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*Protein
The Journal of Immunology, 2004, 173: 5704-5711.
Copyright © 2004 by The American Association of Immunologists

N-Terminal Residues of the Chemotaxis Inhibitory Protein of Staphylococcus aureus Are Essential for Blocking Formylated Peptide Receptor but Not C5a Receptor

Pieter-Jan Haas1,*, Carla J. C. de Haas*, Wendy Kleibeuker*, Miriam J. J. G. Poppelier*, Kok P. M. van Kessel*, John A. W. Kruijtzer{dagger}, Rob M. J. Liskamp{dagger} and Jos A. G. van Strijp*

* Eijkman Winkler Laboratory, University Medical Center Utrecht, Utrecht, The Netherlands; and {dagger} Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Staphylococcus aureus excretes a factor that specifically and simultaneously acts on the C5aR and the formylated peptide receptor (FPR). This chemotaxis inhibitory protein of S. aureus (CHIPS) blocks C5a- and fMLP-induced phagocyte activation and chemotaxis. Monoclonal anti-CHIPS Abs inhibit CHIPS activity against one receptor completely without affecting the other receptor, indicating that two distinct sites are responsible for both actions. A CHIPS-derived N-terminal 6 aa peptide is capable of mimicking the anti-FPR properties of CHIPS but has no effect on the C5aR. Synthetic peptides in which the first 6 aa are substituted individually for all other naturally occurring amino acids show that the first and third residue play an important role in blocking the FPR. Using an Escherichia coli expression system, we created mutant CHIPS proteins in which these amino acids are substituted. These mutant proteins have impaired or absent FPR- but still an intact C5aR-blocking activity, indicating that the loss of the FPR-blocking activity is not caused by any structural impairment. This identifies the first and third amino acid, both a phenylalanine, to be essential for CHIPS blocking the fMLP-induced activation of phagocytes. The unique properties of CHIPS to specifically inhibit the FPR with high affinity (kd = 35.4 ± 7.7 nM) could be an important new tool to further stimulate the fundamental research on the mechanisms underlying the FPR and its role in disease processes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human neutrophils play an important role in host defense against infections and in the pathogenesis of inflammatory processes. These cells are activated in direct response to chemoattractants produced at the site of inflammation. A large number of chemoattractants have been identified. These consist of the "classical" chemoattractants (N-formylated peptides, the activated complement fragments C5a and C3a, leukotriene B4, and the platelet-activating factor (PAF)2), and a class of 8–10 kDa chemotactic chemokines that have characteristic conserved cysteine residues (1, 2, 3). Activation of phagocytes includes directed cell movement, activation of integrins, generation of superoxide anions, and release of granule content (3, 4, 5). The mechanisms of chemoattractant-mediated phagocytic activation and migration involve a cascade of events initiated by the binding of chemotactic factors to seven transmembrane G-protein-coupled-receptors with subsequent activation of multiple signal transduction cascades. G-protein-coupled receptors couple to guanine nucleotide-binding proteins, activating phospholipase-C, resulting in the activation of protein kinase C and an increase in intracellular calcium concentration (1, 6). The migration of neutrophils from the blood to the sites of inflammation in tissues is a hallmark of the innate immune response and a key event in physiological defense against invading microorganisms and tissue damage (7, 8). Evading this first line of defense would benefit every invading microorganism. Research of the invading mechanisms of Staphylococcus aureus identified a chemotaxis inhibitory protein of S. aureus (CHIPS) by Veldkamp et al. (9). CHIPS is a 14.1-kDa protein and is found in over 60% of clinical S. aureus isolates. CHIPS is excreted into the supernatant of growing S. aureus culture and is produced in mice infected with S. aureus and reduces the neutrophil recruitment toward C5a in a mouse peritonitis model (10). CHIPS binds directly to the C5aR and formylated peptide receptor (FPR), consequently, blocking these receptors for their natural ligands. CHIPS binds the C5aR and the FPR with high affinity (kd of 1.1 ± 0.2 nM and 35.4 ± 7.7 nM, respectively; Ref. 11). This makes CHIPS a very potent inhibitor of C5a- and fMLP-induced activation of phagocytes. C5a is an exceptionally important and potent proinflammatory mediator, because it has both anaphylatoxic and chemotactic effects. Therefore, it is active in stimulating both the vascular and cellular phases of the inflammatory response. Because C5a is a plasma protein and is almost instantly available at the site of inflammation, it is an important mediator for initiating the complex series of events that result in augmentation and amplification of the primary inflammatory stimulus (12). The only natural sources of N-formyl-peptides are bacterial and mitochondrial proteins. The prototype N-formyl-peptide, fMLP, contains only 3 aa and is the smallest known chemotactic peptide. Yet, fMLP is one of the most potent chemoattractants, being able to induce all major phagocyte functions. These properties have allowed fMLP as a model chemoattractant for the study of phagocyte functions (3, 13). Inhibiting C5a- and fMLP-mediated phagocyte chemotaxis may be a significant virulence factor for the invasion of S. aureus into the human host. Chemokine receptors are obvious targets for drug development because they have presumed roles in disease processes. The potent capacity of CHIPS to inhibit neutrophil chemotaxis, in vitro and in vivo, makes this new protein a promising candidate anti-inflammatory drug for those diseases in which neutrophil activation plays an important role (e.g., rheumatoid arthritis and ischemic diseases; Refs. 5 and 14, 15, 16, 17, 18). In this report, we use anti-CHIPS mAbs, synthetic peptides, and truncated and mutated rCHIPS molecules to illustrate that two distinct active sites within the CHIPS protein are responsible for anti-C5aR and anti-FPR activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Monoclonal Abs

