FPRL1 Inhibitory Protein) That Inhibits FPRL1 and FPR This Information Is Current As of October 7, 2021

A Homolog of Formyl Peptide Receptor-Like 1 (FPRL1) Inhibitor from Staphylococcus aureus (FPRL1 Inhibitory Protein) That Inhibits FPRL1 and FPR This information is current as of October 7, 2021. Cristina Prat, Pieter-Jan Haas, Jovanka Bestebroer, Carla J. C. de Haas, Jos A. G. van Strijp and Kok P. M. van Kessel J Immunol 2009; 183:6569-6578; Prepublished online 21 October 2009;

doi: 10.4049/jimmunol.0801523 Downloaded from http://www.jimmunol.org/content/183/10/6569

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Homolog of Formyl Peptide Receptor-Like 1 (FPRL1) Inhibitor from Staphylococcus aureus (FPRL1 Inhibitory Protein) That Inhibits FPRL1 and FPR1

Cristina Prat,*† Pieter-Jan Haas,* Jovanka Bestebroer,* Carla J. C. de Haas,* Jos A. G. van Strijp,* and Kok P. M. van Kessel2*

The members of the formyl peptide receptor (FPR) family are involved in the sensing of chemoattractant substances, including bacteria-derived N-formylated peptides and host-derived peptides and proteins. We have recently described two chemoattractant receptor inhibitors from Staphylococcus aureus. Chemotaxis inhibitory protein of S. aureus (CHIPS) blocks the formyl peptide receptor (FPR) and the receptor for complement C5a (C5aR), while FPR-like 1 (FPRL1) inhibitory protein (FLIPr) blocks the FPRL1. Here, we describe another staphylococcal chemoattractant-inhibiting protein with 73% overall homology to FLIPr and identical first 25 aa, which we termed FLIPr-like. This protein inhibits neutrophil calcium mobilization and chemotaxis induced Downloaded from by the FPRL1-ligand MMK-1 and FPR-ligand fMLP. While its FPRL1-inhibitory activity lies in the comparable nanomolar range of FLIPr, its antagonism of the FPR is ϳ100-fold more potent than that of FLIPr and comparable to that of CHIPS. The second N-terminal phenylalanine was required for its inhibition of the FPR, but it was dispensable for the FPRL1. Furthermore, the deletion of the first seven amino acids reduced its antagonism of the FPRL1, and the exchange of the first six amino acids with that of CHIPS-conferred receptor specificity. Finally, studies with cells transfected with several chemoattractant receptors confirmed that FLIPr-like specifically binds to the FPR and FPRL1. In conclusion, the newly described excreted protein from S. aureus, http://www.jimmunol.org/ FLIPr-like, is a potent inhibitor of the FPR- and FPRL1-mediated neutrophil responses and may be used to selectively modulate these chemoattractant receptors. The Journal of Immunology, 2009, 183: 6569–6578.

eutrophils are crucial in the initial host defense against chemoattractants for human leukocytes, and they mediate directed microorganisms, but they also contribute to the patho- motility, phagocytosis, exocytosis, and superoxide anion genera- N genesis of inflammatory diseases. To migrate from the tion (5, 6). In addition to formylated peptides, the FPR can be blood stream toward the site of infection, they are guided by a activated by peptides derived from several viral proteins (7, 8). gradient of chemotactic factors. These chemoattractants are de- The FPRL1, on other hand, recognizes a variety of ligands of dif- by guest on October 7, 2021 rived from pathogens, the complement system, host cells and tis- ferent origin and structure. These include the endogenous antiin- sues, or even phagocytes themselves and signal via a family of flammatory lipid mediator lipoxin A4 and amyloid-related pep- 3 seven-transmembrane, G protein-coupled receptors (GPCRs). tides, bacterial and viral peptides, and peptides derived from The formyl peptide receptor (FPR) gene cluster contains the synthetic libraries (1–3, 7). Initially the FPR expression has been FPR (formylated peptide receptor; FPR1), FPRL1 (formyl peptide described in monocytes, neutrophils, and in microglial and den- receptor-like 1; FPR2/ALX), and FPRL2 (formyl peptide receptor- dritic cells but it is now also found in nonhematopoietic cells and like 2; FPR3) (1–3). The FPR is the high-affinity receptor for tissues. The FPRL1 is also expressed in a large variety of cells and formylated peptides leaking from growing bacteria and therefore organs including monocytes, neutrophils, macrophages, and mi- serves as an important innate immune recognition receptor (4). croglial cells. Neutrophils express FPR and FPRL1 but not FPRL2, Formylated peptides, such as fMLP, are some of the most potent while monocytes express all three members (1, 7). FPRL1 does not only play a role during different stages of the innate immune response, but increasing evidence shows its implication in the patho- *Medical Microbiology, University Medical Center Utrecht, Utrecht, the Netherlands; and †Microbiology Department, Hospital Universitari Germans Trias i Pujol, Univer- genesis of amyloidogenic diseases (9). Both the FPR and FPRL1 sitat Autonoma de Barcelona, Barcelona, Spain trigger many neutrophil functions, including chemotaxis, exocy- Received for publication May 9, 2008. Accepted for publication September 14, 2009. tosis, and superoxide generation. A transient rise in intracellular The costs of publication of this article were defrayed in part by the payment of page calcium is required, albeit not sufficient, for triggering optimal charges. This article must therefore be hereby marked advertisement in accordance chemoattractant-induced responses (5). with 18 U.S.C. Section 1734 solely to indicate this fact. Staphylococcus aureus is a commensal of the human skin but 1 This work was supported by Grants Fondo de Investigacion Sanitaria, Instituto de Salud Carlos III (expediente 01/F062) and Sociedad Espanõla de Enfermedades In- can cause a wide spectrum of infections spanning from trivial skin fecciosas y Microbiologia Clinica (both to C. P.). infections to severe septic diseases. This pathogen can produce 2 Address correspondence and reprint requests to Dr. Kok P. M. van Kessel, Medical infections in essentially every human organ or tissue. It exports a Microbiology, University Medical Center Utrecht, room G04.614, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail address: [email protected] variety of virulence factors to the cell surface and extracellular milieu that contribute to the pathogenesis of infection. These pro- 3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; A␤, amyloid ␤; CHIPS, chemotaxis inhibitory protein of S. aureus; FLIPr, FPRL1 inhibitory proteins cause direct cellular damage and/or interact with host defense tein; FPR, formyl peptide receptor; FPRL, FPR-like receptor; HSA, human serum factors (10, 11). To cause deep infections and survive in the host, albumin; LTB , leukotriene B ; PAF, platelet-activating factor; PrP, prion protein. 4 4 the bacteria must evade the immune system by producing mole- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 cules that target critical processes in nearly every stage of the www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801523 6570 STAPHYLOCOCCAL PROTEIN THAT ANTAGONIZES FPRL1 AND FPR

