IAI Accepts, published online ahead of print on 31 March 2014 Infect. Immun. doi:10.1128/IAI.01624-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

1

Title: ‘Citrullination and proteolytic processing of chemokines by Porphyromonas gingivalis’

2 3 4

Eva A.V. Moelants,a Gitte Loozen,b Anneleen Mortier,a Erik Martens,c Ghislain Opdenakker,c

5

Danuta Mizgalska, d Borys Szmigielski, d Jan Potempa,d,e Jo Van Damme,a Wim Teughelsb and

6

Paul Proosta#

7 8 9

Laboratory of Molecular Immunology, Department of Microbiology and Immunology, Rega

10

Institute, KU Leuven, Leuven, Belgiuma; Research Group for Microbial Adhesion, Department of

11

Oral Health Sciences, KU Leuven, 3000 Leuven, Belgiumb; Laboratory of Immunobiology,

12

Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgiumc;

13

Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology,

14

Jagiellonian University, Krakow, Polandd; Oral Health and Systemic Diseases Research Group,

15

School of Dentistry, University of Louisville, KY, USAe

16 17 18

Running title: ‘Processing of chemokines by Porphyromonas gingivalis’

19 20 21

#Address correspondence to Paul Proost, [email protected]

22 23 24 1

25

ABSTRACT

26

Outgrowth of Porphyromonas gingivalis within the inflammatory subgingival plaque is

27

associated with periodontitis characterized by periodontal tissue destruction, loss of alveolar

28

bone, periodontal pocket formation and eventually tooth loss. Potential virulence factors of P.

29

gingivalis are peptidylarginine deiminase (PPAD), an enzyme modifying free or peptide-bound

30

arginine to citrulline, and the bacterial proteases referred to as gingipains (Rgp and Kgp).

31

Chemokines attract leukocytes during inflammation. However, posttranslational modification

32

(PTM) of chemokines by proteases or human peptidylarginine deiminases may alter their

33

biological activities. Since chemokine processing may be important in microbial defense

34

mechanisms, we investigated whether PTM of chemokines occurs by P. gingivalis enzymes.

35

Upon incubation of interleukin-8 (IL-8/CXCL8) with PPAD, only minor enzymatic citrullination

36

was detected. In contrast, Rgp rapidly cleaved CXCL8 in vitro. Subsequently, different P.

37

gingivalis strains were incubated with the chemokines CXCL8 or CXCL10 and their PTM were

38

investigated. No significant CXCL8 citrullination was detected for the tested strains.

39

Interestingly, although considerable differences in efficiency of CXCL8 degradation were

40

observed with full cultures of various strains, similar rates of chemokine proteolysis were exerted

41

by cell-free culture supernatants. Sequencing of CXCL8 incubated with supernatant or bacteria

42

showed that CXCL8 is processed into its more potent 6-77 and 9-77 forms. In contrast, CXCL10

43

was entirely and rapidly degraded by P. gingivalis with no transient chemokine forms being

44

observed. In conclusion, this study demonstrates PTM of CXCL8 and CXCL10 by gingipains of

45

P. gingivalis and that strain differences may particularly affect the activity of these bacterial

46

membrane-associated proteases.

47

2

48

KEYWORDS: chemokine, citrullination, Porphyromonas gingivalis, proteolytic processing,

49

peptidylarginine deiminase

50 51

ABBREVIATIONS

52

ACPA, anti-citrullinated peptides antibody; biotCXCL8, biotinylated CXCL8; CFU, colony-

53

forming units; Cit, citrulline; [Cit5]CXCL8, citrullinated CXCL8; [Cit5]CXCL10, citrullinated

54

CXCL10; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; E/S, enzyme-

55

substrate; Fmoc, fluorenyl methoxycarbonyl; IL-8/CXCL8, interleukin-8; IP-10/CXCL10,

56

interferon-γ-inducible protein-10; MIP-1α/CCL3, macrophage inflammatory protein-1α; PAD,

