitors, including j-casein peptide (109–137)34 and histatin 5,28 have also been reported. Kinetic analysis of Rgp inhibition revealed that the j-casein peptide (109–137) acts as an uncompetitive inhibitor with Ki0 of 4.0 3 1025 M, whereas histatin 5 exhibits competitive inhibition with Ki of 1.5 3 1025 M. The j-casein peptide (109–137), which contains no arginine residues, is an uncompetitive inhibitor and only binds to the Rgp–substrate complex, probably leading to weak inhibition of product formation.34 Histatin 5 possesses low Rgp-inhibitory activity because it is susceptible to digestion by Rgp as described previously.28 CL(14–25) inhibited Rgp in a mixed-type inhibitory mode (Figure 1), which is different from the inhibition pattern seen with other Rgp-inhibitors described above, suggesting that it binds to free Rgp and Rgp– substrate complex and represses Rgp activity. Ki and Ki0 of CL(14–25) were evaluated to be 1.4 3 1026 M and 4.3 3 1026 M, respectively (Figure 1 and Table II). Rgp-inhibitory activity of CL(14–25) was higher than that of peptide inhibitors, including j-casein peptide (109–137) and histatin 5. CL(14–25) has three arginine residues and therefore it can be digested by Rgp at the three potential cleavage sites. We detected slight degradation of CL(14–25) by Rgp after 3 h (Figure 7), but in reactions in which CL(14–25) was incubated with Rgp for