innate host response to infection (12–14). We have previously re- phenylalanines, and cloning in pRSETB vector by overlap extension ported the molecular properties of several secreted proteins from S. PCR as described above. Three chimeras were also constructed. For CHIPS -FLIPr-like and CHIPS -FLIPr , the first six N-termi- aureus that contribute to the evasion of the human defense system. 1–6 7–104 1–6 7–105 nal amino acids of FLIPr-like and FLIPr were substituted for the first six These newly described proteins interfere with several arms of the amino acids of CHIPS, and for the reverse chimera FLIPr1–6-CHIPS7–121, innate immune response, such as initial rolling of neutrophils (15), the amino acids 1–6 of CHIPS were exchanged for those of FLIPr. The complement activation (16), and chemoattractant-mediated activa- competent cells BL21(DE3) E. coli (Novagen) were used to express the tion and migration of phagocytes (17). The FPRL1-inhibitory pro- mutants and chimeras. After verification of the correct sequence, all HIS- tagged proteins were expressed and purified using the Pro-Bond resin (In- tein or FLIPr (18) is a secreted staphylococcal antiinflammatory vitrogen) or HiTrap chelating HP column following the manufacturer’s protein that potently inhibits FPRL1-specific ligands, such as the instructions and cleaved afterward with enterokinase as described above. synthetic peptides MMK-1 (19) and WKYMVM (20), and the en- ␤ ␤ Synthetic peptides dogenous proteins amyloid 1–42 (A 42) (21, 21) and prion protein fragment PrP106–126 (22). FLIPr binds directly to the GPCR The peptides of the N-terminal amino acids 1–6 of FLIPr as well as FLIPr-

FPRL1, thereby acting as an antagonist. like (H-FFSYEW-NH2) were synthesized by Dr. R. van der Zee (Institute A BLAST search through sequenced S. aureus genomes re- of Infectious Diseases and Immunology, Utrecht University) and of CHIPS (H-FTFEPF-NH ) by Dr. John A.W. Kruijtzer as described before (23). vealed that S. aureus encodes for a protein with 73% homology to 2 FLIPr. This protein, which we named FLIPr-like, consists of 104 Leukocyte isolation aa and is preceded by a classical signal peptide with an AXA Venous blood was collected from healthy volunteers into tubes containing motif. In this study we show that FLIPr-like inhibits FPRL1-me- sodium heparin. Blood was diluted with an equal volume of PBS and diated neutrophil activation as was determined by intracellular cal- layered onto a gradient of Ficoll (Amersham Biosciences) and Histopaque Downloaded from cium mobilization and chemotaxis. Moreover, FLIPr-like also ef- (Sigma-Aldrich). After centrifugation for 20 min at 397 ϫ g and 21°C, ﬁciently inhibits fMLP-induced responses comparable to CHIPS. mononuclear cells and polymorphonuclear neutrophils were collected sep- This newly described excreted protein of S. aureus is an antagonist arately from Ficoll and Histopaque interfaces, respectively. Cells were washed with cold RPMI 1640 containing 25 mM HEPES, L-glutamine of both the FPR and FPRL1. Deletion and substitution mutants (Biowhittaker), and 0.05% human serum albumin (HSA; Sanquin) (RPMI- indicate that the dual activity of FLIP-like is allocated to different HSA). For elimination of erythrocytes, the neutrophil pellet was subjected ϫ sites within the protein. to a hypotonic shock with distilled H2O for 30 s followed by 10 con-