57

peptidylarginine deiminase; PBS, phosphate buffered saline; PPAD, PAD of Porphyromonas

58

gingivalis; PTM, posttranslational modification; RA, rheumatoid arthritis; TFA trifluoroacetic

59

acid; TMB, 3,3',5,5'-tetramethylbenzidine

3

60

1. Introduction

61

Cytokine and chemokine activity is regulated at multiple levels including posttranslational

62

modification (PTM) (1-3). Reduced or enhanced receptor affinity/specificity and chemokine

63

activity have been reported, depending on the chemokine and on the type of PTM (4,5). Most

64

PTM on inflammatory chemokine ligands depend on proteolytic cleavage with highly specific

65

proteases mainly affecting the NH2-terminal region of the protein (2,5-7). Many metalloproteases

66

such as aminopeptidase N and various matrix metalloproteases, and a number of serine proteases

67

including thrombin, plasmin, cathepsin G and the dipeptidylpeptidase CD26 were reported to

68

cleave specific chemokines in the NH2-terminal region. The biological effect of such proteolytic

69

processing varies depending on the chemokine and protease involved. First, limited NH2-terminal

70

proteolytic processing of chemokines by proteases can result in enhanced, e.g. CXCL8 by

71

thrombin (8,9) or matrix metalloproteinase (MMP)-9/gelatinase B (10) and CCL3L1 by

72

DPPIV/CD26 (11), or decreased, e.g. CCL7 by MMP-2/gelatinase A (12) and CXCL10 by CD26

73

(13), biological activity. For some chemokines, like CXCL7 and CCL14, more extensive NH2-

74

terminal truncation is even mandatory to obtain receptor signaling and chemotactic properties

75

(14-16). A detailed description of chemokine-protease interactions and consequences for the

76

biological functions of chemokines has been published in a number of recent reviews (2,5,6,17).

77

Also, naturally occurring N- and O-glycosylated forms of chemokines with altered lectin binding

78

properties have been identified (18-20). In addition to NH2- and COOH-terminal proteolytic

79

processing and glycosylation, citrullination, i.e. deimination of arginine (Arg) to citrulline (Cit),

80

is a recently discovered PTM on the natural chemokines interleukin-8 (IL-8/CXCL8) and

81

interferon-γ-inducible protein-10 (IP-10/CXCL10) (8,13,21-24). CXCL8, a ligand for CXC

82

chemokine receptors CXCR1 and CXCR2, is known to be the major human neutrophil

83

chemoattractant. The CXCR3 ligand CXCL10 predominantly chemoattracts activated 4

84

lymphocytes. For both chemokines Arg at position 5 (Arg5) was converted into Cit. The

85

mammalian enzymes responsible for the conversion are peptidylarginine deiminases (PADs)

86

(25). Chemokines were the first identified PAD substrates that signal in a receptor-dependent

87

manner. Most other PAD substrates are structural proteins (e.g. keratin and fillagrin) (26-31).

88

Citrullination may considerably influence the biological activity of proteins since it may change

89

ionic interactions in macromolecules resulting in altered protein folding and may cause altered

90

binding of proteins to other macromolecules such as DNA, glycosaminoglycans and other

91

proteins (28,32).

92

Currently available in vitro and in vivo data are in line with an anti-inflammatory role for PAD in

93

local acute inflammation by citrullinating and thereby inactivating chemokines such as CXCL5,

94

CXCL8, CXCL10, CXCL11 and CXCL12 and consequently dampening leukocyte migration

95

(33). In addition, chemokine citrullination may be important, in particular in microbial defense

96

mechanisms, since peptidylarginine deiminating activity has been reported in one prokaryotic

97

organism Porphyromonas gingivalis (25,34). It is well known that an outgrowth of P. gingivalis

98

within the periodontal pocket is associated with periodontitis, an inflammatory disorder

99

characterized by periodontal tissue destruction, loss of alveolar bone and eventually tooth loss. In

100

contrast to other mammalian PADs, PAD of P. gingivalis (PPAD) preferentially citrullinates C-

