A Homolog of Formyl Peptide Receptor-Like 1 (FPRL1 ... · variety of virulence factors to the cell surface and extracellular ... in Rosetta-gami(DE3)pLysS E. coli ... competent cells

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Inhibits FPRL1 and FPR (FPRL1 Inhibitory Protein) Thataureus

Staphylococcus1 (FPRL1) Inhibitor from A Homolog of Formyl Peptide Receptor-Like

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

http://www.jimmunol.org/content/183/10/6569doi: 10.4049/jimmunol.0801523October 2009;

2009; 183:6569-6578; Prepublished online 21J Immunol 

Referenceshttp://www.jimmunol.org/content/183/10/6569.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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A Homolog of Formyl Peptide Receptor-Like 1 (FPRL1)Inhibitor from Staphylococcus aureus (FPRL1 InhibitoryProtein) 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, includingbacteria-derived N-formylated peptides and host-derived peptides and proteins. We have recently described two chemoattractantreceptor inhibitors from Staphylococcus aureus. Chemotaxis inhibitory protein of S. aureus (CHIPS) blocks the formyl peptidereceptor (FPR) and the receptor for complement C5a (C5aR), while FPR-like 1 (FPRL1) inhibitory protein (FLIPr) blocks theFPRL1. Here, we describe another staphylococcal chemoattractant-inhibiting protein with 73% overall homology to FLIPr andidentical first 25 aa, which we termed FLIPr-like. This protein inhibits neutrophil calcium mobilization and chemotaxis inducedby the FPRL1-ligand MMK-1 and FPR-ligand fMLP. While its FPRL1-inhibitory activity lies in the comparable nanomolar rangeof FLIPr, its antagonism of the FPR is �100-fold more potent than that of FLIPr and comparable to that of CHIPS. The secondN-terminal phenylalanine was required for its inhibition of the FPR, but it was dispensable for the FPRL1. Furthermore, thedeletion of the first seven amino acids reduced its antagonism of the FPRL1, and the exchange of the first six amino acids with thatof CHIPS-conferred receptor specificity. Finally, studies with cells transfected with several chemoattractant receptors confirmedthat FLIPr-like specifically binds to the FPR and FPRL1. In conclusion, the newly described excreted protein from S. aureus,FLIPr-like, is a potent inhibitor of the FPR- and FPRL1-mediated neutrophil responses and may be used to selectively modulatethese chemoattractant receptors. The Journal of Immunology, 2009, 183: 6569–6578.

N eutrophils are crucial in the initial host defense againstmicroorganisms, but they also contribute to the patho-genesis of inflammatory diseases. To migrate from the

blood stream toward the site of infection, they are guided by agradient of chemotactic factors. These chemoattractants are de-rived from pathogens, the complement system, host cells and tis-sues, or even phagocytes themselves and signal via a family ofseven-transmembrane, G protein-coupled receptors (GPCRs).3

The formyl peptide receptor (FPR) gene cluster contains theFPR (formylated peptide receptor; FPR1), FPRL1 (formyl peptidereceptor-like 1; FPR2/ALX), and FPRL2 (formyl peptide receptor-like 2; FPR3) (1–3). The FPR is the high-affinity receptor forformylated peptides leaking from growing bacteria and thereforeserves as an important innate immune recognition receptor (4).Formylated peptides, such as fMLP, are some of the most potent

chemoattractants for human leukocytes, and they mediate directedmotility, phagocytosis, exocytosis, and superoxide anion genera-tion (5, 6). In addition to formylated peptides, the FPR can beactivated by peptides derived from several viral proteins (7, 8).The FPRL1, on other hand, recognizes a variety of ligands of dif-ferent origin and structure. These include the endogenous antiin-flammatory lipid mediator lipoxin A4 and amyloid-related pep-tides, bacterial and viral peptides, and peptides derived fromsynthetic libraries (1–3, 7). Initially the FPR expression has beendescribed in monocytes, neutrophils, and in microglial and den-dritic cells but it is now also found in nonhematopoietic cells andtissues. The FPRL1 is also expressed in a large variety of cells andorgans including monocytes, neutrophils, macrophages, and mi-croglial cells. Neutrophils express FPR and FPRL1 but not FPRL2,while monocytes express all three members (1, 7). FPRL1 does notonly play a role during different stages of the innate immune re-sponse, but increasing evidence shows its implication in the patho-genesis of amyloidogenic diseases (9). Both the FPR and FPRL1trigger many neutrophil functions, including chemotaxis, exocy-tosis, and superoxide generation. A transient rise in intracellularcalcium is required, albeit not sufficient, for triggering optimalchemoattractant-induced responses (5).

Staphylococcus aureus is a commensal of the human skin butcan cause a wide spectrum of infections spanning from trivial skininfections to severe septic diseases. This pathogen can produceinfections in essentially every human organ or tissue. It exports avariety of virulence factors to the cell surface and extracellularmilieu that contribute to the pathogenesis of infection. These pro-teins cause direct cellular damage and/or interact with host defensefactors (10, 11). To cause deep infections and survive in the host,the bacteria must evade the immune system by producing mole-cules that target critical processes in nearly every stage of the

*Medical Microbiology, University Medical Center Utrecht, Utrecht, the Netherlands;and †Microbiology Department, Hospital Universitari Germans Trias i Pujol, Univer-sitat Autonoma de Barcelona, Barcelona, Spain