centrated PBS to restore isotonicity. After washing, cells were resuspended http://www.jimmunol.org/ Materials and Methods in RPMI-HSA. Reagents Calcium mobilization MMK-1 (H-LESIFRSLLFRVM-OH) was synthesized by Sigma-Genosys. To determine activation of neutrophils by chemoattractants the transient The hexapeptide WKYMVM-NH2 was from Bachem AG (Switzerland), increase in free intracellular calcium concentration was measured by flow its D-conformer WKYMVm was synthesized by J. A.W. Kruijtzer (De- cytometry (18, 24). For this purpose, neutrophils (5 ϫ 106 cells/ml) were partment of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sci- ␮ 4 loaded with 2 M Fluo-3-AM for 20 min at room temperature, protected ences, Utrecht, The Netherlands), and WRWWWW-NH2 (WRW ) and from light with gentle agitation. The cells were washed, resuspended in carboxyfluorescein (FAM)-labeled WKYMVM were from Phoenix Phar- RPMI-HSA to 5 ϫ 106 cells/ml, and incubated with buffer or protein for at 6 maceuticals. fMLP, recombinant C5a, propidium iodide, and anti-FLAG least 1 min. Before stimulation, cells were diluted to 1 ϫ 10 cells/ml in a by guest on October 7, 2021 mAb were from Sigma-Aldrich. The fluorescein conjugate of formyl-Nle- volume of 180 ␮l while maintaining the inhibitory protein concentration. Leu-Phe-Nle-Tyr-Lys (abbreviated as FITC-fMLP) and Fluo-3-AM (ace- The basal fluorescence level for Fluo-3 was monitored at 530 nm for toxymethyl ester) were obtained from Molecular Probes/Invitrogen. IL-8 ϳ8 s after which 12.5 ␮lof10ϫ concentrated stimulus was added. The and neutrophil-activating protein 2 were from PeproTech. Platelet-activat- sample tube was rapidly placed back to the sample holder and the fluo- ing factor (PAF)-16 and purified human C3a were from Calbiochem rescence measurement continued up to 52 s. Neutrophils were gated based (Merck Biosciences). LTB4 (leukotriene B4) was purchased from Cascade on scatter parameters to exclude cell debris, and the mean fluorescence Biochem. Anti-HA mAb (clone 12CA5) was from Roche Applied Science. value at basal level was subtracted from the value at peak level (at 30 s). Allophycocyanin-labeled goat anti-mouse IgG was from BD Biosciences. The different stimulus concentrations were expressed relative to the max- Cloning, expression, and purification of FLIPr-like imal response for each individual stimulus. Primers were designed according to the published sequence of the gene Chemotaxis MW1038 (from S. aureus subsp. aureus MW2) for the cloning of FLIPr- like into pRSETB vector (Invitrogen) and were manufactured by Invitro- Neutrophil migration was measured in a 96-multiwell transmembrane sys- ␮ gen. Recombinant protein was generated by PCR and cloned into the tem (ChemoTX; Neuro Probe) using an 8- m pore size polycarbonate ␮ EcoRI and XbaI site of the pRSETB vector by overlap extension PCR and membrane (18). Cells were labeled with 2 M calcein-AM for 20 min, ϫ 6 propagated in TOP10FЈ Escherichia coli (Novagen) as described before washed, and resuspended to a concentration of 2.5 10 cells/ml in HBSS ␮ (18). After verification of the correct sequence, the protein was expressed with 1% HSA. Wells were filled with 29 l of chemoattractant, and the in Rosetta-gami(DE3)pLysS E. coli (Novagen) by induction with 1 mM membrane holder was assembled. Cells were incubated with different con- ␮ isopropyl-thiogalactopyranoside (Roche). HIS-tagged protein was isolated centrations of FLIPr or FLIPr-like and 25 l was placed as a droplet on the under denaturing conditions using a 5-ml HiTrap chelating HP column (GE membrane. After incubation for 30 min at 37°C in a humidified 5% CO2 Healthcare Europe) following the manufacturer’s protocol and cleaved af- atmosphere, the membrane was washed extensively with PBS, and the terward with enterokinase (Invitrogen) to obtain the native protein. To fluorescence was measured in a FlexStation (Molecular Devices). Percent- ␮ remove the HIS-tag, the cleaved protein mixture was passed over a nickel age migration was calculated relative to wells containing 25 l of cells. column for a second time and selectively eluted with phosphate buffers of pH 6 and pH 5.3. The separate fractions were analyzed on a 15% SDS- Binding to leukocytes PAGE gel and showed two different bands of purified protein correspond- To determine the binding of FLIPr-like to different cell types, 50 ␮lof ing to 12 and 11 kDa, respectively. The corresponding fractions were blood leukocytes at 5 ϫ 106 cells/ml was incubated for 30 min on ice with Ϫ pooled and dialyzed separately toward PBS and stored at 20°C. The buffer or a concentration range FITC-labeled protein. Cells were washed native protein FLIPr-like was mixed with 0.1 mg/ml FITC (Sigma-Aldrich) and resuspended in RPMI-HSA. The fluorescence of 17,500 cells was mea- in 0.1 M carbonate buffer (pH 9.5) and subsequently separated from free sured by flow cytometry, and the different leukocyte populations were FITC by a desalting column, as described earlier for FLIPr and CHIPS identified based on forward and sideward scatter parameters. For binding in (17, 18). whole blood, 50 ␮l of EDTA anticoagulated blood was used, and the sam- Construction of mutants and chimeras ples were treated with FACS lysing solution (BD Biosciences) before analysis. To discriminate between different leukocyte subpopulations, leuko- Site-directed mutagenesis was performed on the N-terminus of both FLIPr cytes were labeled with mAbs and selected by specific gating on scatters and FLIPr-like by deletion of the first or the first two amino acids, both and fluorescence. Specific labels used were anti-CD3-RPE/Cy5 for T cells, The Journal of Immunology 6571

leader sequence, and purified using nickel affinity chromatography. To remove the HIS-tag, the protein was treated with enterokinase and the cleaved protein mixture was passed over a nickel column again. Selective elution with phosphate buffers of different pH showed two different bands of purified protein corresponding to 12 and 11 kDa, respectively, on a 15% SDS-PAGE gel (Fig. 1B). N-terminal sequencing of the two proteins identified the 12-kDa band as the native FLIPr-like (first five N-terminal amino acids: FFSYE) and the 11-kDa band as a cleavage product lacking the

first seven amino acids, FLIPr-like8–104 (first five N-terminal amino acids: GLEIA; Fig. 1A, underlined).

FIGURE 1. Sequence and purification of FLIPr-like. A, Sequence align- ment of FLIPr and FLIPr-like using ClustalW. The putatively excreted FLIPr-like inhibits neutrophil activation by MMK-1 and fMLP protein is depicted in bold with black boxes marking mismatched res- As FLIPr antagonizes the FPRL1, we examined whether FLIPr- idues and gray boxes indicating the signal-peptidase cleavage site. B, like also blocks the activation of this receptor. For this purpose, Coomassie blue-stained SDS-PAGE gel (15%) showing purified FLIPr- ␮ like protein (lane 1; 12 kDa), mixture after enterokinase cleavage (lane neutrophils were preincubated with 3 g/ml FLIPr-like, FLIPr, or CHIPS and compared with control cells for mobilization of intra- 2), FLIPr-like8–104 (lane 3; 11 kDa), and HIS-tagged protein (lane 4; 15.5 kDa). M indicates marker. cellular calcium in response to a concentration range of the FPRL1-specific ligand MMK-1. Fig. 2A shows a representative Downloaded from change in intracellular calcium concentration of control and FLIPr- anti-CD19-PE for B cells, anti-CD16/CD56-PE (plus negative for anti- like-treated neutrophils stimulated with MMK-1 as measured by CD3-RPE/Cy5) for NK cells, and anti-CD14-PE for monocytes. an increase in fluorescence. Increasing concentrations of MMK-1 gradually increased the mobilization of intracellular calcium in Binding to HEK293 and HL60 transfectants control cells. Both FLIPr and FLIPr-like completely inhibited the Human embryonic kidney cells (HEK293; obtained from the American cell response to MMK-1, while CHIPS did not (Fig. 2B). As FLIPr http://www.jimmunol.org/ Type Culture Collection) were transfected with plasmids containing a is also a moderate inhibitor of the FPR, the effect of FLIPr-like on FLAG-tagged FPR, FPRL1, or C5aR or HA-tagged FPRL2, as described fMLP-induced neutrophil activation was examined. Incubation of before (18, 25). After 2 days, transfected cells were harvested with EDTA and incubated with 10 ␮g/ml mouse anti-FLAG or anti-HA mAb for 45 neutrophils with FLIPr-like shifted the fMLP-induced calcium mo- min on ice. Cells were then washed and subsequently incubated with sat- bilization curve toward higher concentrations (Fig. 2, C and D). In urating concentration of allophycocyanin-labeled goat anti-mouse IgG to- comparison to FLIPr, its homolog FLIPr-like was a more potent gether with different concentrations of FITC-labeled protein, FLIPr-like, inhibitor of fMLP-induced neutrophil activation and was almost as FLIPr, or CHIPS. Cells were incubated for 45 min on ice, washed, and resuspended in 200 ␮l of RPMI-HSA containing 5 ␮g/ml propidium io- active as CHIPS (Fig. 2D). Because FLIPr-like mimicked the activity of CHIPS on fMLP, the ability of FLIPr-like to block the dide. FL1 (FITC), FL2 (propidium iodide) and FL4 (allophycocyanin) by guest on October 7, 2021 were measured in a FACSCalibur flow cytometer (BD Biosciences). As a C5a-induced calcium mobilization was tested as well. FLIPr-like control, HEK293 cells transfected with an empty pcDNA3.1 vector were and its homolog FLIPr did not affect the C5a-mediated calcium used. The binding of FITC-labeled proteins was determined by selecting mobilization, while CHIPS effectively inhibited this response (Fig. live cells (propidium iodide-negative) expressing the receptor as indicated by positive staining with anti-FLAG or anti-HA mAb (18). Binding to 2, E and F). Additionally, FLIPr-like and FLIPr did not inhibit the nontransfected cells within the same sample was used to establish back- calcium mobilization in neutrophils stimulated with optimal con- ground values. Human promyelocytic leukemia HL60 cells stable trans- centrations of C3a (C3aR), IL-8 (CXCR1 and CXCR2), neutro- fected with the FPR, FPRL1, and FPRL2 were provided by F. Boulay phil-activating protein 2 (CXCR2), PAF (PAFR), or LTB (BLTR) (Laboratoire Biochimie et Biophysique des Systemes Integres, Grenoble, 4 France) (26, 27). Cells were cultured in RPMI 1640 medium containing (data not shown). Finally, to examine the effects of these proteins G418 (omitted for the parent HL60 cells), and the maximal density was on the FPRL2, we performed experiments with F2L, the acetylated maintained below 2 ϫ 106 cells/ml. Cells were centrifuged at each passage. peptide derived from cleaved heme-binding protein with high af- Fresh cells (5 ϫ 106 cells/ml in RPMI-HSA) were incubated with fluores- finity for FPRL2. Although neutrophils do not express FPRL2, a cent-labeled ligands for 45 min on ice under gentle agitation, washed once, concentration of 4 ␮M F2L induced a significant calcium mobili- and fixed with 1% paraformaldehyde. For competition experiments, cells ␮ were first incubated for 15 min with unlabeled protein or peptide. Curve zation that was inhibited by 3 g/ml FLIPr-like and FLIPr but not

