From the Departments of Microbiology and
¶ Chemistry and Biochemistry, Montana State University,
Bozeman, Montana 59717-3520
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A novel fluorescent photoaffinity
cross-linking probe,
formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys--N-fluorescein
(fMBpaFYK-fl), was synthesized and used to identify binding site
residues in recombinant human phagocyte chemoattractant formyl peptide
receptor (FPR). After photoactivation, fluorescein-labeled membranes
from Chinese hamster ovary cells were solubilized in octylglucoside and
separated by tandem anion exchange and gel filtration chromatography. A
single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein
immunoaffinity matrix. Matrix-assisted laser desorption ionization mass
spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to
Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues
83-87). This peptide was further cleaved with trypsin, repurified by
antifluorescein immunoaffinity, and subjected to matrix-assisted laser
desorption ionization mass spectrometry. A tryptic fragment with
mass = 1582 was observed, which is the mass of fMBpaFYK-fl
cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin
cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger,
B. L., and Jesaitis, A. J. (1997) J. Immunol.
159, 4045-4054) and the photochemical cross-linking data.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The phagocyte chemotactic receptors, including the formyl peptide receptor (FPR),1 the lipoxin A4 receptor, the C5a receptor, the platelet-activating factor receptor, and the interleukin-8 receptor are involved in inflammation and are all members of the G protein-coupled receptor (GPCR) superfamily. Among the most studied in this inflammatory receptor family is neutrophil FPR (1). FPR binds N-formyl peptides, such as formyl-Met-Leu-Phe (fMLF), with nanomolar affinity (2). Such N-formyl peptides are indicators of the presence of bacteria (3) or damage to host cell mitochondria (4, 5). Binding of N-formyl peptides to FPR thus provides phagocytes with signals for infection or injury and results in activation of chemotaxis and other host defensive processes including lysosomal enzyme secretion, stimulation of production of inflammatory mediators, and generation of superoxide.
The effects of amino acid substitutions and modifications of fMLF peptides on binding to FPR and activation have been studied extensively (6-8). The formyl group, the methionine at position 1, and phenylalanine at position 3 have been shown to be necessary for high affinity binding. Decarboxylation of the C-terminal phenylalanine markedly reduces activity, but esters or amides of this residue or peptides with C-terminal amino acid additions exhibit similar activity to the tripeptide with the free acid. None of the fMLF functional groups have been shown to be absolutely essential for activity but rather they appear to individually contribute to the overall free energy of binding.
Chemical and photoaffinity cross-linking of fMLF analogs to FPR has
been achieved by a number of groups (9-12). However, none of these
studies have identified the site of labeling. The residue in the second
position of N-formylated peptides appears to be the most
tolerant of modification (6, 13) and both of the flanking residues are
critical for high affinity binding (14); so the second residue was
chosen to accommodate the photoreactive amino acid benzoylphenylalanine
(Bpa). Bpa is chemically stable in the absence of photoexcitation, can
be directly introduced into peptide ligands by solid phase peptide
synthesis, and has been photocross-linked into several peptide
receptors (15). Here we report that a fluorescent photoaffinity analog
of fMLF, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys--N-fluorescein (fMBpaFYK-fl), efficiently photocross-links to FPR residues 83-85. Derivatization of these residues in FPR by Bpa supports recent site-directed mutagenesis studies predicting that the formyl peptide binding site of FPR lies within the transmembrane spanning region, near
the transmembrane-extracellular interface of the receptor (17).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peptide
Synthesis--
fMet-p-Benzoyl-L-phenylalanine-Phe-Tyr-Lys
was synthesized from Fmoc (N-(9-fluorenyl)methoxycarbonyl)
amino acids using a Milligen 9050 peptide synthesizer and purified by
reverse phase HPLC. Peptide (4 µmol) was suspended in
dimethylformamide, 5% triethylamine, and 4 µmol of
hexanoylfluorescein-N-hydroxysuccinimide (Molecular Probes)
was added. The reaction was allowed to proceed for 5 min at 4 °C.
The products were separated by C18 reverse phase HPLC. The desired
peptide, fMBpaFYK-fl was identified by its absorbance and fluorescence
spectra (in 10 mM NaCO3, pH 10.5), and its mass
(m/z = 1340) measured by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-MS) in a
-cyano-4-hydroxycinnamic acid matrix. Samples were stored in amber
vials at
20 °C. The syntheses and all manipulations were carried
out under dim light.