Received for publication May 9, 2008. Accepted for publication September 14, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grants Fondo de Investigacion Sanitaria, Instituto deSalud Carlos III (expediente 01/F062) and Sociedad Espanola de Enfermedades In-fecciosas y Microbiologia Clinica (both to C. P.).2 Address correspondence and reprint requests to Dr. Kok P. M. van Kessel, MedicalMicrobiology, University Medical Center Utrecht, room G04.614, Heidelberglaan100, 3584 CX Utrecht, The Netherlands. E-mail address: [email protected] Abbreviations used in this paper: GPCR, G protein-coupled receptor; A�, amyloid�; CHIPS, chemotaxis inhibitory protein of S. aureus; FLIPr, FPRL1 inhibitory pro-tein; FPR, formyl peptide receptor; FPRL, FPR-like receptor; HSA, human serumalbumin; LTB4, leukotriene B4; PAF, platelet-activating factor; PrP, prion protein.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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innate host response to infection (12–14). We have previously re-ported the molecular properties of several secreted proteins from S.aureus that contribute to the evasion of the human defense system.These newly described proteins interfere with several arms of theinnate immune response, such as initial rolling of neutrophils (15),complement activation (16), and chemoattractant-mediated activa-tion and migration of phagocytes (17). The FPRL1-inhibitory pro-tein or FLIPr (18) is a secreted staphylococcal antiinflammatoryprotein that potently inhibits FPRL1-specific ligands, such as thesynthetic peptides MMK-1 (19) and WKYMVM (20), and the en-dogenous proteins amyloid � 1–42 (A�42) (21, 21) and prion pro-tein fragment PrP106–126 (22). FLIPr binds directly to the GPCRFPRL1, thereby acting as an antagonist.

A BLAST search through sequenced S. aureus genomes re-vealed that S. aureus encodes for a protein with 73% homology toFLIPr. This protein, which we named FLIPr-like, consists of 104aa and is preceded by a classical signal peptide with an AXAmotif. In this study we show that FLIPr-like inhibits FPRL1-me-diated neutrophil activation as was determined by intracellular cal-cium mobilization and chemotaxis. Moreover, FLIPr-like also ef-ficiently inhibits fMLP-induced responses comparable to CHIPS.This newly described excreted protein of S. aureus is an antagonistof both the FPR and FPRL1. Deletion and substitution mutantsindicate that the dual activity of FLIP-like is allocated to differentsites within the protein.

Materials and MethodsReagents

MMK-1 (H-LESIFRSLLFRVM-OH) was synthesized by Sigma-Genosys.The hexapeptide WKYMVM-NH2 was from Bachem AG (Switzerland),its D-conformer WKYMVm was synthesized by J. A.W. Kruijtzer (De-partment of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sci-ences, Utrecht, The Netherlands), and WRWWWW-NH2 (WRW4) andcarboxyfluorescein (FAM)-labeled WKYMVM were from Phoenix Phar-maceuticals. fMLP, recombinant C5a, propidium iodide, and anti-FLAGmAb were from Sigma-Aldrich. The fluorescein conjugate of formyl-Nle-Leu-Phe-Nle-Tyr-Lys (abbreviated as FITC-fMLP) and Fluo-3-AM (ace-toxymethyl ester) were obtained from Molecular Probes/Invitrogen. IL-8and neutrophil-activating protein 2 were from PeproTech. Platelet-activat-ing factor (PAF)-16 and purified human C3a were from Calbiochem(Merck Biosciences). LTB4 (leukotriene B4) was purchased from CascadeBiochem. Anti-HA mAb (clone 12CA5) was from Roche Applied Science.Allophycocyanin-labeled goat anti-mouse IgG was from BD Biosciences.

Cloning, expression, and purification of FLIPr-like

Primers were designed according to the published sequence of the geneMW1038 (from S. aureus subsp. aureus MW2) for the cloning of FLIPr-like into pRSETB vector (Invitrogen) and were manufactured by Invitro-gen. Recombinant protein was generated by PCR and cloned into theEcoRI and XbaI site of the pRSETB vector by overlap extension PCR andpropagated in TOP10F� Escherichia coli (Novagen) as described before(18). After verification of the correct sequence, the protein was expressedin Rosetta-gami(DE3)pLysS E. coli (Novagen) by induction with 1 mMisopropyl-thiogalactopyranoside (Roche). HIS-tagged protein was isolatedunder denaturing conditions using a 5-ml HiTrap chelating HP column (GEHealthcare Europe) following the manufacturer’s protocol and cleaved af-terward with enterokinase (Invitrogen) to obtain the native protein. Toremove the HIS-tag, the cleaved protein mixture was passed over a nickelcolumn for a second time and selectively eluted with phosphate buffers ofpH 6 and pH 5.3. The separate fractions were analyzed on a 15% SDS-PAGE gel and showed two different bands of purified protein correspond-ing to 12 and 11 kDa, respectively. The corresponding fractions werepooled and dialyzed separately toward PBS and stored at �20°C. Thenative protein FLIPr-like was mixed with 0.1 mg/ml FITC (Sigma-Aldrich)in 0.1 M carbonate buffer (pH 9.5) and subsequently separated from freeFITC by a desalting column, as described earlier for FLIPr and CHIPS(17, 18).

Construction of mutants and chimeras

Site-directed mutagenesis was performed on the N-terminus of both FLIPrand FLIPr-like by deletion of the first or the first two amino acids, both

phenylalanines, and cloning in pRSETB vector by overlap extensionPCR as described above. Three chimeras were also constructed. ForCHIPS1–6-FLIPr-like7–104 and CHIPS1–6-FLIPr7–105, the first six N-termi-nal amino acids of FLIPr-like and FLIPr were substituted for the first sixamino acids of CHIPS, and for the reverse chimera FLIPr1–6-CHIPS7–121,the amino acids 1–6 of CHIPS were exchanged for those of FLIPr. Thecompetent cells BL21(DE3) E. coli (Novagen) were used to express themutants and chimeras. After verification of the correct sequence, all HIS-tagged proteins were expressed and purified using the Pro-Bond resin (In-vitrogen) or HiTrap chelating HP column following the manufacturer’sinstructions and cleaved afterward with enterokinase as described above.

Synthetic peptides

The peptides of the N-terminal amino acids 1–6 of FLIPr as well as FLIPr-like (H-FFSYEW-NH2) were synthesized by Dr. R. van der Zee (Instituteof Infectious Diseases and Immunology, Utrecht University) and of CHIPS(H-FTFEPF-NH2) by Dr. John A.W. Kruijtzer as described before (23).