Female BALB/c mice, 6- to 8-wk-old, were immunized s.c. with 50 µg of rCHIPS in 100 µl of PBS containing 50% Freund’s complete adjuvant. After 3 wk, the mice were boosted with 50 µg of rCHIPS s.c. in 100 µl of PBS containing 50% Freund’s incomplete adjuvant. Two months later, the mice were boostered with 50 µg of CHIPS i.v. Three days after the booster, spleen cells were collected and fused with the SP2/O myeloma cell line (19). Spleen cells and myeloma cells were fused in a ratio of 5:1 (20). Wells containing hybrids were screened for the presence of mAbs against CHIPS and a CHIPS-derived synthetic peptide containing the 35 N-terminal amino acids (Pepscan Systems, Lelystad, The Netherlands), by ELISA. Hybridomas were subcloned using limiting dilution (21). Cells were cultured in IMDM (Invitrogen Life Technologies, Paisley, U.K.) containing 15% FCS (Invitrogen Life Technologies) using standard protocols. Supernatants were collected and mAbs were purified using a protein-G Sepharose column (Amersham Biosciences, Piscataway, NJ). Subclass determinations were performed using the One Step Dipstick kit (HyCult Biotech, Uden, The Netherlands). IgG concentrations were determined using an IgG-specific ELISA. The relative affinity of the different mAb for the CHIPS protein was determined by a CHIPS-specific ELISA. A total of 50 µl of CHIPS (3 µg/ml) was coated onto a 96-well Microlon ELISA-plate (Greiner Bio-One, Frickenhausen, Germany) overnight at 4°C. Wells were washed three times with PBS/0.1% Tween and subsequently blocked with 75 µl of 5% human serum albumin (HSA) in PBS/0.1% Tween at 37°C for 45 min. Wells were washed, and different concentrations of anti-CHIPS mAb in PBS/0.1% Tween were added and incubated at 37°C for 45 min. Incubation with mouse-IgG served as control. Thereafter, goat-anti-mouse IgG-PO 0.1 µg/ml in PBS/0.1% Tween was added for 45 min at 37°C. After washing, 50 µl of conjugate (175 µg/ml ureumperoxide and 105 µg/ml tetramethylbenzidine in 0.1 M sodium acetate) was added. The reaction was stopped with 50 µl of 1M H2SO4. Absorbance at 450 nm was determined in a microplate reader, model 3550 (Bio-Rad, Hercules, CA). Animal experiments were approved by the local ethics committee.

Synthetic peptides

Several sets of synthetic peptides were synthesized: 1) The sequence of CHIPS (121 aa) was divided into 22 different 15-mer peptides that progressed along the CHIPS sequence by initiating a new peptide every fifth amino acid resulting in a pep-scan spanning the sequence of CHIPS (Dr. R. van der Zee, Institute of Infectious Diseases and Immunology, Utrecht University, The Netherlands); 2) At the N terminus, a new 15-mer peptide was initiated every next amino acid up to residue number 7 (Dr. R. van der Zee); 3) Peptides with increasing length, containing 5–16 aa based on the N-terminal part of the natural occurring CHIPS molecule (FTFEPFPTNEEIESNK), were created; 4) Peptides derived from the first 10 aa were synthesized. These peptides contained single amino acid substitutions of the first six residues. In succession, all residues were substituted for all other naturally occurring amino acids. (Pepscan Systems); and 5) A peptide containing the first 40 residues (pep 1–40) and a scrambled peptide containing the first 15 aa in reversed order (pep 15–1; Dr. R. van der Zee). Additionally, a 25-mer pep-scan based on the CHIPS sequence was synthesized. The peptides (peptides with increasing length, pep 1–40 and the 25-mer pep-scan) were synthesized on a MultiSynTech Syro II Robot Synthesizer (MultiSynTech, Witten, Germany) on a 0.05 mmol scale in a polypropylene reaction tube equipped with a polypropylene frit. Syntheses were conducted on ArgoGel Rink-NH-9-fluorenylmethyloxycarbonyl (F-moc) resin (0.32 mmol/g–1) to obtain C-terminal amides. Each synthesis started with 156 mg of dry resin. This resin was swollen in 1,2-dichloroethane (twice with 2 ml, each for 4 min) followed by 1-methyl-2-pyrrolidinone (two times with 2 ml, each for 4 min). The F-moc group was removed by a double treatment with 2 ml 20% piperidine in 1-methyl-2-pyrrolidinone during 8 min. Subsequently, the resin was extensively washed with 1-methyl-2-pyrrolidinone (five times with 2.5 ml, each for 2 min). To this deprotected resin, 1 ml of a 0.2 M F-moc-protected amino acid solution in 1-methyl-2-pyrrolidinone was added followed by 0.75 ml of a 0.267 M solution of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole in 1-methyl-2-pyrrolidinone and 0.5 ml of a 0.4 M solution of N,N-diisopropylethylamine in 1-methyl-2-pyrrolidinone. After 45 min, the reaction tube was drained and the resin was washed with 1-methyl-2-pyrrolidinone (five times with 2.5 ml, each 2 min). After the final F-moc removal, the resin was washed with 1-methyl-2-pyrrolidinone (five times with 2.5 ml, each 2 min) and 1,2-dichloroethane (three times with 2.5. ml, each 2 min). Thus, the obtained anchored peptides were cleaved from the resin and deprotected by treatment with 2 ml of trifluoroacetic acid/H2O/triisopropylsilane (90:2.5:2.5, v/v/v) solution for 2 h at room temperature, followed by precipitation with methyl tert-butyl ether (MTBE)/n-hexane (1:1, v/v, 50 ml). After pelleting the precipitate by centrifugation (3000 rpm, 5 min), the supernatant was decanted, resuspended in MTBE/n-hexane (1:1, v/v), and centrifuged again. Finally, the pellet was washed twice with MTBE (50 ml), dissolved in tert-butanol/water (1:1, v/v, 5–10 ml), and lyophilized to give the peptides as white fluffy solids. The identity and purity of the synthesized peptides were verified by HPLC and LC-electrospray ionization-mass spectrometry. All peptides gave accurate electrospray ionization mass spectra, and the purity was ≥90% in all cases. The peptides synthesized have an amide at the C terminus and a free amine at the N terminus.