fitting and calculation of inhibitory concentration value (IC50) was per- by CHIPS. This response was probably mediated via the FPRL1 formed by nonlinear regression analysis of the dose-response curves gen- and could therefore be inhibited by FLIPr-like and FLIPr. While erated using Prism 5 (GraphPad Software). monocytes are described to express FPRL2, we were unable to show stimulation by nanomolar concentrations F2L. FLIPr-like it- Results self at concentrations up to 30 ␮g/ml did not induce a calcium Identification of FLIPr-like mobilization in human neutrophils. The tblastn algorithm search of the sequenced S. aureus genomes with FLIPr identified a protein with 73% homology in two of the nine genomes: hypothetical protein MW1038 (S. aureus subsp. FLIPr-like inhibits neutrophil chemotaxis to MMK-1 and fMLP aureus MW2) and hypothetical protein SAS1089 (S. aureus subsp. Both MMK-1 and fMLP induce chemotactic migration of neutro- aureus MSSA476). The genes encode for an extracellular protein phils. We investigated the effects of FLIPr-like, FLIPr, and CHIPS of 104 aa preceded by a classical signal peptide with an AXA motif on neutrophil chemotaxis in a 96-well transmembrane system. that is cleaved at the bacterial membrane (Fig. 1A). The homology Both FLIPr-like and FLIPr showed a comparable concentration- of this newly identified protein to CHIPS is 27% for the processed dependent inhibition of chemotaxis to MMK-1, while even 10Ϫ6 mature protein with a high degree of homology in the leader pep- M CHIPS had no effect (Fig. 3A). FLIPr-like and CHIPS effec- tides. This homolog of FLIPr, which we named FLIPr-like, was tively inhibited neutrophil chemotaxis toward fMLP (Fig. 3B), and cloned, expressed in E. coli as a HIS-tagged protein without the FLIPr only showed an effect at a high concentration (Fig. 3B). 6572 STAPHYLOCOCCAL PROTEIN THAT ANTAGONIZES FPRL1 AND FPR Downloaded from http://www.jimmunol.org/

FIGURE 2. FLIPr-like inhibits MMK-1- and fMLP-induced calcium mobilization. Fluo-3-loaded neutrophils were incubated with buffer (F), 3 ␮g/ml FLIPr-like (f), FLIPr (ࡗ), or CHIPS (Œ) for 20 min at room temperature. To monitor calcium mobilization by flow cytometry, each sample was first measured for ϳ8 s to determine the basal fluorescence. Subsequently, MMK-1 (A and B), fMLP (C and D), or C5a (E and F) was added, and the sample was immediately placed back in the sample holder to continue the measurement. Examples are shown of the change in fluorescence for control (dark line) and FLIPr-like- (gray line) treated cells stimulated with 3 ϫ 10Ϫ7 M MMK-1 (A), 3 ϫ 10Ϫ9 M fMLP (C), and 3 ϫ 10Ϫ10 M C5a (E). For reasons of clarity, the gray line was set slightly offset. Cells were stimulated with increasing concentrations of agonist, and the difference in fluorescence values after and Ϫ Ϫ Ϫ before stimulation was expressed relative to the maximal response induced in control cells stimulated with 10 6 M MMK-1 (B), 10 6 M fMLP (D), or 10 8 by guest on October 7, 2021 M C5a (F). Data are means Ϯ SEM of three independent experiments.

The first seven amino acids of FLIPr-like are crucial for fMLP cleavage product lacking the first seven amino acids (FLIPr-like8–104). but not MMK-1 inhibition This deletion mutant was also isolated and used to investigate Cleavage of the HIS-tag from the recombinant FLIPr-like protein the importance of the N terminus in FLIPr-like activity. Cells with enterokinase generated the native protein as well as an additional were incubated with increasing concentrations FLIPr-like8–104, native FLIPr-like, FLIPr, or CHIPS and stimulated with an optimal concentration MMK-1 or fMLP. Loss of the first seven

amino acids in FLIPr-like (FLIPr-like8–104) resulted in diminished inhibitory potency of the MMK-1-induced neutrophil activation as compared with native FLIPr-like (Fig. 4A) that showed maximal inhibition up to 10Ϫ10 M. While FLIPr-like

inhibits fMLP-induced neutrophil activation, FLIPr-like8–104 lost all activity (Fig. 4B). These results suggest an active site in the N terminus of FLIPr-like for FPR inhibition.