Leukocyte isolation

Venous blood was collected from healthy volunteers into tubes containingsodium heparin. Blood was diluted with an equal volume of PBS andlayered onto a gradient of Ficoll (Amersham Biosciences) and Histopaque(Sigma-Aldrich). After centrifugation for 20 min at 397 � g and 21°C,mononuclear cells and polymorphonuclear neutrophils were collected sep-arately from Ficoll and Histopaque interfaces, respectively. Cells werewashed with cold RPMI 1640 containing 25 mM HEPES, L-glutamine(Biowhittaker), and 0.05% human serum albumin (HSA; Sanquin) (RPMI-HSA). For elimination of erythrocytes, the neutrophil pellet was subjectedto a hypotonic shock with distilled H2O for 30 s followed by 10� con-centrated PBS to restore isotonicity. After washing, cells were resuspendedin RPMI-HSA.

Calcium mobilization

To determine activation of neutrophils by chemoattractants the transientincrease in free intracellular calcium concentration was measured by flowcytometry (18, 24). For this purpose, neutrophils (5 � 106 cells/ml) wereloaded with 2 �M Fluo-3-AM for 20 min at room temperature, protectedfrom light with gentle agitation. The cells were washed, resuspended inRPMI-HSA to 5 � 106 cells/ml, and incubated with buffer or protein for atleast 1 min. Before stimulation, cells were diluted to 1 � 106 cells/ml in avolume of 180 �l while maintaining the inhibitory protein concentration.The basal fluorescence level for Fluo-3 was monitored at 530 nm for�8 s after which 12.5 �l of 10� concentrated stimulus was added. Thesample tube was rapidly placed back to the sample holder and the fluo-rescence measurement continued up to 52 s. Neutrophils were gated basedon scatter parameters to exclude cell debris, and the mean fluorescencevalue at basal level was subtracted from the value at peak level (at 30 s).The different stimulus concentrations were expressed relative to the max-imal response for each individual stimulus.

Chemotaxis

Neutrophil migration was measured in a 96-multiwell transmembrane sys-tem (ChemoTX; Neuro Probe) using an 8-�m pore size polycarbonatemembrane (18). Cells were labeled with 2 �M calcein-AM for 20 min,washed, and resuspended to a concentration of 2.5 � 106 cells/ml in HBSSwith 1% HSA. Wells were filled with 29 �l of chemoattractant, and themembrane holder was assembled. Cells were incubated with different con-centrations of FLIPr or FLIPr-like and 25 �l was placed as a droplet on themembrane. After incubation for 30 min at 37°C in a humidified 5% CO2

atmosphere, the membrane was washed extensively with PBS, and thefluorescence was measured in a FlexStation (Molecular Devices). Percent-age migration was calculated relative to wells containing 25 �l of cells.

Binding to leukocytes

To determine the binding of FLIPr-like to different cell types, 50 �l ofblood leukocytes at 5 � 106 cells/ml was incubated for 30 min on ice withbuffer or a concentration range FITC-labeled protein. Cells were washedand resuspended in RPMI-HSA. The fluorescence of 17,500 cells was mea-sured by flow cytometry, and the different leukocyte populations wereidentified based on forward and sideward scatter parameters. For binding inwhole blood, 50 �l of EDTA anticoagulated blood was used, and the sam-ples were treated with FACS lysing solution (BD Biosciences) before anal-ysis. To discriminate between different leukocyte subpopulations, leuko-cytes were labeled with mAbs and selected by specific gating on scattersand fluorescence. Specific labels used were anti-CD3-RPE/Cy5 for T cells,

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anti-CD19-PE for B cells, anti-CD16/CD56-PE (plus negative for anti-CD3-RPE/Cy5) for NK cells, and anti-CD14-PE for monocytes.

Binding to HEK293 and HL60 transfectants

Human embryonic kidney cells (HEK293; obtained from the AmericanType Culture Collection) were transfected with plasmids containing aFLAG-tagged FPR, FPRL1, or C5aR or HA-tagged FPRL2, as describedbefore (18, 25). After 2 days, transfected cells were harvested with EDTAand incubated with 10 �g/ml mouse anti-FLAG or anti-HA mAb for 45min on ice. Cells were then washed and subsequently incubated with sat-urating concentration of allophycocyanin-labeled goat anti-mouse IgG to-gether with different concentrations of FITC-labeled protein, FLIPr-like,FLIPr, or CHIPS. Cells were incubated for 45 min on ice, washed, andresuspended in 200 �l of RPMI-HSA containing 5 �g/ml propidium io-dide. FL1 (FITC), FL2 (propidium iodide) and FL4 (allophycocyanin)were measured in a FACSCalibur flow cytometer (BD Biosciences). As acontrol, HEK293 cells transfected with an empty pcDNA3.1 vector wereused. The binding of FITC-labeled proteins was determined by selectinglive cells (propidium iodide-negative) expressing the receptor as indicatedby positive staining with anti-FLAG or anti-HA mAb (18). Binding tonontransfected cells within the same sample was used to establish back-ground values. Human promyelocytic leukemia HL60 cells stable trans-fected with the FPR, FPRL1, and FPRL2 were provided by F. Boulay(Laboratoire Biochimie et Biophysique des Systemes Integres, Grenoble,France) (26, 27). Cells were cultured in RPMI 1640 medium containingG418 (omitted for the parent HL60 cells), and the maximal density wasmaintained below 2 � 106 cells/ml. Cells were centrifuged at each passage.Fresh cells (5 � 106 cells/ml in RPMI-HSA) were incubated with fluores-cent-labeled ligands for 45 min on ice under gentle agitation, washed once,and fixed with 1% paraformaldehyde. For competition experiments, cellswere first incubated for 15 min with unlabeled protein or peptide. Curvefitting and calculation of inhibitory concentration value (IC50) was per-formed by nonlinear regression analysis of the dose-response curves gen-erated using Prism 5 (GraphPad Software).