Preparation of Antifluorescein-Sepharose-- Antibodies to fluorescein isothiocyanate were prepared by injecting rabbits with fluorescein isothiocyanate-labeled keyhole limpet hemocyanin, as described previously (16). Serum was precipitated with 50% ammonium sulfate and antibodies were purified on fluorescein isothiocyanate-labeled aminohexyl-Sepharose. Purified antibodies bound ~2 mol of fluorescein isothiocyanate-ethanolamine/mol of antibody, as assessed by its ability to quench fluorescein fluorescence. The antibodies were reacted with CNBr-activated Sepharose and cross-linked with 50 mM dimethyl pimelimidate (Pierce) in 0.1 M Na2CO3, pH 10.5, for 16 h. The antifluorescein-Sepharose was washed extensively (>72 h) with phosphate-buffered saline, 1.5 M NaCl, and 50% ethylene glycol. It was washed quickly (<5 min) with 1% triethylamine, 1.5 M NaCl, 50% ethylene glycol, and equilibrated in 10 mM HEPES (pH 7.4) and 1% octyl glucoside just prior to use. The antifluorescein-Sepharose bound ~16 pmol of fMBpaFYK-fl per µl of Sepharose.
FACScan Binding of fMBpaFYK-fl to FPR-expressing Chinese Hamster Ovary (CHO) Cells-- FACScan analysis was carried out as described previously by (17). The analysis was carried out in dim light to prevent photolysis of fMBpaFYK-fl.
Photolabeling of fMBpaFYK-fl to FPR and Preparation of CHO Membranes-- CHO cells expressing FPR (17) or WT CHO cells were grown in 15-cm tissue culture dishes and treated with 6 mM sodium butyrate 16 h prior to labeling. The cells were washed 3 times with phosphate-buffered saline and incubated with 100 nM fMBpaFYKf for 5 min at 4 °C. The tissue culture dishes were loaded directly into a Rayonet RPR-100 UV photoreactor and exposed to UV irradiation for 15 min at 4 °C. EDTA, leupeptin, and fMLF were added to give final concentrations of 1 mM, 1 µg/ml, and 10 µM, respectively. The cells were harvested by scraping and centrifuged at 150,000 × g for 30 min. The cell pellets were sonicated in phosphate-buffered saline, 1 mM EDTA, 1 µg/ml leupeptin, 10 µM fMLF, and 200 µM dithiothreitol (Buffer A), washed twice with 10 mM Na2CO3, pH 10.5, 0.8 M NaCl, 1 mM EDTA, 1 µg/ml leupeptin, 10 µM fMLF, and 200 µM dithiothreitol.
HPLC Analysis of Photolabeled FPR-- Membranes from CHO cells expressing FPR and wild type control CHO cell membranes were prepared as described above, resuspended in 0.7 ml of Buffer A containing 3% octyl glucoside, sonicated, and centrifuged at 150,000 × g for 30 min. The supernatant was injected onto anion exchange (Vydac 300VHP) and gel filtration (TSK GW3000) HPLC columns connected in series, and the columns were monitored for fluorescence with excitation at 490 nm and emission at 520 nm. 1 ml of 1 M NaCl was injected after 60 min elution to remove material that bound to the anion exchange column. The HPLC analysis was carried out using a Hitachi 6200 HPLC system with a F-1050 fluorescence detector.
Digestion of Photolabeled FPR with CNBr and Isolation of
Photocross-linked Fragments--
Photolabeled membranes from
FPR-expressing and WT CHO cells were washed with H2O, the
membranes were resuspended in 200 µl of H2O, sonicated,
and dissolved by addition of 800 µl of trifluoroacetic acid. 50 µmol of CNBr was added, and the reaction mixtures were incubated for
16 h at 20 °C. An additional 10 µmol of CNBr was added, and
the incubation continued for an additional 8 h. The samples were
frozen at 80 °C and lyophilized. The samples were resuspended in
10 mM HEPES and 1% octyl glucoside, adjusted to pH 7-8
and centrifuged at 150,000 × g for 30 min. The
supernatant was added to 30 µl of rabbit antifluorescein antibodies
bound to Sepharose and incubated for 16 h at 4 °C. The
Sepharose beads were washed 5 times with H2O, eluted with
1% triethylamine, 40% acetonitrile in water and lyophilized. For
trypsinization, the sample was resuspended in 1% octyl glucoside, 10 mM Tris, pH 8.0, and incubated with 1 µg of trypsin
(sequencing grade, Boehringer Mannheim) for 16 h at 25 °C. 4 µg of soybean trypsin inhibitor was added, followed by the addition
of 30 µl of antifluorescein immunoaffinity matrix, and the isolation
procedure described above was repeated.