CHIPS mutants

To study the FPR-inhibiting activity of CHIPS, four different CHIPS mutants were expressed: CHIPS lacking the first N-terminal amino acid (CHIPS{Delta}1F), CHIPS with an additional alanine at the N terminus (A-CHIPS), and two CHIPS mutants with the first or third amino acid substituted from a phenylalanine to an alanine (CHIPS-F1A and CHIPS-F3A, respectively). For the cloning of CHIPS and CHIPS{Delta}1F, we used the pTrcHISB vector (Invitrogen Life Technologies), for cloning of A-CHIPS, we used the Pet43.1a+ vector (Novagen, Darmstadt, Germany), and the two single mutants were cloned in the pRSET-B vector (Invitrogen Life Technologies). The pTrcHISB and pRSET-B vector were digested with BamH1 (Invitrogen Life Technologies) restriction endonuclease with subsequent S1 nuclease (Invitrogen Life Technologies) treatment and EcoR1 (Invitrogen Life Technologies) digestion to enable ligation of CHIPS genes directly downstream of the enterokinase cleavage site. To achieve the same in the Pet43.1a+ vector, the vector was cleaved with PshA1 (Westburg, Leusden, The Netherlands) and EcoR1 restriction endonuclease. An extra guanine at the 5' end of the CHIPS primer was used to complement the enterokinase cleavage site. The following 5' primers were used to amplify CHIPS, CHIPS{Delta}1F, A-CHIPS, CHIPS-F1A, and CHIPS-F3A, respectively; 5'-TTTACTTTTGAACCGTTTCC-3', ACTTTTGAACCGTTTCCTAC-3', 5'-GGCATTTACTTTTGAACCGTTTCC-3', 5'-GCTACTTTTGAACCGTTTCC-3', 5'-TTTACTGCTGAACCGTTTCC-3'. As 3' primer 5'-CGTCCTGAATTCTTAGTATGCATATTCATTAG-3',was used containing an EcoRI site (underlined). Amplification of wild-type CHIPS and the four CHIPS mutants was performed with Pwo DNA polymerase (Roche, Basel, Switzerland) or Pfu Turbo polymerase (Stratagene, Cedar Creek, TX) to obtain blunt ends. The target for amplification was purified DNA from the S. aureus strain, Newman. CHIPS and CHIPS{Delta}1F were transformed and expressed in a top 10 E. coli, while BL21(DE3) E. coli was used to express the other mutants. All proteins were expressed and purified using the Pro-Bond purification resin according to the manufacturer’s instructions (Invitrogen Life Technologies).

Isolation of human PMN

Blood obtained from healthy volunteers was collected into tubes containing sodium heparin (Greiner Bio-One) as anticoagulant. Heparinized blood was diluted 1/1 (v/v) with PBS and layered onto a gradient of 10 ml Ficoll (Amersham Biosciences, Uppsala, Sweden) and 12 ml Histopaque (density 1.119 g/ml; Sigma-Aldrich, St. Louis, MO). After centrifugation (320 x g, for 20 min at 22°C), the neutrophils were collected from the Histopaque phase and washed with cold RPMI 1640 medium containing 25 mM HEPES buffer, L-glutamine (Invitrogen Life Technologies) and 0.05% HSA (Sanguin, Amsterdam, The Netherlands). The remaining erythrocytes were lysed for 30 s with ice-cold water, after which concentrated PBS (10 x PBS) was added to restore isotonicity. After washing, cells were counted and resuspended in RPMI 1640/0.05% HSA at 107 neutrophils per ml.