ResultsIdentification of FLIPr-like

The tblastn algorithm search of the sequenced S. aureus genomeswith FLIPr identified a protein with 73% homology in two of thenine genomes: hypothetical protein MW1038 (S. aureus subsp.aureus MW2) and hypothetical protein SAS1089 (S. aureus subsp.aureus MSSA476). The genes encode for an extracellular proteinof 104 aa preceded by a classical signal peptide with an AXA motifthat is cleaved at the bacterial membrane (Fig. 1A). The homologyof this newly identified protein to CHIPS is 27% for the processedmature protein with a high degree of homology in the leader pep-tides. This homolog of FLIPr, which we named FLIPr-like, wascloned, expressed in E. coli as a HIS-tagged protein without the

leader sequence, and purified using nickel affinity chromatography.To remove the HIS-tag, the protein was treated with enterokinaseand the cleaved protein mixture was passed over a nickel columnagain. Selective elution with phosphate buffers of different pHshowed two different bands of purified protein corresponding to 12and 11 kDa, respectively, on a 15% SDS-PAGE gel (Fig. 1B).N-terminal sequencing of the two proteins identified the 12-kDaband as the native FLIPr-like (first five N-terminal amino acids:FFSYE) and the 11-kDa band as a cleavage product lacking thefirst seven amino acids, FLIPr-like8–104 (first five N-terminalamino acids: GLEIA; Fig. 1A, underlined).

FLIPr-like inhibits neutrophil activation by MMK-1 and fMLP

As FLIPr antagonizes the FPRL1, we examined whether FLIPr-like also blocks the activation of this receptor. For this purpose,neutrophils were preincubated with 3 �g/ml FLIPr-like, FLIPr, orCHIPS and compared with control cells for mobilization of intra-cellular calcium in response to a concentration range of theFPRL1-specific ligand MMK-1. Fig. 2A shows a representativechange in intracellular calcium concentration of control and FLIPr-like-treated neutrophils stimulated with MMK-1 as measured byan increase in fluorescence. Increasing concentrations of MMK-1gradually increased the mobilization of intracellular calcium incontrol cells. Both FLIPr and FLIPr-like completely inhibited thecell response to MMK-1, while CHIPS did not (Fig. 2B). As FLIPris also a moderate inhibitor of the FPR, the effect of FLIPr-like onfMLP-induced neutrophil activation was examined. Incubation ofneutrophils with FLIPr-like shifted the fMLP-induced calcium mo-bilization curve toward higher concentrations (Fig. 2, C and D). Incomparison to FLIPr, its homolog FLIPr-like was a more potentinhibitor of fMLP-induced neutrophil activation and was almost asactive as CHIPS (Fig. 2D). Because FLIPr-like mimicked the ac-tivity of CHIPS on fMLP, the ability of FLIPr-like to block theC5a-induced calcium mobilization was tested as well. FLIPr-likeand its homolog FLIPr did not affect the C5a-mediated calciummobilization, while CHIPS effectively inhibited this response (Fig.2, E and F). Additionally, FLIPr-like and FLIPr did not inhibit thecalcium mobilization in neutrophils stimulated with optimal con-centrations of C3a (C3aR), IL-8 (CXCR1 and CXCR2), neutro-phil-activating protein 2 (CXCR2), PAF (PAFR), or LTB4 (BLTR)(data not shown). Finally, to examine the effects of these proteinson the FPRL2, we performed experiments with F2L, the acetylatedpeptide derived from cleaved heme-binding protein with high af-finity for FPRL2. Although neutrophils do not express FPRL2, aconcentration of 4 �M F2L induced a significant calcium mobili-zation that was inhibited by 3 �g/ml FLIPr-like and FLIPr but notby CHIPS. This response was probably mediated via the FPRL1and could therefore be inhibited by FLIPr-like and FLIPr. Whilemonocytes are described to express FPRL2, we were unable toshow stimulation by nanomolar concentrations F2L. FLIPr-like it-self at concentrations up to 30 �g/ml did not induce a calciummobilization in human neutrophils.

FLIPr-like inhibits neutrophil chemotaxis to MMK-1 and fMLP

Both MMK-1 and fMLP induce chemotactic migration of neutro-phils. We investigated the effects of FLIPr-like, FLIPr, and CHIPSon neutrophil chemotaxis in a 96-well transmembrane system.Both FLIPr-like and FLIPr showed a comparable concentration-dependent inhibition of chemotaxis to MMK-1, while even 10�6

M CHIPS had no effect (Fig. 3A). FLIPr-like and CHIPS effec-tively inhibited neutrophil chemotaxis toward fMLP (Fig. 3B), andFLIPr only showed an effect at a high concentration (Fig. 3B).

FIGURE 1. Sequence and purification of FLIPr-like. A, Sequence align-ment of FLIPr and FLIPr-like using ClustalW. The putatively excretedprotein is depicted in bold with black boxes marking mismatched res-idues and gray boxes indicating the signal-peptidase cleavage site. B,Coomassie blue-stained SDS-PAGE gel (15%) showing purified FLIPr-like protein (lane 1; 12 kDa), mixture after enterokinase cleavage (lane2), FLIPr-like8–104 (lane 3; 11 kDa), and HIS-tagged protein (lane 4; 15.5kDa). M indicates marker.

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The first seven amino acids of FLIPr-like are crucial for fMLPbut not MMK-1 inhibition

Cleavage of the HIS-tag from the recombinant FLIPr-like proteinwith enterokinase generated the native protein as well as an additional

cleavage product lacking the first seven amino acids (FLIPr-like8–104).This deletion mutant was also isolated and used to investigatethe importance of the N terminus in FLIPr-like activity. Cellswere incubated with increasing concentrations FLIPr-like8 –104,native FLIPr-like, FLIPr, or CHIPS and stimulated with an op-timal concentration MMK-1 or fMLP. Loss of the first sevenamino acids in FLIPr-like (FLIPr-like8 –104) resulted in dimin-ished inhibitory potency of the MMK-1-induced neutrophil ac-tivation as compared with native FLIPr-like (Fig. 4A) thatshowed maximal inhibition up to 10�10 M. While FLIPr-likeinhibits fMLP-induced neutrophil activation, FLIPr-like8 –104

lost all activity (Fig. 4B). These results suggest an active site inthe N terminus of FLIPr-like for FPR inhibition.