Function of FLIPr and FLIPr-like N-terminal mutants and chimeras To further investigate which residues are important in the activity of FLIPr-like, calcium mobilization assays were per- FIGURE 3. FLIPr-like inhibits neutrophil chemotaxis toward MMK-1 formed with several mutants, chimeras, and peptides. Deletion and fMLP. Calcein-labeled neutrophils were incubated with different con- of the ﬁrst N-terminal amino acid, a phenylalanine, in FLIPr- centrations FLIPr-like (f), FLIPr (ࡗ), or CHIPS (Œ) and allowed to mi- Ϫ like (FLIPr-like ) did not affect the inhibition of MMK-1- grate across a 8-␮m pore membrane toward 3.3 ϫ 10 8 M MMK-1 (A)or ⌬F1 3.3 ϫ 10Ϫ9 M fMLP (B). Results are expressed as percentage inhibition of and fMLP-stimulated neutrophils (Fig. 5, A and B). However, the migration of buffer-treated cells, and all samples were run in triplicate the mutant lacking the ﬁrst two N-terminal phenylalanines for two experiments. Percentage migration of control cells toward fMLP (FLIPr-like⌬F1F2) lost the inhibition for the fMLP-induced ac- was 44.4 Ϯ 4.6 (mean Ϯ SD) and toward MMK-1 was 51.8 Ϯ 3.6, and tivation (Fig. 5B) but retained full inhibition for the MMK-1- spontaneous migration was 3.4 Ϯ 1.3. induced response (Fig. 5A). It was remarkable that both mutants The Journal of Immunology 6573

FIGURE 4. The ﬁrst seven N-terminal amino acids of FLIPr-like are crucial for fMLP- but not the MMK- 1-induced calcium mobilization. The activity of FLIPr- like8–104 that lacks the ﬁrst seven amino acids was compared with FLIPr-like, FLIPr, and CHIPS in a calcium mobilization assay with neutrophils. Fluo-3-loaded cells were incubated with different concentrations of inhibitory protein and challenged with the FPRL1 agonists MMK-1 (3 ϫ 10Ϫ7 M; A) and FPR agonist fMLP (3 ϫ 10Ϫ9 M; B). Data are expressed as percentage inhibition of buffer-treated cells and are the mean of three independent experiments.

showed a slightly more potent activity toward MMK-1 as com- FLIPr-like, FLIPr, or CHIPS. The chimera CHIPS1–6-FLIPr7–105 pared with the parent FLIPr-like protein. The same N-terminal showed no inhibition of MMK-1- (Fig. 5E) or fMLP-induced deletion mutants were constructed for FLIPr. Both mutants, (Fig. 5F) neutrophil activation. Surprisingly, the chimera

FLIPr⌬F1 and FLIPr⌬F1F2, retained all inhibitory activity toward CHIPS1–6-FLIPr-like7–104 showed only limited inhibition of the Downloaded from MMK-1 (Fig. 5C). As shown for FLIPr-like, deletion of both MMK-1 response (Fig. 5E) and no inhibition of the fMLP re- phenylalanines (FLIPr⌬F1F2) eliminates the inhibitory activity sponse (Fig. 5F). The reverse chimera FLIPr1–6-CHIPS7–121 for fMLP (Fig. 5D). To further elucidate the importance of the retained inhibition of the fMLP response (Fig. 5F) and, like N terminus, chimeras were constructed in which the ﬁrst six CHIPS, did not inhibit the MMK-1 response (Fig. 5E). These amino acids were exchanged with the remaining sequence of results indicate the N terminus as the active site of both FLIPr http://www.jimmunol.org/ by guest on October 7, 2021

FIGURE 5. Importance of the N- terminal phenylalanines in FLIPR- like and FLIPr. The activities of different N-terminal-deletion mutants ⌬ ⌬ ( F1, F1F2, and FLIPr-like8–104) and chimeras of the ﬁrst six amino acids of CHIPS with FLIPr (CHIPS- FLIPr), CHIPS with FLIPr-like (CHIPS-FLIPR-like), and FLIPr (equal to FLIPr-like) with CHIPS (FLIPr- CHIPS) were tested in a calcium mobilization assay with neutrophils stimulated with MMK-1 (3 ϫ 10Ϫ7 M; A, C, and E) or fMLP (3 ϫ 10Ϫ9 M; B, D, F). Cells were preincubated with increasing concentrations of antagonists. Data are the means of three or more experiments. 6574 STAPHYLOCOCCAL PROTEIN THAT ANTAGONIZES FPRL1 AND FPR

even more potently when compared with the CHIPS1–6 peptide. Complete FLIPr-like protein was ϳ100 times more potent than

the peptides (Fig. 6). Neither FLIPr1–6 nor CHIPS1–6 peptide inhibited the MMK-1-induced response (data not shown).

FLIPr-like binds to cells expressing FPR or FPRL1 To verify the direct association of FLIPr-like with leukocytes expressing the FPRL1, the binding of FLIPr-likeFITC to neutrophils, monocytes, and lymphocytes was measured by flow cytometry. Fig. 7A shows a concentration-dependent binding of FLIPr-likeFITC to neutrophils, monocytes, and a proportion of lymphocytes. Also, in whole blood, FLIPr-likeFITC bound to neutrophils and monocytes (data not shown). Further analysis of lym- FIGURE 6. Synthetic N-terminal peptide inhibits the FPR. The syn- phocyte subpopulations revealed a strong binding to B and NK thetic peptides FLIPr and CHIPS were compared with native FLIPr- 1–6 1–6 cells and not to T cells, as determined by double labeling with like for the inhibition of fMLP-induced calcium mobilization in neutro- specific mAbs (Fig. 7B). These results are similar to those previ- phils. Results are expressed as percentage inhibition of cells stimulated FITC with 5 ϫ 10Ϫ10 M fMLP and are the mean of two experiments. ously observed for FLIPr (18). Because B and NK cells are not known to definitively express members of the FPR family, cells