Cell culture

U937 cells (human promonocytic cell line) transfected with C5aR (U937/C5aR) or FPR (U937/FPR) were a generous gift from Dr. E. Prossnitz (University of New Mexico, Albuquerque, NM). Cells were grown in 75 cm2 cell culture flasks with 2 µm vent caps (Corning, Acton, MA) placed in a 5% CO2 incubator at 37°C. Cells were maintained in RPMI 1640 medium with L-glutamine (Invitrogen Life Technologies) including 1 mM sodium pyruvate (Invitrogen Life Technologies), 2.5 g/L glucose (Sigma-Aldrich), 10% FCS (Invitrogen Life Technologies) and 10 µg/ml gentamycin (Invitrogen Life Technologies). Cells were diluted 1/10 (v/v) twice a week.

CHIPS activity assays

CHIPS and CHIPS mutants were tested for their ability to inhibit the fMLP- and C5a-induced calcium mobilization in neutrophils or U937/FPR and U937/C5aR. Cells (5 x 106 ml–1 in RPMI 1640/0.05% HSA) were incubated with 2 x 10–6 M Fluo-3AM (Molecular Probes, Eugene, OR) at room temperature for 20 min, washed twice, and suspended in RPMI 1640/0.05% HSA (106 ml–1). The cells were preincubated with buffer, or 1 µg/ml CHIPS, CHIPS{Delta}1F, A-CHIPS, CHIPS-F1A, CHIPS-F3A, or 100 µM peptide (with the exception of the peptides with single amino acid substitutions that were tested in an elastase release assay) at room temperature for 30 min. Incubation with buffer served as blanc control. Triggering neutrophils with fMLP or C5a initiates a rapid and transient increase in the free intracellular calcium concentration that was measured as an increase in a Fluo-3 fluorescence signal detected in a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ). Cells were stimulated with an increasing concentration of fMLP (Sigma-Aldrich) or C5a (Sigma-Aldrich). Each sample of 250 µl of cells was first measured for ~15 s at a fluorescence of 530 nm, to determine the basal calcium level. Next, 20 µl of a 10-fold-concentrated reagent was added (final concentration of fMLP, 10–12 M to 10–5 M, and a final concentration of C5a, 10–12 M to 10–7 M) while vortexing and quickly placed back in the sample holder to continue the measurement. Samples were analyzed after gating the cell population, thereby excluding cell debris and background noise. To check for cell viability of cells incubated with peptides, the stimulated cells were restimulated after ~20 min with PAF (Calbiochem, Darmstadt, Germany; final concentration 10–10 M).

Elastase release essay

Human neutrophils contain enzymes in their granules, among which is elastase. Furthermore, the granules contain the FPR ready for quick recruitment to the cell surface. Cytochalasin B is an actin-polymerization inhibitor that enhances the release of granules after the addition of a stimulus like fMLP. Upon cytochalasin B treatment and subsequent stimulation of neutrophils with fMLP, the cells will effectively excrete their granule content into the medium, thereby releasing elastase and increasing the amount of FPR on the cell surface. CHIPS will inhibit the activation of the neutrophils with fMLP, which can be measured via a decrease in elastase release. The amount of elastase is determined via a specific enzymatic reaction using the fluorescent substrate methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Val-MAC (elastase substrate V) (Calbiochem). The effect of CHIPS on the fMLP-induced elastase release was measured as follows. In a round-bottom microtiter plate, neutrophils (104 per well) were incubated for 15 min with 5 µg/ml cytochalasin B together with single amino acid substituted peptides (10 µM) at room temperature. Subsequently, fMLP (10–8 M) was added. After a 1-h incubation at 37°C, the microtiter plate was centrifuged and a fluorescent substrate (250 µM) was added to each well. The elastase response was measured for 30 min at 37°C in a FluostarII multiwell fluorometer (Isogen Life-Science, Maarssen, The Netherlands). Results are expressed as a percentage of cytochalasin B- and buffer-treated cells stimulated with fMLP after subtraction of the values for nonstimulated cells. The results are compared with inhibition of elastase release of unsubstituted peptide. When the activity is >70% of unsubstituted peptide activity, the peptide is marked inhibitory.

Inhibition of CHIPS activity by mAbs

To test the inhibition of CHIPS by anti-CHIPS mAbs, 6 µg/ml CHIPS was incubated with 100 µg/ml mAb for 30 min at room temperature in a 50 µl of volume. Next, 50 µl of Fluo-3-labeled U937/FPR or U937/C5aR (5 x 106 ml–1) was added and incubated for 30 min at room temperature. Then 150 µl of RPMI 1640/0.05% HSA was added and fluorescence was measured immediately for ~10 s at 530 nm using a FACScan flow cytometer (BD Biosciences). Subsequently cells were stimulated with 3 x 10–9 M fMLP or 10–9 M C5a, and measurement at 530 nm was continued.