Function of FLIPr and FLIPr-like N-terminal mutants andchimeras

To further investigate which residues are important in the ac-tivity of FLIPr-like, calcium mobilization assays were per-formed with several mutants, chimeras, and peptides. Deletionof the first N-terminal amino acid, a phenylalanine, in FLIPr-like (FLIPr-like�F1) did not affect the inhibition of MMK-1-and fMLP-stimulated neutrophils (Fig. 5, A and B). However,the mutant lacking the first two N-terminal phenylalanines(FLIPr-like�F1F2) lost the inhibition for the fMLP-induced ac-tivation (Fig. 5B) but retained full inhibition for the MMK-1-induced response (Fig. 5A). It was remarkable that both mutants

FIGURE 2. FLIPr-like inhibits MMK-1- and fMLP-induced calcium mobilization. Fluo-3-loaded neutrophils were incubated with buffer (F), 3 �g/mlFLIPr-like (f), FLIPr (�), or CHIPS (Œ) for 20 min at room temperature. To monitor calcium mobilization by flow cytometry, each sample was firstmeasured for �8 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 samplewas 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 � 10�7 M MMK-1 (A), 3 � 10�9 M fMLP (C), and 3 � 10�10 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 andbefore 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

M C5a (F). Data are means � SEM of three independent experiments.

FIGURE 3. FLIPr-like inhibits neutrophil chemotaxis toward MMK-1and fMLP. Calcein-labeled neutrophils were incubated with different con-centrations FLIPr-like (f), FLIPr (�), or CHIPS (Œ) and allowed to mi-grate across a 8-�m pore membrane toward 3.3 � 10�8 M MMK-1 (A) or3.3 � 10�9 M fMLP (B). Results are expressed as percentage inhibition ofthe migration of buffer-treated cells, and all samples were run in triplicatefor two experiments. Percentage migration of control cells toward fMLPwas 44.4 � 4.6 (mean � SD) and toward MMK-1 was 51.8 � 3.6, andspontaneous migration was 3.4 � 1.3.

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showed a slightly more potent activity toward MMK-1 as com-pared with the parent FLIPr-like protein. The same N-terminaldeletion mutants were constructed for FLIPr. Both mutants,FLIPr�F1 and FLIPr�F1F2, retained all inhibitory activity towardMMK-1 (Fig. 5C). As shown for FLIPr-like, deletion of bothphenylalanines (FLIPr�F1F2) eliminates the inhibitory activityfor fMLP (Fig. 5D). To further elucidate the importance of theN terminus, chimeras were constructed in which the first sixamino acids were exchanged with the remaining sequence of

FLIPr-like, FLIPr, or CHIPS. The chimera CHIPS1–6-FLIPr7–105

showed no inhibition of MMK-1- (Fig. 5E) or fMLP-induced(Fig. 5F) neutrophil activation. Surprisingly, the chimeraCHIPS1– 6-FLIPr-like7–104 showed only limited inhibition of theMMK-1 response (Fig. 5E) and no inhibition of the fMLP re-sponse (Fig. 5F). The reverse chimera FLIPr1– 6-CHIPS7–121

retained inhibition of the fMLP response (Fig. 5F) and, likeCHIPS, did not inhibit the MMK-1 response (Fig. 5E). Theseresults indicate the N terminus as the active site of both FLIPr

FIGURE 4. The first seven N-terminal amino acidsof 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 com-pared with FLIPr-like, FLIPr, and CHIPS in a calciummobilization assay with neutrophils. Fluo-3-loaded cellswere incubated with different concentrations of inhibi-tory protein and challenged with the FPRL1 agonistsMMK-1 (3 � 10�7 M; A) and FPR agonist fMLP (3 �10�9 M; B). Data are expressed as percentage inhibitionof buffer-treated cells and are the mean of three inde-pendent experiments.

FIGURE 5. Importance of the N-terminal phenylalanines in FLIPR-like and FLIPr. The activities of dif-ferent N-terminal-deletion mutants(�F1, �F1F2, and FLIPr-like8–104)and chimeras of the first six amino ac-ids of CHIPS with FLIPr (CHIPS-FLIPr), CHIPS with FLIPr-like(CHIPS-FLIPR-like), and FLIPr (equalto FLIPr-like) with CHIPS (FLIPr-CHIPS) were tested in a calcium mobi-lization assay with neutrophils stimu-lated with MMK-1 (3 � 10�7 M; A, C,and E) or fMLP (3 � 10�9 M; B, D, F).Cells were preincubated with increas-ing concentrations of antagonists. Dataare the means of three or moreexperiments.

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and FLIPr-like for the FPR as was shown before for CHIPS(16). Additionally, part of the N terminus seems to be importantfor the activity toward the FPRL1. The N-terminal HIS-taggedforms of the proteins were also tested for their inhibitory ca-pacity. Both HIS-tagged FLIPr and FLIPr-like retained theiractivity on MMK-1-induced responses but lost their effect onfMLP-induced responses (data not shown). We have previouslydescribed the importance of the first phenylalanine in CHIPSfor the FPR-blocking activity and showed that a synthetic pep-tide compromising the first six N-terminal amino acids blocksfMLP-induced activation (23). Because the first six amino acidsof FLIPr closely resemble the allowed substitutions within thissix-amino acid CHIPS peptide (23), synthetic peptides ofCHIPS and FLIPr were compared. The FLIPr1– 6 peptide, rep-resenting the first six amino acids of FLIPr as well as FLIPr-like(see Fig. 1A), effectively inhibited the fMLP-induced response,

even more potently when compared with the CHIPS1– 6 peptide.Complete FLIPr-like protein was �100 times more potent thanthe peptides (Fig. 6). Neither FLIPr1– 6 nor CHIPS1– 6 peptideinhibited 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 ex-pressing the FPRL1, the binding of FLIPr-likeFITC to neutro-phils, monocytes, and lymphocytes was measured by flow cy-tometry. Fig. 7A shows a concentration-dependent binding ofFLIPr-likeFITC to neutrophils, monocytes, and a proportion of lym-phocytes. Also, in whole blood, FLIPr-likeFITC bound to neutro-phils and monocytes (data not shown). Further analysis of lym-phocyte subpopulations revealed a strong binding to B and NKcells and not to T cells, as determined by double labeling withspecific mAbs (Fig. 7B). These results are similar to those previ-ously observed for FLIPrFITC (18). Because B and NK cells are notknown to definitively express members of the FPR family, cellswere preincubated with unlabeled FLIPr-like and FPR agonists todisplace FLIPR-likeFITC binding (Fig. 7C). FLIPr-like displacedthe binding of FILR-likeFITC to all leukocyte subtypes. For B andNK cells none of the FPR agonists inhibited the binding of FILR-likeFITC. For monocytes and neutrophils that express all FPRs,only WKYMVm showed displacement of FLIPr-likeFITC bindingwhile the high-affinity FPR ligand fMLP and FPRL1-specific li-gand MMK-1 were ineffective (Fig. 7C).