were preincubated with unlabeled FLIPr-like and FPR agonists to Downloaded from and FLIPr-like for the FPR as was shown before for CHIPS displace FLIPR-likeFITC binding (Fig. 7C). FLIPr-like displaced (16). Additionally, part of the N terminus seems to be important the binding of FILR-likeFITC to all leukocyte subtypes. For B and for the activity toward the FPRL1. The N-terminal HIS-tagged NK cells none of the FPR agonists inhibited the binding of FILR- forms of the proteins were also tested for their inhibitory ca- likeFITC. For monocytes and neutrophils that express all FPRs, pacity. Both HIS-tagged FLIPr and FLIPr-like retained their only WKYMVm showed displacement of FLIPr-likeFITC binding

activity on MMK-1-induced responses but lost their effect on while the high-affinity FPR ligand fMLP and FPRL1-specific li- http://www.jimmunol.org/ fMLP-induced responses (data not shown). We have previously gand MMK-1 were ineffective (Fig. 7C). described the importance of the first phenylalanine in CHIPS The FITC-labeled protein was also used in binding experi- for the FPR-blocking activity and showed that a synthetic pep- ments with HEK293 cells transiently transfected with FLAG- or tide compromising the first six N-terminal amino acids blocks HA-tagged versions of FPR, FPRL1, FPRL2, or C5aR. HEK293 fMLP-induced activation (23). Because the first six amino acids cells expressing the different receptors were analyzed for bind- of FLIPr closely resemble the allowed substitutions within this ing of FLIPr-likeFITC, while the nontransfected cells within the six-amino acid CHIPS peptide (23), synthetic peptides of same sample were used to determine background binding. FITC CHIPS and FLIPr were compared. The FLIPr1–6 peptide, rep- FLIPr-like bound clearly to HEK293 cells transfected with resenting the first six amino acids of FLIPr as well as FLIPr-like FPRL1, with some binding to cells transfected with FPR and by guest on October 7, 2021 (see Fig. 1A), effectively inhibited the fMLP-induced response, FPRL2 and not with C5aR (Fig. 8A). As a control, CHIPSFITC

FIGURE 7. FLIPr-like binds to neutrophils, monocytes, and a subpopu- lation of lymphocytes. A, Peripheral blood leukocytes were incubated with FLIPr-likeFITC (0.03–2.60 ␮g/ml) for 30 min on ice. Binding was analyzed with a flow cytometer, and the different cells (neutrophils (F), monocytes (f), lymphocytes (Œ) were identified based on scatter parameters and anti- CD14-PE staining for monocytes. Data are from a representative experiment. B, Binding of 2 ␮g/ml FLIPr-likeFITC to T cells (CD3-positive), B cells (CD19-positive), NK cells (CD3-negative and CD16/56-positive), monocytes (CD14-positive), and neutrophils. Re- sults are mean fluorescence Ϯ SEM of three experiments. C, Competitive binding of 1 ␮g/ml (ϳ83 nM) FLIPr- likeFITC to B cells, NK cells, monocytes, and neutrophils by unlabeled FLIPr-like (250 nM), WKYMVm (1 ␮M), MMK-1 (10 ␮M), and fMLP (10 ␮M). Data are from a representative experiment. The Journal of Immunology 6575

FIGURE 8. Binding of ﬂuorescent FLIPr-like to HEK293 and HL60 cells transfected with the FPR and FPRL1. A, HEK293 cells were transiently transfected with a vector containing FLAG-tagged human FPR, FPRL1, or C5aR or HA-tagged FPRL2. Binding of 3 ␮g/ml FLIPr- likeFITC to transfectants was determined by selecting cells stained positive for anti-FLAG or anti-HA mAb and allophycocyanin-labeled goat anti- mouse IgG Ab (open histogram). The binding to nontransfected cells was determined in the same sample (gray histogram). Data are represen- Downloaded from tative of three experiments. B, Con- centration-dependent binding of FLIPr-likeFITC to HL60 cells stably expressing the FPR, FPRL1, or FPRL2 as well as control parent cells. Results are presented as mean

fluorescence after subtraction of http://www.jimmunol.org/ autofluorescence from a representative experiment. C, Mean fluorescence values Ϯ SEM (n ϭ 3) for binding of 3 ␮g/ml (250 nM) FLIPr- likeFITC,(D) 100 nM fMLPFITC, and (E) 300 nM WKYMVMFAM to the different HL60 transfectants. ND indicates not done. by guest on October 7, 2021

bound only to FPR- and C5aR-transfected cells as previous de- expressing the FPRL1, FLIPr-like, and FLIPr as well as the spe- scribed (18) (data not shown). Additionally, undifferentiated HL60 cific agonists WKYMVM, WKYMVm, and MMK-1 and the an- cells stably transfected with the FPR, FPRL1, or FPRL2 were tagonist WRW4 efficiently prevented binding of FLIPr-likeFITC used. Fig. 8B shows a concentration-dependent binding of FLIPr- (Fig. 9D). CHIPS did not inhibit this binding. Also, the binding of likeFITC to all HL60-transfected cells with minimal association the fluorescent-labeled ligand WKYMVMFAM to HL60/FPRL1 with control cells. The most prominent binding was observed for cells was prevented by FLIPr-like, and the specific ligands, and not FPRL1-expressing cells incubated with 3 ␮g/ml (ϳ250 nM) by fMLP (Fig. 9E). The concentration dependently displacement FLIPr-likeFITC (Fig. 8C). The binding of the high-affinity FPR ligand of FLIPr-likeFITC binding to HL60/FPRL1 by unlabeled FLIPr- FITC fMLP (10 nM) to the same panel of HL60 transfectants is shown like (Fig. 9F) results in an IC50 of 2.5 nM. in Fig. 8D. The fluorescent FPRL1 ligand WKYMVMFAM bound not only to HL60/FPRL1 cells, but also to the HL60/FPR cells but not to the parent undifferentiated HL60 cells (Fig. 8E). To show spec- Discussion ificity, binding of fluorescent-labeled ligands to HL60/FPR and The activation and migration of phagocytes to the site of inflam- HL60/FPRL1 cells was measured in the presence of various un- mation is a key event in host defense against invading microor- labeled agonists or antagonists. Binding of FLIPr-likeFITC to ganisms and in the pathogenesis of several inflammatory diseases HL60/FPR cells was effectively prevented by FLIPr-like, not its (28). We have previously described a secreted protein from S. homolog FLIPr or CHIPS. The FPR agonistic peptides fMLP, aureus, FLIPr, which is a potent antagonist of the FPRL1 (18). WKYMVM, and WKYMVm only partly inhibited this binding Here, we identify a highly homologous protein from S. aureus, (Fig. 9A). Binding of WKYMVMFAM to HL60/FPR cells was named FLIPr-like, which inhibits both FPR and FPRL1. FLIPr- partly prevented by FLIPr-like and efficiently by the FPR ligands like has an overall homology of 73% with FLIPr, and their first 25 fMLP, WKYMVM, and WKYMVm and not by the specific aa are identical (Fig. 1). The homology with CHIPS, which inhibits FPRL1 ligand MMK-1 (Fig. 9B). Binding of the high-affinity li- the FPR and C5aR, is only 27% for the native excreted and pro- gand fMLPFITC to HL60/FPR cells was prevented by the peptide cessed protein. The genes for both FLIPr and FLIPr-like encode an ligands fMLP, WKYMVM, and WKYMVm and by CHIPS but identical signal peptide with an AXA cleavage motive and shows not by FLIPr-like (both at 250 nM) (Fig. 9C). For HL60 cells high homology with the signal peptide encoding CHIPS. CHIPS 6576 STAPHYLOCOCCAL PROTEIN THAT ANTAGONIZES FPRL1 AND FPR Downloaded from http://www.jimmunol.org/