101

terminal arginine residues and is also able to convert free Arg into Cit. In contrast to mammalian

102

enzymes, PPAD deiminates peptidylarginine residues in a calcium independent manner

103

(25,34,35). Its ammonia-producing properties are well studied as a response to acidic cleansing

104

cycles in the mouth (34,36,37). Though the substrates for the action of PPAD are unidentified and

105

the exact role of PPAD in assisting the bacterium in circumventing the host immune defense is

106

unknown, PPAD has been suggested to function as an additional virulence factor (25,34). In the

107

context of an innate immune response, chemokine citrullination by PPAD may establish a 5

108

negative feedback on local leukocyte-mediated inflammation and hence bacterial clearance.

109

Together with inflammation-associated and PPAD-exerted citrullination of host proteins this may

110

contribute to breakdown of immunotolerance to citrullinated epitopes and eventual development

111

of rheumatoid arthritis (38).

112

Gingipains, including arginine-specific gingipains (RgpA and RgpB) and the lysine-specific

113

gingipain (Kgp) are considered to be essential virulence factors of P. gingivalis (39). Theses

114

proteinases are either cell-surface associated or secreted. Gingipains play an important role in the

115

evasion and dysregulation of the host’s immune response by the degradation of proinflammatory

116

cytokines, complement factors, antimicrobial peptides and immunoglobulins at the site of

117

infection (39-41). Since both PPAD and gingipains were found to significantly contribute to P.

118

gingivalis virulence we investigated whether PTM of chemokines by these enzymes represents a

119

way by which P. gingivalis regulates the inflammatory response.

120 121

2. Materials and methods

122

Reagents and materials

123

The 77 amino acid forms of the chemokines CXCL8, CXCL10 and macrophage inflammatory

124

protein-1α (MIP-1α/CCL3) were obtained from PeproTech (Rocky Hill, NJ, USA).

125

N-Acetylo-L-Arg, L-Cit, 2,3-butanedione monoxime, thiosemicarbazide, antipyrine and 2,3-

126

butanedione (>99.4% [GC]) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

127

RgpB was purified as previously described (42,43). To obtain PPAD, P. gingivalis W83 was

128

engineered to secrete a soluble form of PPAD with a C-terminal hexahistidine affinity tag using

129

the same molecular strategy as was reported previously for the RgpB protease in the mutant

130

662i6H (44). Subsequently, PPAD was separated from the culture medium via ion-exchange and

131

gel filtration chromatography as described for purification of soluble Kgp from the growth 6

132

medium (45). Briefly, bacteria were removed by centrifugation and proteins in the medium were

133

precipitated with acetone, resuspended in phosphate buffer (pH 6.5), dialyzed and passed through

134

a DE-52 column (Whatman, GE Healthcare, Pittsburgh, PA, USA) to remove excess hemin. The

135

flow-through was dialyzed against 50 mM Tris-HCl, 0.02% NaN3, pH 8.0 and loaded on a Mono

136

Q column (GE Healthcare). Adsorbed proteins were eluted with a NaCl gradient and fractions

137

containing PPAD activity were pooled. The final PPAD purification was achieved by gel

138

filtration chromatography using a Superdex 75 column (GE Healthcare). The purity of PPAD was

139

evaluated by SDS-PAGE followed by silver staining. Gingipain-specific inhibitors, KYT-1 and

140

KYT-36, were obtained from PeptaNova (Sandhausen, Germany).

141

Biotinylated CXCL8 (biotCXCL8) was synthesized, folded and purified in our lab. Briefly,

142

CXCL8 was synthesized as previously described (23). After removal of the final fluorenyl

143

methoxycarbonyl (Fmoc) group of the most NH2-terminal amino acid, the free NH2-terminus was

144

biotinylated with biotin-p-nitrophenyl ester (Novabiochem, Darmstadt, Germany) on a 433A

145

solid phase peptide synthesizer (Applied Biosystems, Foster City, CA, USA) using standard

146

coupling chemistry. The synthetic biotinylated CXCL8 peptide was cleaved from the resin,

147

purified, folded and repurified as described for intact CXCL8 (23). Purity of biotinylated CXCL8

148

was confirmed after purification using RP-HPLC by ion trap mass spectrometry (Figure 1).