Detection of receptor-bound CHIPS using mAbs

To show competition between the binding of CHIPS to its receptors and mAbs, we studied CHIPS-binding to U937/FPR cells or U936/C5aR cells in the presence of anti-CHIPS mAbs. Isolated U937 cells (5 x 106 ml–1) in RPMI 1640/0.05% HSA were incubated with 1 µg/ml CHIPS for 40 min. Cells were washed with RPMI 1640/0.05%HSA and incubated with different concentrations anti-CHIPS mAb. Thereafter, cells were washed again and incubated with goat anti-mouse-FITC (DakoCytomation, Glostrup, Denmark) according to the manufacturer’s instructions. Mean fluorescence was measured at 530 nm using a FACScan flow cytometer (BD Biosciences). The experiment was performed at 4°C.

Iodination of CHIPS

CHIPS was iodinated using the chloramine-T method (11, 22). Briefly, 500 µCi of Na125I was added to 12.5 µg of CHIPS in 0.04 M phosphate buffer, pH 7.4. Chloramine-T (1 µg) was added for 90 s, and the reaction was quenched by the addition of 6 µg of sodium thiosulfate for 60 s. Then, 2% of HSA was added as carrier and bound 125I was separated from free 125I by chromatography on a PD-10 desalting column (Amersham Biosciences) using RPMI 1640/0.2% HSA. The 125I-labeled CHIPS (125I-CHIPS) had a specific activity of 25–55 µCi/µg.

Competition experiments using U937 transfectants

U937/C5aR cells (106 ml–1) or U937/FPR cells (6 x 106 ml–1) were incubated with 0.5 or 20 nM 125I-CHIPS, respectively, together with buffer or 0.1 nM to 10 µM of unlabeled CHIPS, CHIPS{Delta}1F, or A-CHIPS in a final volume of 100 µl. The assay buffer consisted of RPMI 1640/0.05% HSA. Different concentrations of cells and 125I-CHIPS were used to compensate for differences in receptor expression and ligand affinity. After a 1-h incubation at room temperature, 90 µl was applied to a 150-µl mixture of 20% olive oil/80% dibutylphthalate (Sigma-Aldrich) in a microsediment-winning 0.3 ml tube (Sarstedt, Newton, NC). To separate the cell-bound 125I-CHIPS from free 125I-CHIPS, the tubes were centrifuged for 5 min at 1800 x g, after which the tip of the tubes containing the cell pellet were cut and counted in a COBRA gamma counter (Packard Instrument, Meriden, CT) for 5 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Monoclonal Abs

The relative affinity of the anti-CHIPS mAbs were determined by a CHIPS-specific ELISA. The affinity of mAb 6A5 for the CHIPS protein is 10 times lower compared with the other mAbs tested (Fig. 1). The affinity of mAbs 2H7, 2G8, and 5F7 for CHIPS appear equal in this ELISA.



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  		FIGURE 1. mAb 6A5 reacts less in CHIPS-specific ELISA. CHIPS 3 µg/ml was coated on the bottom of a 96-well ELISA-plate. After blocking with 5% HSA, PBS/0.1% Tween wells were incubated with different concentrations anti-CHIPS mAb or mouse IgG in PBS/0.1% Tween, washed, and incubated with goat-anti-mouse-IgG-PO 0.1 µg/ml in PBS/0.1% Tween. Wells were washed and conjugate (0.1 M sodium acetate, 175 µg/ml ureumperoxide, 105 µg/ml tetramethylbenzidine) was added. The reaction was stopped with 50 µl of 1 M H2SO4. Absorbance at 450 nm was determined.

 
Inhibition of CHIPS activity by mAbs

To evaluate the effects of the different mAbs on CHIPS activity, all mAbs were tested for their ability to block the inhibitory activity of CHIPS on the fMLP- and C5a-induced calcium mobilization in U937/FPR and U937/C5aR cells, respectively. Different groups can be distinguished. mAb 2H7 blocks CHIPS activity on U937/FPR cells while leaving the CHIPS activity on U937/C5aR cells completely intact. mAbs 2G8 an 5F7 were not able to block CHIPS activity on U937/FPR, but blocked CHIPS activity on U937/C5aR cells, and thus showed complete opposite action to mAb 2H7 (Fig. 2). This indicates that both actions are located on different sites within the CHIPS protein. Also a mAb (6A5) that interfered with CHIPS activity on both receptors was identified. Incubating cells with mAb in the absence of CHIPS had no effect on cell activation (data not shown).



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  		FIGURE 2. CHIPS activity can be specifically blocked by mAbs. Different mAbs were tested in their ability to inhibit CHIPS activity on the fMLP- and C5a-induced calcium mobilization in U937/FPR and U937/C5aR cells. A total of 6 µg/ml CHIPS was incubated for 30 min with 100 µg/ml mAb in a total volume of 50 µl. Fifty microliters of Fluo-3-labeled U937 cells (5 x 106 ml–1) were added and incubated for 30 min. Increase in fluorescence upon stimulation with fMLP or C5a was measured in a FACScan flow cytometer (BD Biosciences). Control are cells that are not incubated with CHIPS and mAb and activated with fMLP or C5a. The figure shows the activation of cells relative to control. The data show the mean ± SEM of at least three independent experiments.