The FITC-labeled protein was also used in binding experi-ments with HEK293 cells transiently transfected with FLAG- orHA-tagged versions of FPR, FPRL1, FPRL2, or C5aR. HEK293cells expressing the different receptors were analyzed for bind-ing of FLIPr-likeFITC, while the nontransfected cells within thesame sample were used to determine background binding.FLIPr-likeFITC bound clearly to HEK293 cells transfected withFPRL1, with some binding to cells transfected with FPR andFPRL2 and not with C5aR (Fig. 8A). As a control, CHIPSFITC

FIGURE 6. Synthetic N-terminal peptide inhibits the FPR. The syn-thetic peptides FLIPr1–6 and CHIPS1–6 were compared with native FLIPr-like for the inhibition of fMLP-induced calcium mobilization in neutro-phils. Results are expressed as percentage inhibition of cells stimulatedwith 5 � 10�10 M fMLP and are the mean of two experiments.

FIGURE 7. FLIPr-like binds toneutrophils, monocytes, and a subpopu-lation of lymphocytes. A, Peripheralblood leukocytes were incubated withFLIPr-likeFITC (0.03–2.60 �g/ml) for30 min on ice. Binding was analyzedwith a flow cytometer, and the differentcells (neutrophils (F), monocytes (f),lymphocytes (Œ) were identified basedon scatter parameters and anti-CD14-PE staining for monocytes. Dataare from a representative experiment.B, Binding of 2 �g/ml FLIPr-likeFITC

to T cells (CD3-positive), B cells(CD19-positive), NK cells (CD3-nega-tive and CD16/56-positive), monocytes(CD14-positive), and neutrophils. Re-sults are mean fluorescence � SEMof three experiments. C, Competitivebinding of 1 �g/ml (�83 nM) FLIPr-likeFITC to B cells, NK cells, mono-cytes, and neutrophils by unlabeledFLIPr-like (250 nM), WKYMVm (1�M), MMK-1 (10 �M), and fMLP (10�M). Data are from a representativeexperiment.

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bound only to FPR- and C5aR-transfected cells as previous de-scribed (18) (data not shown). Additionally, undifferentiated HL60cells stably transfected with the FPR, FPRL1, or FPRL2 wereused. Fig. 8B shows a concentration-dependent binding of FLIPr-likeFITC to all HL60-transfected cells with minimal associationwith control cells. The most prominent binding was observed forFPRL1-expressing cells incubated with 3 �g/ml (�250 nM)FLIPr-likeFITC (Fig. 8C). The binding of the high-affinity FPR ligandfMLPFITC (10 nM) to the same panel of HL60 transfectants is shownin Fig. 8D. The fluorescent FPRL1 ligand WKYMVMFAM bound notonly to HL60/FPRL1 cells, but also to the HL60/FPR cells but notto the parent undifferentiated HL60 cells (Fig. 8E). To show spec-ificity, binding of fluorescent-labeled ligands to HL60/FPR andHL60/FPRL1 cells was measured in the presence of various un-labeled agonists or antagonists. Binding of FLIPr-likeFITC toHL60/FPR cells was effectively prevented by FLIPr-like, not itshomolog FLIPr or CHIPS. The FPR agonistic peptides fMLP,WKYMVM, and WKYMVm only partly inhibited this binding(Fig. 9A). Binding of WKYMVMFAM to HL60/FPR cells waspartly prevented by FLIPr-like and efficiently by the FPR ligandsfMLP, WKYMVM, and WKYMVm and not by the specificFPRL1 ligand MMK-1 (Fig. 9B). Binding of the high-affinity li-gand fMLPFITC to HL60/FPR cells was prevented by the peptideligands fMLP, WKYMVM, and WKYMVm and by CHIPS butnot by FLIPr-like (both at 250 nM) (Fig. 9C). For HL60 cells

expressing the FPRL1, FLIPr-like, and FLIPr as well as the spe-cific agonists WKYMVM, WKYMVm, and MMK-1 and the an-tagonist WRW4 efficiently prevented binding of FLIPr-likeFITC

(Fig. 9D). CHIPS did not inhibit this binding. Also, the binding ofthe fluorescent-labeled ligand WKYMVMFAM to HL60/FPRL1cells was prevented by FLIPr-like, and the specific ligands, and notby fMLP (Fig. 9E). The concentration dependently displacementof FLIPr-likeFITC binding to HL60/FPRL1 by unlabeled FLIPr-like (Fig. 9F) results in an IC50 of 2.5 nM.

DiscussionThe activation and migration of phagocytes to the site of inflam-mation is a key event in host defense against invading microor-ganisms and in the pathogenesis of several inflammatory diseases(28). We have previously described a secreted protein from S.aureus, FLIPr, which is a potent antagonist of the FPRL1 (18).Here, we identify a highly homologous protein from S. aureus,named FLIPr-like, which inhibits both FPR and FPRL1. FLIPr-like has an overall homology of 73% with FLIPr, and their first 25aa are identical (Fig. 1). The homology with CHIPS, which inhibitsthe FPR and C5aR, is only 27% for the native excreted and pro-cessed protein. The genes for both FLIPr and FLIPr-like encode anidentical signal peptide with an AXA cleavage motive and showshigh homology with the signal peptide encoding CHIPS. CHIPS