GTPS Binding to FPR-expressing CHO Cells--
Membranes from
FPR-expressing CHO cells were harvested, sonicated, washed in Buffer A
and resuspended in GTP
S binding buffer. Binding assays were
performed in 1 ml of 10 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM Mg2+, 1 µM GDP. Samples containing 30 µg of protein were
preincubated with formyl peptide or analog at 30 °C for 10 min, 0.05 µCi of [35S]GTP
S (1000 Ci/mmol) was added, and the
reaction continued for 6 min. The samples were filtered through BA 85 0.45-µm filters (Schleicher & Schuell), the filters were washed with
5 ml of 10 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM Mg2+ buffer, and the bound
[35S]GTP
S was measured by liquid scintillation
counting.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The novel synthetic fluorescent photoaffinity analog fMBpaFYK-fl
activates the formyl peptide receptor more efficiently than does fMLF.
Fig. 1A shows that fMBpaFYK-fl
enhanced [35S]GTPS binding to CHO cell membranes
expressing FPR with an EC50 of 3 nM. This
EC50 was about 2-fold lower than that observed with fMLF
(EC50 ~ 6 nM). At saturation, both ligands
increased GTP
S binding to a similar extent (~100% increase in
binding). Fig. 1B shows FACScan measurement of the binding
of fMBpaFYK-fl to FPR expressed in CHO cells. Analysis of the FACScan
binding data by nonlinear least squares fit of the data to a sigmoidal
curve, using Prism 2 (Graph Pad Software, Inc.), indicated that
fMBpaFYK-fl bound to FPR in intact cells with a Kd
of 37 nM. This photoaffinity analog had a binding affinity
similar to that observed with the hexapeptide,
formyl-Nle-Leu-Phe-Tyr-Nle-Lys-fluorescein, a similar fluorescent
analog of fMLF that had been previously studied (17-19).
|
UV irradiation of FPR occupied with fMBpaFYK-fl, produced extensive nonreversible binding of the photoactivatable ligand, as shown in Fig. 2. After 10 min of irradiation, greater than 40% (n = 2) of the fMBpaFYK-fl was no longer displaced from FPR by fMLF under conditions that completely displaced fMBpaFYK-fl, which had not been exposed to UV light.
|
To determine if fMBpaFYK-fl specifically photolabeled FPR, octyl glucoside-solubilized membranes from CHO cells expressing FPR or WT control CHO cells, which had both been exposed to fMBpaFYK-fl and UV irradiated, were injected onto anion exchange and gel filtration HPLC columns connected in series. The columns were monitored for fluorescein fluorescence (excitation 490 nm/emission 520 nm). 1 ml of 1 M NaCl was injected at time = 60 min to remove material that bound to the anion exchange column and subject that material to size separation by gel filtration. Fig. 3 shows a large peak of fluorescein labeled protein from FPR-expressing CHO cells that was not retained by the anion exchange column (consistent with an isoelectric point of 9.4 for FPR calculated from its amino acid sequence). This peak was found to FPR by Western blot (not shown) and was completely absent in the WT controls, indicating that the fMBpaFYK-fl was specifically labeling FPR. The area under the FPR peak in Fig. 3 represented 60-70% of the eluted fluorescein fluorescence and indicated that most of the photolabeled material was in FPR.
|
To determine the site of photocross-linking between fMBpaFYK-fl and FPR, photolabeled FPR was cleaved with CNBr and the fMBpaFYK-fl-containing adducts were identified by MALDI mass spectrometry. Fig. 4 shows a model of the transmembrane topology of FPR (17) where the positions of the methionine CNBr cleavage sites are indicated by gray shading. The table inset shows the expected masses of the 11 possible cross-linked adducts between CNBr-digested FPR and benzoylphenylalanine-FYK-fl (BpaFYK-fl), the CNBr cleavage product of fMBpaFYK-fl. All of the expected masses of the CNBr cleavage products are sufficiently different that they can be resolved by MALDI-MS, which has a mass accuracy of about 0.1%.
|
Since the HPLC analysis indicated that most of the photoaffinity label was incorporated into FPR, CNBr digestions were performed directly on CHO cell membranes to maximize the yield of photocross-linked peptides. To obtain sufficient photolabeled FPR for identification of CNBr fragment adduct(s) by MALDI-MS and to show that these adduct(s) were derived from FPR, 20 15- × 1-cm tissue culture plates from WT CHO cells and CHO cells expressing FPR were photolabeled with 100 nM fMBpaFYK-fl. Membranes from an equal number of cells of each type were solubilized in 1 ml of 80% trifluoroacetic acid and cleaved with CNBr.