 
Detection of receptor-bound CHIPS with mAbs

The capacity of all mAbs to detect receptor-bound CHIPS was assessed. Fig. 3A shows that the CHIPS bound to U937/FPR can only be detected with mAbs 2G8 and 5F7. These are the two mAbs that were unable to inhibit CHIPS function on U937/FPR. Consistent with the inhibition experiments mAb 2H7 can only detect CHIPS bound to U937/C5aR (Fig. 3B). mAb 6A5 is not capable of detecting receptor-bound CHIPS at all. This is not caused by the decreased affinity of this mAb because it does inhibit CHIPS function on both receptors in a calcium mobilization assay.



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  		FIGURE 3. mAbs specifically detect CHIPS bound to FPR or C5aR. Isolated U937 cells (5 x 106 ml–1) in RPMI 1640/0.05% HSA were incubated with 1 µg/ml CHIPS for 40 min. Cells were washed with RPMI 1640/0.05% HSA and incubated with different concentrations of mAb. Cells were washed in RPMI 1640/0.05% HSA and optimal diluted goat anti-mouse-FITC was added. Mean fluorescence was measured at 530 nm using a FACScan flow cytometer (BD Biosciences). The experiment was performed at 4°C.

 
Peptides

To investigate what part of the CHIPS sequence was responsible for the FPR- and C5aR-blocking activity, we synthesized and tested different panels of CHIPS-based peptides. Peptides were tested for their ability to inhibit the fMLP- or C5a-induced calcium mobilization of neutrophils or elastase release (peptides with single amino acid substitutions). We tested a pep-scan of overlapping 15-mer peptides. Only the peptide (1–15), consisting of the first 15 N-terminal amino acids, showed FPR-blocking activity with complete inhibition at a concentration of 100 µM (Fig. 4). There is also a slightly decreased activity for peptides 45–60 to 60–75 when stimulated with fMLP. However, the same decrease is seen when cells are stimulated with PAF, indicating that this decrease in activity is not specific for fMLP stimulation. Similar results are obtained when the stimuli are added in reversed order. Fig. 5 focuses on the N-terminal-derived peptides. Peptide 1–15 is the only peptide that inhibits fMLP activation. This shows that the first residue, a phenylalanine, is essential for activity. Also, a control peptide (15–1) containing all residues in reversed order showed no activity. To find the smallest N-terminal peptide with FPR-blocking activity, we created a set of N-terminal peptides with decreasing length from 15 to 5 aa. Peptide 1–6 still showed FPR-blocking activity where peptide 1–5 lost this capacity completely (Fig. 6). To determine the importance of each single amino acid in this six amino acid peptide, we successively substituted each single residue with all the naturally occurring amino acids (Table I). Consistent with the pep-scan, this showed that the first amino acid is important for the inhibition of fMLP-induced elastase release in neutrophils. In addition, the third amino acid, also a phenylalanine, seems to be important for blocking this fMLP-induced release as well. All pep-scan peptides were tested in their ability to inhibit C5aR. No peptide was found that could inhibit the activation of neutrophils by C5a. The same results were observed when testing a 25-mer pep-scan (data not shown). The active peptides were compared in their FPR-blocking activity with CHIPS. This showed a 10,000-fold decrease in activity of the peptides compared with the wild-type protein. Increasing the length of the N-terminal peptide up to 40 residues did not improve activity (Fig. 7).



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  		FIGURE 4. N-terminal 15-mer CHIPS peptide inhibits fMLP-induced activation of neutrophils. A pep-scan of 22 overlapping 15-mer peptides was tested for the ability to inhibit fMLP-induced calcium mobilization in neutrophils. Fluo-3-labeled neutrophils were incubated with 100 µM peptide for 30 min at room temperature. Cells were stimulated with 10–10 M fMLP and activation was assessed by FACScan analysis (BD Biosciences). After ~20 min, cells were restimulated with 10–10 M PAF. Control are cells incubated with buffer and stimulated with fMLP or PAF. Activation of control cells is set to 100%. Each bar shows the mean ± SEM of three independent experiments.

 


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  		FIGURE 5. The first N-terminal residue, a phenylalanine, is essential for activity. Different 15-mer peptides (100 µM), derived from CHIPS N terminus, and also a peptide containing the first 15 aa in reversed order, were tested for the ability to inhibit fMLP-induced calcium mobilization in neutrophils. Fluo-3-labeled neutrophils were incubated with 100 µM peptide for 30 min at room temperature. Cells were stimulated with 10–10 M fMLP and activation was assessed by FACS scan analysis (BD Biosciences). Activation of cells incubated with buffer was set to 100%.

 


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  		FIGURE 6. A peptide containing the first 6 N-terminal residues is the smallest still able to inhibit fMLP-induced neutrophil response. N-terminal peptides of increasing length, varying from 5 to 16 residues, were tested in their ability to inhibit fMLP-induced activation of neutrophils. Fluo-3-labeled neutrophils were incubated with 100 µM peptide for 30 min at room temperature. Cells were stimulated with 10–10 M fMLP and the increase in fluorescence was measured by FACScan analysis (BD Biosciences). Activation of cells incubated with buffer is set to 100%.