149 150

Strains and culture conditions

151

P. gingivalis ATCC 33277 was used as a reference strain. Five capsule-typed strains (K1-5)

152

(46,47) and five clinical isolates with non-typified capsule (Pg1-5) (48) were included in the

153

analysis (Table 1). P. gingivalis strains were grown on blood agar plates (Oxoid, Basingstoke,

154

UK) supplemented with 5µg/ml hemin (Sigma-Aldrich), 1µg/ml menadione and 5% sterile horse

155

blood (Biotrading, Keerbergen, Belgium). Bacteria were collected from blood agar plates, 7

156

transferred to 10ml tryptone soya broth (Oxoid) and incubated overnight at 37°C in an anaerobic

157

atmosphere. The bacterial concentration was adjusted by measuring optical density at 600nm to

158

obtain bacterial suspensions with starting concentrations of 5 x 107 colony-forming units

159

(CFU)/ml. Supernatants were obtained by centrifuging for 3min at 13200rpm.

160 161

PPAD activity assay

162

PPAD activity was tested by a colorimetric method for the determination of enzymatically

163

produced L-Cit as described by Knipp et al. (49). A standard for L-Cit ranging from 0-1200µM

164

was prepared in 0.1M Tris-HCl buffer (pH 7.5) and subsequently 50µL was pipetted in a 96-well

165

plate. Ten microliters of each sample was mixed with 40µL 0.1M Tris-HCl buffer (pH 7.5)

166

supplemented with 10mM N-acetylo-L-Arg before pipetting into the 96-well plate. The 96-well

167

plate containing standard and samples was incubated for 1h at 37°C. Finally, 150µL of freshly

168

prepared citrulline detection reagent (1 volume of solution A [80mM 2,3-butanedione monoxime

169

and 2mM thiosemicarbazide] and 3 volumes of solution B [3M H3PO4, 6M H2SO4 and 2mM

170

NH4Fe(SO4)2.12H2O]) were added and the 96-well plate was incubated for 15min at 95°C. Cit

171

was detected by measuring the absorbance at 535nm.

172 173

In vitro incubation of CXCL8 and CCL3 with PPAD

174

CXCL8 or CCL3 were incubated with purified activated PPAD and gingipain-specific inhibitors

175

(10µM KYT-1 and KYT-36) for 1.5h at 37°C at an enzyme-substrate (E/S) molar ratio of 1/10

176

and a substrate concentration of 7.6µM. Incubations with PPAD were carried out in 50mM Tris-

177

HCl 150mM NaCl (pH7.8). Enzymatic citrullination was stopped with 0.1% (v/v) trifluoroacetic

178

acid (TFA).

179 8

180

In vitro incubation of CXCL8, CXCL10 and [Cit5]CXCL8 with gingipains

181

RgpB was first activated for 10 min at 37°C in activation buffer (0.2M HEPES 10mM L-Cys

182

10mM CaCl2 pH8). CXCL8, CXCL10 or [Cit5]CXCL8 were then incubated with activated RgpB

183

for 1h at 37°C at an enzyme-substrate (E/S) molar ratio of 1/1 and a substrate concentration of

184

1µM. Incubations with RgpB were carried out in 20mM Tris-HCl 5mM CaCl2 0.1M NaCl (pH8).

185

Enzymatic processing was stopped with 0.1% (v/v) trifluoroacetic acid (TFA) and investigated by

186

Edman degradation on a 491 Procise cLC protein sequencer (Applied Biosystems, Foster City,

187

CA, USA).