FIGURE 9. Specific binding of FLIPr-like to FPR and FPRL1. A, HL60 cells transfected with FPR (A–C) or FPRL1 (D and E) were incubated for 15 min at 4°C with unlabeled FLIPr-like (250 nM), FLIPr (250 nM), CHIPS (220 nM), fMLP (10 ␮M), WKYMVM (1 ␮M), WKYMVm (1 ␮M), MMK-1 (10 ␮M), or WRW4 (10 ␮M). Subsequently, binding of FLIPr-likeFITC (83 nM) (A and D), WKYMVMFAM (10 nM) (B and E), and fMLPFITC (C) was measured for 45 min at 4°C. Data are expressed relative to binding of fluorescent ligand in the absence of a competitor. F, Displacement of FLIPr-likeFITC (83 nM) binding to FPRL1/HL60 by increasing concentrations unlabeled FLIPr-like. Results are the means Ϯ SD of two to four experiments. ND indicates not done. by guest on October 7, 2021 can be identified in the supernatant of growing S. aureus by West- has been reported to inhibit calcium mobilization, chemotaxis, and ern blot analysis (17). Also, FLIPr (SA1001) can be identified in superoxide generation (34, 35). The antiinflammatory drug piroxi- the extracellular proteome of S. aureus, as was determined by 2D- cam also inhibits neutrophil activation by FPR but not by FPRL1 PAGE and MALDI-TOF mass spectrometry (11). The presence of agonists due to the competition with the natural ligand (36, 37). a leader peptide in the gene encoding FLIPr-like that is identical to Furthermore, through screening of hexapeptide libraries, WRW4 that of FLIPr and is highly homologous to the leader peptide of was identified as a novel, potent, FPRL1 antagonist (37). This CHIPS makes it likely that the processed protein can be found in peptide inhibits the increase in intracellular calcium induced by the secretome of S. aureus. Additionally, a specific rabbit anti- several FPRL1 agonists. WRW4 also inhibits the activation of serum identified FLIPr-like in the secretome of S. aureus strain FPRL2 by the specific ligand F2L, a heme-binding protein frag- MW2 by Western blot (data not shown). The genes for FLIPr ( flr) ment peptide (38). Recently, modifications of a substituted quina- or FLIPr-like ( fll) cluster with other known and potential immune zoline compound (Quin-C1) convert this selective nonpeptide evasion molecules on the genomes of all sequenced S. aureus FPRL1 ligand into an antagonists (39). We have previously de- strains. These include SCIN (staphylococcal complement inhibi- scribed a potent FPRL1 antagonist that is secreted by S. aureus. tor) homologs, Efb (extracellular fibrinogen-binding protein), and FLIPr inhibits the chemotaxis and intracellular calcium mobiliza- its homolog Ecb (29). This region represents a novel immune eva- tion induced by the synthetic FPRL1 agonists WKYMVM, sion cluster (IEC-2) in S. aureus, whereas CHIPS is located on a WKYMVm, and MMK-1 as well as the endogenous peptides A␤ bacteriophage-localized cluster IEC-1 (30). 42 and prion protein fragment PrP (18). Additionally, FLIPr Phagocytes, but also many other cell types including cells of the 106–126 nervous system, express the formyl peptide receptors FPR (FPR1), modestly inhibits the fMLP-induced activation of neutrophils. FPRL1 (FPR2/ALX), and FPRL2 (FPR3) (3). Expression of Here, we show that the newly identified S. aureus protein FLIPr- FPRL2 is restricted to myeloid cells, including monocytes and like inhibits both FPR and FPRL1 agonist-induced calcium dendritic cells, and human neutrophils only express FPR and mobilization and chemotaxis in human neutrophils at nanomolar 4 FPRL1 (2, 3, 7). This receptor family plays a crucial role in the concentrations. The hexapeptide WRW inhibits the WKYMVm- recognition of microorganisms and inflammatory responses, but induced calcium mobilization in FPRL1-expressing RBL-2H3 only a few antagonists have been reported. Replacement of the cells at micromolar concentrations (37). Ligands acting on other formyl group of fMLP with butyloxylcarbonyl resulted in an an- neutrophil GPCRs are not affected by FLIPr-like. The FPRL2 se- tagonistic peptide (31), and other modifications of such peptides lective agonist F2L (40) induced a calcium flux in neutrophils at can also influence their antagonistic power (32, 33). Cyclosporine micromolar range and could be inhibited by both FLIPr and FLIPr- H has been developed as a potent and selective FPR antagonist and like. F2L is described to activate human FPRL1 as well and to The Journal of Immunology 6577 target the mouse low-affinity receptor Fpr2 (41). Because neutro- not essential for inhibition of the C5aR, as a variant lacking those phils do not express FPRL2, we think that the response was me- amino acids retains full activity (45). In contrast, the N-terminal diated by FPRL1 and could therefore be inhibited by FLIPr-like region of CHIPS is critical for inhibition of the FPR. In particular, and FLIPr. Others have indeed shown that F2L is a chemotactic the first amino acid, a phenylalanine, is crucial (23). This study stimulus for neutrophils that binds to the FPRL1 and FPR. In con- demonstrates that an N-terminal phenylalanine of FLIPr-like and trast to our data, they observed that F2L fails to stimulate the FLIPr is also crucial in the inhibition of the FPR. Both proteins intracellular calcium increase and superoxide generation (42). start with two phenylalanines, the first of which is dispensable. It FLIPr-like is an antagonist that directly binds to the FPRL1 and must be noted that the initial six N-terminal amino acids of FLIPr- FPR, thereby preventing peptide ligands from binding and induc- like and FLIPr resemble the allowed substitutions within the ing subsequent signaling. Specific binding was demonstrated using hexapeptide FTFEPF, the minimal N-terminal peptide structure of HEK293 transfectants expressing appropriate GPCRs. HL60 cells CHIPS that inhibits FPR activation (23). A peptide resembling the stably expressing each of the FPRs were used to investigate the first six amino acids of FLIPr-like and FLIPr indeed inhibited receptor specificity of FLIPr-like. Undifferentiated HL60 cells, a fMLP-induced calcium mobilization, even more potently than did cell line of myeloid origin that does not express these receptors the corresponding CHIPS peptide. To further