 

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  		Table I. Amino acid substitutions in the CHIPS proteina

 


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  		FIGURE 7. The N-terminal CHIPS peptides are 10,000-fold less active than CHIPS in inhibiting fMLP-induced calcium mobilization in neutrophils. Different concentrations of CHIPS and N-terminal CHIPS peptides were compared in their ability to inhibit fMLP-induced Ca2+ flux in neutrophils. Fluo-3-labeled neutrophils incubated for 30 min with different concentrations of CHIPS or peptide were stimulated with 10–10 M fMLP. Increase in fluorescence was measured in an FACScan flow cytometer (BD Biosciences). Activation of cells incubated with buffer was set to 100%. The data represent a mean ± SEM of three independent experiments.

 
CHIPS mutants

To further investigate the importance of the first and third amino acid and to confirm the peptide results, we created different CHIPS mutants. These mutants contained a substitution of the first (CHIPS-F1A) or third (CHIPS-F3A) amino acid for an alanine. Also, a mutant lacking the first amino acid (CHIPS{Delta}1F) and a mutant with an added N-terminal alanine (A-CHIPS) were created. These proteins were tested in their ability to block the fMLP- and C5a-mediated calcium mobilization in neutrophils. All mutants showed a decreased FPR-blocking activity (Fig. 8A). In particular, the first amino acid mutants showed a complete absence of this activity, again confirming the importance of the first phenylalanine. Adding an extra alanine to the N terminus (A-CHIPS) gave a minor but significant decrease in fMLP-induced activation. All mutants were able to completely inhibit activation of neutrophils by C5a (Fig. 8B). This showed that the C5aR-blocking activity is still completely intact, indicating that the loss of FPR-blocking activity is not caused by any structural effect.



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  		FIGURE 8. CHIPS mutants show a decrease or absence of FPR-blocking activity but a complete preservation of C5aR-blocking activity. CHIPS and several CHIPS mutants were tested for their ability to inhibit the fMLP- and C5a-induced calcium mobilization in neutrophils. Fluo-3-labeled neutrophils (106 ml–1) were incubated with CHIPS (1 µg/ml), CHIPS mutants (1 µg/ml), or buffer for 30 min at room temperature. Cells are stimulated with an increasing concentration of fMLP (A) or C5a (B). Increase of fluorescence upon stimulation was measured in a FACScan flow cytometer (BD Biosciences). Activation of cells incubated with buffer and stimulated with 10–7 M fMLP or 3 x 10–9 M C5a was set to 100%. The data represent mean ± SEM of three independent experiments.

 
125I competition experiments on U937/FPR and U937/C5aR cells

To determine whether the decrease in fMLP-induced calcium mobilization is caused by an altered binding affinity, competition experiments with 125I-CHIPS were performed. 125I-CHIPS can be easily displaced on both the FPR (Fig. 9A) and C5aR (Fig. 9B) by CHIPS. A-CHIPS and CHIPS{Delta}1F mutants were able to compete with 125I-CHIPS on the U937/C5aR cells equally effective as CHIPS. In contrast, competition of 125I-CHIPS with both CHIPS mutants on U937/FPR cells showed a marked decrease in affinity of these mutants for the FPR. This proves that the first amino acid is essential in CHIPS binding to the FPR and that the loss of activity is caused by a decreased affinity.



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  		FIGURE 9. CHIPS mutants show a decreased affinity for the FPR but a complete preservation of affinity for the C5aR. Figure shows results of competition experiments between CHIPS or CHIPS mutants and 125I-CHIPS on U937/FPR (A) or U937/C5aR (B) cells. U937/C5aR cells (106 ml–1) or U937/FPR cells (6 x 106 ml–1) were incubated for 1 h with 0.5 nM or 20 nM 125I-CHIPS, respectively, together with buffer or 0.1 nM to 10 µM of unlabeled CHIPS, CHIPS{Delta}1F, or A-CHIPS. Cells were washed and counted in a COBRA gamma counter for 5 min. The data represent the mean ± SEM of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Different anti-CHIPS mAbs were able to inhibit CHIPS function on the C5aR and the FPR independently. This clearly indicates that different sites within the CHIPS protein are responsible for binding the FPR and C5aR, respectively. The fact that mAb 6A5 was able to inhibit the CHIPS function on both the FPR and C5aR could implicate that these different active sites are closely spatially related and might be overlapping. This could be of future importance in separating the two functions. Compared with an IgG molecule, CHIPS is a small protein (14.1 kDa). mAb 6A5 can be directed against a part of CHIPS that is not involved in binding any of the receptors but still causes an inhibitory effect by steric hindrance.

Despite its relatively low affinity, mAb 6A5 is still able to inhibit both CHIPS functions. This shows that we used an excess mAb in the inhibition experiments. Therefore, the noninhibitory properties of the mAbs are not caused by concentration or affinity effects.