188 189

PTM of CXCL8 and CXCL10 by different P. gingivalis strains

190

Bacterial suspensions with starting concentrations of 5 x 107 CFU/ml were first grown for 24h in

191

96-well plates (200µl/well) at 37°C in an anaerobic atmosphere. Then, the strains were treated

192

with 5µg/ml CXCL8 or CXCL10 for 2h and supernatants were harvested and stored at -20°C

193

after centrifuging for 3min at 13200rpm.

194

To investigate the potential binding of CXCL8 to the bacterial membrane, strains were treated

195

with 500ng/ml biotCXCL8 and protease inhibitor (cOmplete ULTRA Mini EDTA-free

196

EASYpack, Roche) supplemented with 5mM EDTA for 2h. Supernatants and pellets were

197

harvested and stored at -20°C after centrifuging for 3min at 13200rpm.

198 199

Proteolytic processing of CXCL8 and CXCL10 by secreted versus membrane-bound

200

gingipains

201

Bacterial suspensions of 5 x 107 CFU/ml were cultured for 24h in 96-well plates (200µl/well) at

202

37°C in an anaerobic atmosphere and supernatants were harvested. To examine the proteolytic

203

processing of chemokines by P. gingivalis proteases, CXCL8 and CXCL10 were incubated with 9

204

5µl of supernatants or with complete bacterial culture at 37°C. Samples were separated by SDS-

205

PAGE using 14% ProSieve®50 gels (Lonza, Basel, Switzerland) with a Tris/Tricine electrode

206

buffer (0.1M Tris, 0.1M Tricine, 0.1% SDS). Gels were blotted onto a PVDF membrane that was

207

subsequently stained using Coomassie Brilliant Blue. Finally, the NH2-terminal protein sequences

208

of the protein bands visible on the blot were determined by Edman degradation on a 491 Procise

209

cLC protein sequencer (Applied Biosystems).

210 211

Detection of total CXCL8, biotCXCL8 and CXCL10

212

Levels of human CXCL8 and CXCL10 were quantified by specific sandwich enzyme-linked

213

immunosorbent assays (ELISAs) (50-52).

214

For human CXCL8 a 96-well plate was coated with goat polyclonal anti-human CXCL8 antibody

215

generated in our laboratory (53), followed by blocking with phosphate buffered saline (PBS)

216

(pH7.4) containing 0.1% (w/v) casein and 0.05% (v/v) Tween 20. Human CXCL8 bound to the

217

coating antibody was detected with mouse monoclonal anti-human CXCL8 antibody (R&D

218

Systems, Abingdon, UK), followed by a secondary peroxidase-conjugated anti-mouse IgG

219

(Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Peroxidase activity was

220

quantified by measuring the conversion of 3,3',5,5'-tetramethylbenzidine (TMB) (Sigma-Aldrich)

221

at 450nm.

222

To measure biotCXCL8 plates were coated with goat polyclonal anti-human CXCL8 antibody

223

(vide supra) and biotCXCL8 detected with peroxidase-conjugated streptavidin (R&D Systems).

224

The sandwich ELISA for human CXCL10 consisted of mouse monoclonal anti-human CXCL10

225

as coating antibody, biotinylated rabbit polyclonal anti-human CXCL10 as detecting antibody

226

and peroxidase-conjugated streptavidin as secondary reagent (all R&D Systems).

227 10

228

Detection of citrullinated chemokines

229

The procedure to chemically modify and quantify a citrulline-containing protein of interest was

230

performed as described before (54). Briefly, peptidylcitrulline residues in samples were first

231

chemically modified by addition of 50mM antipyrine, 16% (v/v) TFA and 12.5mM 2,3-

232

butanedione (55) followed by incubation for 2h at 37°C in the dark. Then, the strongly acidic

233

reaction mixture was dialyzed against PBS containing 0.05% Tween 20 (pH7.4) overnight at

234

room temperature and protected from light using Slide-A-Lyzer®MINI Dialyse Units (Pierce,

235

Rockford, IL, USA).

236

To detect citrullinated CXCL8 ([Cit5]CXCL8), a specific sandwich ELISA was developed (54).