FIGURE 8. Binding of fluorescentFLIPr-like to HEK293 and HL60cells transfected with the FPR andFPRL1. A, HEK293 cells were tran-siently transfected with a vector con-taining FLAG-tagged human FPR,FPRL1, or C5aR or HA-taggedFPRL2. Binding of 3 �g/ml FLIPr-likeFITC to transfectants was deter-mined by selecting cells stained pos-itive for anti-FLAG or anti-HA mAband allophycocyanin-labeled goat anti-mouse IgG Ab (open histogram).The binding to nontransfected cellswas determined in the same sample(gray histogram). Data are represen-tative of three experiments. B, Con-centration-dependent binding ofFLIPr-likeFITC to HL60 cells stablyexpressing the FPR, FPRL1, orFPRL2 as well as control parentcells. Results are presented as meanfluorescence after subtraction ofautofluorescence from a representa-tive experiment. C, Mean fluores-cence values � SEM (n � 3) forbinding of 3 �g/ml (250 nM) FLIPr-likeFITC, (D) 100 nM fMLPFITC, and(E) 300 nM WKYMVMFAM to thedifferent HL60 transfectants. ND in-dicates not done.

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can be identified in the supernatant of growing S. aureus by West-ern blot analysis (17). Also, FLIPr (SA1001) can be identified inthe extracellular proteome of S. aureus, as was determined by 2D-PAGE and MALDI-TOF mass spectrometry (11). The presence ofa leader peptide in the gene encoding FLIPr-like that is identical tothat of FLIPr and is highly homologous to the leader peptide ofCHIPS makes it likely that the processed protein can be found inthe secretome of S. aureus. Additionally, a specific rabbit anti-serum identified FLIPr-like in the secretome of S. aureus strainMW2 by Western blot (data not shown). The genes for FLIPr ( flr)or FLIPr-like ( fll) cluster with other known and potential immuneevasion molecules on the genomes of all sequenced S. aureusstrains. These include SCIN (staphylococcal complement inhibi-tor) homologs, Efb (extracellular fibrinogen-binding protein), andits homolog Ecb (29). This region represents a novel immune eva-sion cluster (IEC-2) in S. aureus, whereas CHIPS is located on abacteriophage-localized cluster IEC-1 (30).

Phagocytes, but also many other cell types including cells of thenervous system, express the formyl peptide receptors FPR (FPR1),FPRL1 (FPR2/ALX), and FPRL2 (FPR3) (3). Expression ofFPRL2 is restricted to myeloid cells, including monocytes anddendritic cells, and human neutrophils only express FPR andFPRL1 (2, 3, 7). This receptor family plays a crucial role in therecognition of microorganisms and inflammatory responses, butonly a few antagonists have been reported. Replacement of theformyl group of fMLP with butyloxylcarbonyl resulted in an an-tagonistic peptide (31), and other modifications of such peptidescan also influence their antagonistic power (32, 33). CyclosporineH has been developed as a potent and selective FPR antagonist and

has been reported to inhibit calcium mobilization, chemotaxis, andsuperoxide generation (34, 35). The antiinflammatory drug piroxi-cam also inhibits neutrophil activation by FPR but not by FPRL1agonists due to the competition with the natural ligand (36, 37).Furthermore, through screening of hexapeptide libraries, WRW4

was identified as a novel, potent, FPRL1 antagonist (37). Thispeptide inhibits the increase in intracellular calcium induced byseveral FPRL1 agonists. WRW4 also inhibits the activation ofFPRL2 by the specific ligand F2L, a heme-binding protein frag-ment peptide (38). Recently, modifications of a substituted quina-zoline compound (Quin-C1) convert this selective nonpeptideFPRL1 ligand into an antagonists (39). We have previously de-scribed a potent FPRL1 antagonist that is secreted by S. aureus.FLIPr inhibits the chemotaxis and intracellular calcium mobiliza-tion induced by the synthetic FPRL1 agonists WKYMVM,WKYMVm, and MMK-1 as well as the endogenous peptides A�42

and prion protein fragment PrP106–126 (18). Additionally, FLIPrmodestly inhibits the fMLP-induced activation of neutrophils.Here, we show that the newly identified S. aureus protein FLIPr-like inhibits both FPR and FPRL1 agonist-induced calciummobilization and chemotaxis in human neutrophils at nanomolarconcentrations. The hexapeptide WRW4 inhibits the WKYMVm-induced calcium mobilization in FPRL1-expressing RBL-2H3cells at micromolar concentrations (37). Ligands acting on otherneutrophil GPCRs are not affected by FLIPr-like. The FPRL2 se-lective agonist F2L (40) induced a calcium flux in neutrophils atmicromolar range and could be inhibited by both FLIPr and FLIPr-like. F2L is described to activate human FPRL1 as well and to

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 15min 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 45min 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 toFPRL1/HL60 by increasing concentrations unlabeled FLIPr-like. Results are the means � SD of two to four experiments. ND indicates not done.