evaluate this effect, (26), showed some minor association of FLIPr-like. This observa- chimeras of FLIPr-like, FLIPr, and CHIPS were constructed in tion can be attributed to the low endogenous expression of FPRL1 which the 6 N-terminal amino acids were exchanged between the or another unknown receptor. However, the specific FPRL1 ligand proteins. However, introduction of the N terminus of CHIPS in the MMK-1 only induced a calcium response in HL60/FPRL1 cells FLIPr-like or FLIPr backbone abolished FPR activity, which may (data not shown). Strong binding with FPRL1-expressing HL60 have been caused by nonproperly folded proteins or the need of cells confirms the data obtained with transiently expression of the additional structural elements that were absent or masked. In con- Downloaded from receptor in HEK293 cells. FLIPr-like also binds to the FPR- and trast, the reverse substitution resulting in a CHIPS mutant with FPRL2-expressing HL60 cells. Furthermore, competitive binding N-terminal FLIPr-like and FLIPr led to a protein with slightly assays with FPR and FPRL1 demonstrated that all of the FPRL1 diminished activity toward the FPR that was comparable to the specific peptides, WKYMVM, WKYMVm, MMK-1 and WRW4, activity of wild-type FLIPr-like. As FLIPr-like and FLIPr have displace FLIPR-likeFITC as well as WKYMVMFAM bound to identical N-terminal 25 aa, other parts of FLIPr-like contribute to HL60/FPRL1 cells. A different type of interaction may exist for the higher efficacy toward the FPR as compared with FLIPr. In http://www.jimmunol.org/ the FPR where only a partial competition by high-affinity peptide addition to its importance in FPR antagonism, the N terminus of ligands for FLIPr-likeFITC was observed. In line with these FLIPr-like and FLIPr is also important in the inhibition of the observations unlabeled FLIPr-like efficiently competes with FPRL1. The phenylalanines were dispensable, but the truncation WKYMVMFAM binding to the FPRL1 and partly with the FPR of the first seven amino acids did result in a 100-fold drop in while the high-affinity ligand fMLPFITC was only displaced by FPRL1-inhibitory activity. Replacing the first six amino acids of CHIPS. In our hands, the fluorescent WKYMVMFAM effectively FLIPr-like with those of CHIPS resulted in a 10-fold drop in in- bound to HL60/FPR cells indicative for a high-affinity binding (as hibition and may be due to masking of the active site. shown for CHO cells expressing the different FPRs) (40), whereas The FPRL1 shares 69% amino acid identity with FPR but in- by guest on October 7, 2021 published data indicate specificity for the FPRL1 (20, 26, 27, 43). teracts with a variety of ligands (7). Distinct ligands induce dif- Apparently both FPR and FPRL1 contain structures necessary and ferent biological responses and have different modes of receptor sufficient for binding of FLIPr-like, resulting in prevention of cal- activation (5, 46). Interestingly, two homologous S. aureus pro- cium mobilization by fMLP and MMK-1, respectively. The dis- teins antagonize these two members of the FPR family with a crepancy between displacement of fluorescent ligands and ability differential profile. FLIPr-like and FLIPr both inhibit the MMK- to block functional responses could indicate that nonoverlapping 1-induced calcium mobilization and chemotaxis within nanomolar binding sites are involved. FLIPr-like binds to neutrophils and range, but they differ in their affinity for the FPR. fMLP-induced monocytes, which express the FPR as well as FPRL1 (1, 7). In- calcium mobilization is inhibited by nanomolar concentrations of terestingly, FLIPr-like also binds to subpopulations of lympho- FLIPr-like and low micromolar concentrations of FLIPr. The ac- cytes. Both CD19-positive B cells and CD16/CD56-positive NK tivity toward these two related GPCRs probably resides in different cells show strong binding of FLIPr-like that is comparable to its domains of FLIPR-like and FLIPr. Structural information on both binding to CD14-positive monocytes and neutrophils. Binding is FLIPr-like and FLIPr will contribute to the elucidation of their specific and effectively displaced by unlabeled FLIPr-like. How- inhibitory activity toward the FPR and FPRL1 and may serve as a ever, only the peptide WKYMVm, with high affinity for FPRL1 as nonpeptide structural basis for future design of potential therapeu- well as FPR, inhibited FLIPr-likeFITC binding to monocytes and tic agents for FPR/FPRL1-related pathophysiology. Alternatively, neutrophils but not with B and NK cells. Because peripheral blood based on the WKYM peptide structure, the core sequence of a lymphocytes are not known to definitively express one or more func- potent agonist was identified that provides important information tional FPRs when unstimulated (7, 44), it is likely that FLIPr-like in designing future peptidomimetic agents for therapeutic use (43). recognizes yet another unknown receptor (or receptors) on these Formyl peptide receptors mediate immune responses to infection, cells that is restricted to certain leukocyte subpopulations. Also on but the identification of novel endogenous agonists in recent years monocytes and neutrophils, FLIPr-like binds probably to another expands the spectrum of biological significance and potential ther- unknown receptor (or receptors) next to members of the FPR apeutical approaches. family. The homologous protein FLIPr also mimics the profile for this unknown receptors (or receptors) as previously described (18). Acknowledgments In addition to FLIPr-like and FLIPr, S. aureus excretes CHIPS The authors thank Miriam Poppelier and Maartje Ruyken for excellent technical assistance with experiments and protein purification. that effectively and specifically inhibits two GPCRs involved in early neutrophil migration, namely the C5aR and FPR (17). For CHIPS, structurally important motifs were identified that partici- Disclosures pate in the interaction with the C5aR and FPR. The first 30 aa are The authors have no financial conflicts of interest. 6578 STAPHYLOCOCCAL PROTEIN THAT ANTAGONIZES FPRL1 AND FPR

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