237

A 96-well plate was coated with mouse monoclonal anti-human CXCL8 antibody (R&D

238

Systems), followed by blocking with PBS containing 0.1% casein and 0.05% Tween 20. Human

239

[Cit5]CXCL8 in chemically modified samples was detected by specific rabbit antibodies against

240

chemically modified citrulline residues and by a secondary peroxidase-conjugated anti-rabbit IgG

241

antibody (Jackson ImmunoResearch Laboratories). Peroxidase activity was quantified by

242

measuring the conversion of TMB (Sigma-Aldrich) at 450nm. Citrullination of CCL3 was

243

detected in a similar way by using a mouse monoclonal anti-human CCL3 antibody (R&D

244

Systems) instead of the mouse monoclonal anti-human CXCL8 antibody.

245

In addition, for the in vitro incubation of CXCL8 with PPAD the NH2-terminal sequences of

246

CXCL8 were determined by Edman degradation on a 491 Procise cLC protein sequencer

247

(Applied Biosystems) to verify if citrullination occurred on the more NH2-terminally located Arg

248

residues.

249 250

3. Results

251

Enzymatic modification of chemokines by PPAD and gingipains 11

252

Before incubating chemokines with PPAD in vitro, the activation of the enzyme by different

253

reducing agents was evaluated with a PPAD activity assay (49). In contrast to 10mM reduced

254

glutathione, both L-cysteine (L-Cys) (10mM, 30mM and 90mM) and dithiothreitol (DTT) (5mM)

255

were able to activate PPAD (Figure 2).

256

Upon incubation of intact CXCL8 with activated PPAD (30mM L-Cys) at an E/S molar ratio of

257

1/10 for 1.5h, no citrullination was detected by the specific ELISA. However, NH2-terminal

258

sequencing revealed about 10% citrullination on the most NH2-terminal Arg, but no citrullination

259

of the Arg in the ELR motif of CXCL8. Besides CXCL8, also CCL3 is reported to be an

260

important inflammatory chemokine mediator in periodontitis (56,57). However, upon incubation

261

of intact CCL3 with activated PPAD (30mM L-Cys) at an E/S molar ratio of 1/10 for 1.5h, no

262

citrullination could be detected by the specific ELISA to measure citrullinated CCL3. In contrast

263

to CXCL8, the NH2-terminus of CCL3 contains no Arg, so NH2-terminal sequencing was not

264

performed. Activated RgpB efficiently cleaved CXCL8 and CXCL10 in vitro. After 30min no

265

intact CXCL8 was present anymore as only NH2-terminally processed forms of CXCL8 (e.g. 6-

266

77, 12-77 and 32-77) were detected. Also for CXCL10, cleavage occurred very rapidly, as after

267

5min of incubation, less than 25% intact chemokine could be detected (data not shown).

268

Interestingly, citrullination of CXCL8 prevented this chemokine from being cleaved by RgpB.

269

This confirms the high specificity of RgpB for Arg-X bonds.

270 271

Enzymatic citrullination of chemokines by P. gingivalis

272

CXCL8 and CXCL10 were incubated with full cultures of different P. gingivalis strains for 2h.

273

Subsequently, total amounts of CXCL8 and CXCL10 were determined by standard ELISA and

274

citrullinated chemokine was quantified by a specific sandwich ELISA as recently described (54).

12

275

CXCL8 was recovered after incubation with most tested strains, however, only a small amount of

276

CXCL8 (up to 5%) was detected to be citrullinated in samples for all tested strains of P.

277

gingivalis (Figure 3). At first, an obvious explanation for this could be the NH2-terminal cleavage

278

of CXCL8 at Lys8 by Kgp, which potentially removes the first 8 amino acids including the Arg

279

in position 5, i.e. the Arg, which is most susceptible to citrullination by human PAD2 and PAD4

280

(8). However, detected citrullination levels upon incubation with PPAD in vitro were also very

281

low. Therefore, a more plausible reason for the low citrullination of CXCL8 for all tested strains

282

of P. gingivalis is that PPAD citrullinates CXCL8 inefficiently.