Using diverse synthetic CHIPS-derived peptides and four different recombinant CHIPS mutants, we deduced the CHIPS active binding site toward the FPR to its first and third phenylalanine. Although experiments with the synthetic peptides showed equal importance of both phenylalanines, the CHIPS mutants, CHIPS-1FA and CHIPS-3FA, demonstrated that the first phenylalanine of CHIPS is the most crucial for its FPR-blocking activity. The small decrease in activity observed with the A-CHIPS mutant indicates that this first amino acid has to be freely available at the N terminus. Competition experiments using 125I-CHIPS show that the loss of activity is caused by a loss of binding capacity for the receptor. One might speculate about the presence of another FPR binding site within CHIPS as all CHIPS-derived peptides showed a 10,000-fold decrease in activity. This will be subject to future research. None of the CHIPS-derived peptides or CHIPS mutants showed any inhibiting effect on the C5aR. Even when a pep-scan of 25-mer overlapping CHIPS-derived peptides was used, no CHIPS activity on the C5aR was found. It could be that the three-dimensional structure of CHIPS, necessary for C5aR activity, is not mimicked by the peptides. Additionally, a concerted action of more than one C5aR binding site could be necessary for the CHIPS activity. Currently, we are investigating the C5aR-blocking activity of larger protein fragments, obtained via chemical cleavage of CHIPS. We also are determining the three-dimensional structure of CHIPS via NMR. Hopefully, in the future, this will provide us information about the exact C5aR-blocking sites within CHIPS. The differences in the FPR- and C5aR-activity of the CHIPS-derived peptides and CHIPS mutants suggests that two distinct sites within the CHIPS protein are responsible for both actions. These sites might be closely related to each other as was strongly suggested by the anti-CHIPS mAbs data.

Leukocytes accumulate at sites of inflammation and immunological reaction in response to locally existing chemotactic mediators. N-formyl peptides, such as fMLP, are some of the first identified and most potent chemoattractants for phagocytic leukocytes (16). In all organisms, protein synthesis is initiated with a methionine residue which can be removed during protein maturation. In bacteria and mitochondria, methionyl-tRNA-formyltransferase, the fmt gene product, adds a formyl group to the amino group of the methionine esterified to tRNA (tRNAfmet). Consequently, nascent polypeptides have a formylated methionine at their N termini. Following initiation of protein synthesis and addition of several amino acid residues to the growing peptide chain, the formyl group is almost invariably removed by the action of peptide deformylase encoded by the def gene. Two deformylase homologues, defA and defB, were identified in S. aureus, but only defB is an active enzyme (23). The system is by no means perfect and it has been known for a long time that microbial metabolic activity is associated with a low level of N-formyl peptide release (8, 24, 25, 26). This may be the result of a "leaky" metabolic system, but it has also been suggested that such peptides represent N-terminal signal peptides from newly synthesized bacterial proteins, which are cleaved by prokaryotic signal peptidases present in the bacterial cell wall (24). The efficiency and rate of deformylation are most probably also affected by the sequence of amino acids in the N terminus, as well as their susceptibility to oxidation. N-formyl peptides are products of bacterial metabolism, and their binding to the FPR induces chemotaxis and activation of phagocytes. Because phagocytes are critical effector cells in our innate immune system, it is reasonable to assume that the interaction between these two counterparts (formylated peptides and FPR) is important in the antimicrobial host defense. It has been demonstrated that mice with a disrupted FPR gene, display an impaired antibacterial immunity (27, 28). Inhibiting this response would benefit any invading organism, and therefore, CHIPS could be an important virulence strategy (29) of S. aureus and a new potential target in the treatment of S. aureus infections. Also, chemokine receptors are obvious targets for drug development because they have presumed roles in disease processes (16). During the past few years, progress has been made in the understanding of the biological roles of once elusive FPR. The identification of novel and host-derived agonists broadens the spectrum of functional significance of such receptors. A full understanding of the role of the FPR in disease states requires further investigation, in which formyl peptide antagonists play an important role (30, 31). Well-known FPR antagonists include cyclosporine H and Boc-PLPLP that are used extensively in FPR research. Compared with these molecules, the affinity and potency of CHIPS to bind and block the FPR is at least 1000-fold higher (32). The unique properties of CHIPS to specifically inhibit the FPR with high affinity could be an important new tool to further stimulate the fundamental research on the mechanisms underlying the FPR and its role in disease processes (16, 17, 32, 33).


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Pieter-Jan Haas, Eijkman Winkler Laboratory, University Medical Center Utrecht, HP G04-614, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands. E-mail address: P.J.A.Haas{at}azu.nl Back

2 Abbreviations used in this paper: PAF, platelet activating factor; CHIPS, chemotaxis inhibitory protein of Staphylococcus aureus; FPR, formylated peptide receptor; HSA, human serum albumin; F-moc, 9-fluorenylmethyloxycarbonyl; MTBE, methyl tert-butyl ether; 125I-CHIPS, 125I-labeled CHIPS. Back

Received for publication March 8, 2004. Accepted for publication August 13, 2004.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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