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target the mouse low-affinity receptor Fpr2 (41). Because neutro-phils do not express FPRL2, we think that the response was me-diated by FPRL1 and could therefore be inhibited by FLIPr-likeand FLIPr. Others have indeed shown that F2L is a chemotacticstimulus for neutrophils that binds to the FPRL1 and FPR. In con-trast to our data, they observed that F2L fails to stimulate theintracellular calcium increase and superoxide generation (42).FLIPr-like is an antagonist that directly binds to the FPRL1 andFPR, thereby preventing peptide ligands from binding and induc-ing subsequent signaling. Specific binding was demonstrated usingHEK293 transfectants expressing appropriate GPCRs. HL60 cellsstably expressing each of the FPRs were used to investigate thereceptor specificity of FLIPr-like. Undifferentiated HL60 cells, acell line of myeloid origin that does not express these receptors(26), showed some minor association of FLIPr-like. This observa-tion can be attributed to the low endogenous expression of FPRL1or another unknown receptor. However, the specific FPRL1 ligandMMK-1 only induced a calcium response in HL60/FPRL1 cells(data not shown). Strong binding with FPRL1-expressing HL60cells confirms the data obtained with transiently expression of thereceptor in HEK293 cells. FLIPr-like also binds to the FPR- andFPRL2-expressing HL60 cells. Furthermore, competitive bindingassays with FPR and FPRL1 demonstrated that all of the FPRL1specific peptides, WKYMVM, WKYMVm, MMK-1 and WRW4,displace FLIPR-likeFITC as well as WKYMVMFAM bound toHL60/FPRL1 cells. A different type of interaction may exist forthe FPR where only a partial competition by high-affinity peptideligands for FLIPr-likeFITC was observed. In line with theseobservations unlabeled FLIPr-like efficiently competes withWKYMVMFAM binding to the FPRL1 and partly with the FPRwhile the high-affinity ligand fMLPFITC was only displaced byCHIPS. In our hands, the fluorescent WKYMVMFAM effectivelybound to HL60/FPR cells indicative for a high-affinity binding (asshown for CHO cells expressing the different FPRs) (40), whereaspublished data indicate specificity for the FPRL1 (20, 26, 27, 43).Apparently both FPR and FPRL1 contain structures necessary andsufficient for binding of FLIPr-like, resulting in prevention of cal-cium mobilization by fMLP and MMK-1, respectively. The dis-crepancy between displacement of fluorescent ligands and abilityto block functional responses could indicate that nonoverlappingbinding sites are involved. FLIPr-like binds to neutrophils andmonocytes, which express the FPR as well as FPRL1 (1, 7). In-terestingly, FLIPr-like also binds to subpopulations of lympho-cytes. Both CD19-positive B cells and CD16/CD56-positive NKcells show strong binding of FLIPr-like that is comparable to itsbinding to CD14-positive monocytes and neutrophils. Binding isspecific and effectively displaced by unlabeled FLIPr-like. How-ever, only the peptide WKYMVm, with high affinity for FPRL1 aswell as FPR, inhibited FLIPr-likeFITC binding to monocytes andneutrophils but not with B and NK cells. Because peripheral bloodlymphocytes are not known to definitively express one or more func-tional FPRs when unstimulated (7, 44), it is likely that FLIPr-likerecognizes yet another unknown receptor (or receptors) on thesecells that is restricted to certain leukocyte subpopulations. Also onmonocytes and neutrophils, FLIPr-like binds probably to anotherunknown receptor (or receptors) next to members of the FPRfamily. The homologous protein FLIPr also mimics the profilefor this unknown receptors (or receptors) as previously described(18).

In addition to FLIPr-like and FLIPr, S. aureus excretes CHIPSthat effectively and specifically inhibits two GPCRs involved inearly neutrophil migration, namely the C5aR and FPR (17). ForCHIPS, structurally important motifs were identified that partici-pate in the interaction with the C5aR and FPR. The first 30 aa are

not essential for inhibition of the C5aR, as a variant lacking thoseamino acids retains full activity (45). In contrast, the N-terminalregion of CHIPS is critical for inhibition of the FPR. In particular,the first amino acid, a phenylalanine, is crucial (23). This studydemonstrates that an N-terminal phenylalanine of FLIPr-like andFLIPr is also crucial in the inhibition of the FPR. Both proteinsstart with two phenylalanines, the first of which is dispensable. Itmust be noted that the initial six N-terminal amino acids of FLIPr-like and FLIPr resemble the allowed substitutions within thehexapeptide FTFEPF, the minimal N-terminal peptide structure ofCHIPS that inhibits FPR activation (23). A peptide resembling thefirst six amino acids of FLIPr-like and FLIPr indeed inhibitedfMLP-induced calcium mobilization, even more potently than didthe corresponding CHIPS peptide. To further evaluate this effect,chimeras of FLIPr-like, FLIPr, and CHIPS were constructed inwhich the 6 N-terminal amino acids were exchanged between theproteins. However, introduction of the N terminus of CHIPS in theFLIPr-like or FLIPr backbone abolished FPR activity, which mayhave been caused by nonproperly folded proteins or the need ofadditional structural elements that were absent or masked. In con-trast, the reverse substitution resulting in a CHIPS mutant withN-terminal FLIPr-like and FLIPr led to a protein with slightlydiminished activity toward the FPR that was comparable to theactivity of wild-type FLIPr-like. As FLIPr-like and FLIPr haveidentical N-terminal 25 aa, other parts of FLIPr-like contribute tothe higher efficacy toward the FPR as compared with FLIPr. Inaddition to its importance in FPR antagonism, the N terminus ofFLIPr-like and FLIPr is also important in the inhibition of theFPRL1. The phenylalanines were dispensable, but the truncationof the first seven amino acids did result in a 100-fold drop inFPRL1-inhibitory activity. Replacing the first six amino acids ofFLIPr-like with those of CHIPS resulted in a 10-fold drop in in-hibition and may be due to masking of the active site.

The FPRL1 shares 69% amino acid identity with FPR but in-teracts with a variety of ligands (7). Distinct ligands induce dif-ferent biological responses and have different modes of receptoractivation (5, 46). Interestingly, two homologous S. aureus pro-teins antagonize these two members of the FPR family with adifferential profile. FLIPr-like and FLIPr both inhibit the MMK-1-induced calcium mobilization and chemotaxis within nanomolarrange, but they differ in their affinity for the FPR. fMLP-inducedcalcium mobilization is inhibited by nanomolar concentrations ofFLIPr-like and low micromolar concentrations of FLIPr. The ac-tivity toward these two related GPCRs probably resides in differentdomains of FLIPR-like and FLIPr. Structural information on bothFLIPr-like and FLIPr will contribute to the elucidation of theirinhibitory activity toward the FPR and FPRL1 and may serve as anonpeptide structural basis for future design of potential therapeu-tic agents for FPR/FPRL1-related pathophysiology. Alternatively,based on the WKYM peptide structure, the core sequence of apotent agonist was identified that provides important informationin designing future peptidomimetic agents for therapeutic use (43).Formyl peptide receptors mediate immune responses to infection,but the identification of novel endogenous agonists in recent yearsexpands the spectrum of biological significance and potential ther-apeutical approaches.

AcknowledgmentsThe authors thank Miriam Poppelier and Maartje Ruyken for excellenttechnical assistance with experiments and protein purification.

DisclosuresThe authors have no financial conflicts of interest.

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