283

To further investigate whether inflammatory chemokines become citrullinated by PPAD, we

284

selected CXCL10, which is a major attractant for natural killer cells and activated T cells and is

285

citrullinated by human PAD2 and PAD4, but which has no NH2-terminal lysine residues (Lys or

286

K). Thus, the NH2-terminus of CXCL10 (NH2-1VPLSRTVRCT10...) is resistant to proteolysis by

287

Kgp leaving Arg5 and Arg8 available for citrullination by PPAD. In this case, however, only very

288

low levels of CXCL10 were recovered immediately after treatment with P. gingivalis and no

289

CXCL10 was detected for any strain after 2h of incubation. This suggests that P. gingivalis

290

directly interacts with CXCL10 by binding, degrading or internalizing the chemokine. When

291

CXCL10 was incubated with supernatants of P. gingivalis, already after 5 min, no intact

292

CXCL10 was present, indicating very fast degradation of CXCL10 by P. gingivalis (vide infra).

293 294

Proteolytic cleavage of chemokines by P. gingivalis

295

CXCL8 was incubated with different P. gingivalis strains for 2h and CXCL8 levels were

296

measured by ELISA. For all strains the amount of CXCL8 still present after 2h was lower than

297

the initial quantity added, indicating that clearance of CXCL8 from the bacterial medium

298

occurred (Figure 4). However, some strains seemed more efficient in degrading CXCL8, since for 13

299

Pg1, Pg2 and K1-4 only a very small amount of CXCL8 was detected after 2h. Samples in which

300

CXCL8 was still present after 2h were examined by gel electrophoresis and NH2-terminal

301

sequencing (Table 2). Besides intact CXCL8, three NH2-terminally processed forms of CXCL8,

302

i.e. the more potent CXCL8(6-77) and CXCL8(9-77) variants and further truncated

303

CXCL8(16-77), could be detected, indicating that NH2-terminal truncation by cleavage at Arg5

304

and Lys8 precedes further degradation of the chemokine.To investigate the potential binding of

305

CXCL8 to the bacterial membrane, biotCXCL8was incubated with different strains and protease

306

inhibitor for 2h. CXCL8 was specifically biotinylated at the NH2-terminus by chemical synthesis.

307

Thus modification of the side chains of Lys was avoided and potential cleavage sites for Kgp

308

remained unaltered. Supernatants and bacteria were harvested and biotCXCL8 in both fractions

309

measured by ELISA (Figure 5). The amount of biotCXCL8 recovered in the cell pellets was

310

neglectable compared to chemokine levels present in the supernatants. These data confirm the

311

importance of the degradation by proteases (e.g. gingipains) (and not by binding to the bacterial

312

cell surface) in the removal of CXCL8 from the medium.

313

When CXCL8 was incubated for 2h with supernatants of P. gingivalis, cleavage clearly occurred

314

(Figure 6). In contrast to the difference in cleavage measured by ELISA for CXCL8 incubated

315

with P. gingivalis cultures (Figure 4), no quantitative difference in processing of CXCL8 by cell-

316

free culture supernatants of strains ATCC 33277, Pg1-5 and K1-5 could be seen (Figure 6).

317

Sequencing of the CXCL8 incubated with supernatant showed that CXCL8 is processed first into

318

its more potent 6-77 and 9-77 forms before being totally degraded. The highly active truncated

319

CXCL8(9-77) was clearly the most dominant form found in all incubations (57 ± 5.4%).

320

Also CXCL10 was incubated with different P. gingivalis strains for 2h and CXCL10 levels were

321

detected by ELISA. However, none of the CXCL10 was recovered when incubated for 2h with

322

whole bacteria (vide supra). When CXCL10 was incubated for 5min, 30min or 2h with 14

323

supernatants of P. gingivalis, CXCL10 was only detectable after 5min as evidenced by gel

324

electrophoresis, blotting and Edman degradation. However, even after 5min of incubation, no

325

intact CXCL10 was detected, indicating fast degradation of CXCL10 by P. gingivalis. Two

326

processed forms of CXCL10 (e.g. 23-77 and 27-77) were detected.

327 328

4. Discussion

329

Enzymatic citrullination of chemokines by P. gingivalis

330

PTMs affect the biological activity of cytokines and chemokines (2,4,58). Recently, citrullination,

331

a new PTM of chemokines, has been discovered (8,21). The conversion of peptidylarginine to

332

peptidylcitrulline is performed by the mammalian PAD enzymes (25). The biological function of

333

chemokines is influenced by citrullination. Citrullination of CXCL8 by PAD protects CXCL8

334

from NH2-terminal activation by thrombin and plasmin, reduces its glycosaminoglycan binding

335

properties, moderately alters binding of CXCL8 to the CXC chemokine receptors CXCR1 and

336

CXCR2, reduces CXCL8-induced neutrophil extravasation to the peritoneal cavity and increases

337

chemokine ability to mobilize neutrophils to the blood circulation (8,59). Not only eukaryotes,

338

but also the prokaryotic organism P. gingivalis is known to express ‘PAD’. Colonization of a

339

biofilm on the tooth surface by P. gingivalis and its subgingival proliferation leads to immune

340

reactions eroding teeth-supporting tissues. Recently, a potential correlation between periodontitis

341

and the autoimmune disease rheumatoid arthritis (RA), which is linked to protein citrullination,

342

has been proposed (32,60-62). It is suggested that PPAD is responsible for the citrullination of

343

proteins like fibrinogen and α-enolase, leading to the production of anti-citrullinated peptides

344

antibodies (ACPAs) and immune complexes. This results in inflammation and may finally cause

345

chronic RA (63-65). The occurrence of chemokine citrullination by bacterial PPAD, however,

346

was not investigated up to now. Therefore, we hypothesized that chemokines are possibly 15

347

citrullinated by PPAD during P. gingivalis infection. In the gingival crevicular fluid of

348

periodontitis patients, however, CXCL8 is only present in pg/ml concentrations (66) in a milieu

349

containing mg/ml of other proteins and peptides, which makes detection of [Cit5]CXCL8 with the

350

currently available techniques a great challenge. Future studies should elucidate if CXCL8 is

351

citrullinated by PPAD in vivo by the detection of [Cit5]CXCL8 in gingival crevicular fluid of

352

periodontitis patients.

353

We only observed a low level of enzymatic citrullination of CXCL8 upon incubation with

354

purified PPAD at an E/S molar ratio of 1/10. In comparison, human PAD2 and PAD4 were able

355

to citrullinate 50% of the NH2-terminal Arg in CXCL8 in 10 min at an E/S molar ratio of 1/200

356

(8). Also for CCL3, another important inflammatory chemokine mediator in periodontitis (56,57),

357

no citrullination could be detected. Furthermore, no significant citrullination of CXCL8 could be

358

measured when testing several P. gingivalis strains by a recently developed detection method for

359

citrullinated proteins (54). In contrast, rapid cleavage of CXCL8 by the gingipains expressed by

360

P. gingivalis occurred. The processing of CXCL8(1-77) into CXCL8(9-77) by secreted

361

gingipains removes the NH2-terminal octapeptide including the Arg5, which is the residue known

362

to be citrullinated by human PAD (8). Another potential explanation for the failure to detect

363

[Cit5]CXCL8 can be related to low citrullinating activity of PPAD on Arg5 of CXCL8. Indeed,

364

previous research indicated that PPAD preferentially targets COOH-terminal Arg, in contrast to

365

human PAD which efficiently citrullinates NH2-terminal or internal Arg (34). However, as only

366

Citrullination and proteolytic processing of chemokines by Porphyromonas gingivalis.

The outgrowth of Porphyromonas gingivalis within the inflammatory subgingival plaque is associated with periodontitis characterized by periodontal tis...
1MB Sizes 25 Downloads 4 Views