Fluoresceinated Adducts from Both Wild Type and FPR-expressing CHO Cell Membranes-- Fluoresceinated adducts from both wild type and FPR-expressing CHO cell membranes were isolated with antifluorescein immunoaffinity chromatography, as described under "Materials and Methods." 20 and 8 pmol of cross-linked fluoresceinated peptides were isolated from FPR-expressing and WT CHO cell membranes, respectively, indicating that ~60% (i.e. 12 of 20 pmol total) was specifically photocross-linked to FPR, in agreement with the HPLC analysis shown in Fig. 2. The material eluted from the antifluorescein antibodies was analyzed by MALDI-MS (Fig. 5, inset). Mass spectra obtained from WT cells was subtracted from that observed for FPR-expressing cells, and a typical difference spectrum is shown in Fig. 5.
|
|
Three-dimensional Placement of the fMLF Binding Site in
FPR--
The ability to photoaffinity cross-link FPR with a high
affinity analog of fMLF provides an opportunity to experimentally test
the intramembrane ligand binding hypothesis for G protein-coupled peptide receptors (17, 18). A three-dimensional model of FPR was
generated from a rhodopsin template designed by Herzyk and Hubbard
(20), and a space filling model of fMet-Bpa-Phe (fMBpaF) was placed
between the seven transmembrane helices (Fig.
7). Two stereo views are shown in Fig. 7,
a view from the extracellular space and a transmembrane view. fMBpaF
was positioned between the helices to allow maximum contact with
residues previously shown to be important in ligand binding (17). By
modeling fMBpaF in an extended conformation, the Bpa moiety could be
placed near the site of cross-linking while maintaining interactions
between fMBpaF and residues that affect binding. The methionine and
phenylalanine side chains of fMBpaF, which are essential for high
affinity binding, were positioned deep within the binding pocket
(relative to the extracellular side), whereas the Bpa side chain was
positioned nearer the extracellular space, so as to be consistent with
the cross-linking data. Since studies with model compounds suggest the
carbonyl carbon of the benzophenone moiety of Bpa generally reacts with
another carbon within a distance of approximately 3 Å and the carbon
adjacent to the amino group of lysine is reported to be one of more
reactive side chains toward photocross-linking with benzophenone (15),
the
carbon of Lys-85 was placed within 3 Å of the carbonyl oxygen
of the benzophenone moiety of fMBpaF. In this position, the amine
nitrogen of Lys-85 (II-24) is within 2-3 Å of the methionine carbonyl
oxygen on fMBpaF, sufficiently close to form a hydrogen bond.
Alternatively, Lys-85 could ion pair with Asp-284 and still be close
enough to the carbonyl oxygen of the benzophenone moiety of fMBpaF to
photocross-link effectively. In the present model, neither of the
sidechains of Val-83 or Arg-84 appear to be sufficiently close to the
benzophenone carbonyl of fMBpaF to be likely sites of cross-linking,
but their backbone moieties might be close enough to cross-link
efficiently. The present data does not distinguish between
cross-linking to the three residues (83-85), and additional studies
will be needed to delineate the exact site(s) of interaction.
|
|
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Pawel Herzyk and Rod E. Hubbard for establishing the 7TM receptor modeling program at Swiss Pro on the World Wide Web (Expasy.hcuge.ch/CAI-bin/Promod-GPCR.pl).
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Science Foundation EPSCoR Grant RII-891879 (to E. A. D.), a Grant from the Pittsburgh Supercomputing Centers through the National Institutes of Health resource Grant 2p41RR06009, a grant from the Rocky Mountain Chapter of the Arthritis Foundation and the Harmon Foundation (to J. S. M.), a grant from the Rocky Mountain Chapter of the Arthritis Foundation (to H. M. M.), and Public Health Service Grants 1RO1A40108-01 and RO122735 (to A. J. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Montana State University, Dept. of Microbiology, 109 Lewis Hall, Bozeman, MT 59717-3520. Tel.: 406-994-6506; Fax: 406-994-4926; E-mail: umbmj{at}gemini.oscs.montana.edu.
Present address: Dept. of Analytical Chemistry, Uppsala
University, Box 531, S-751 21 Uppsala, Sweden.
** Present address: Combined Program in Pediatric Gastroenterology and Nutrition, Enders Bldg. 1209, Children's Hospital, 300 Longwood Ave., Boston, MA 02115.
1
The abbreviations used are: FPR, formyl peptide
receptor; fMBpaFYK-fl,
formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys--N-fluorescein; GPCR, G protein-coupled receptor; fMLF, formyl-Met-Leu-Phe; Bpa, benzoylphenylalanine; HPLC, high performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption ionization mass
spectrometry; CHO, Chinese hamster ovary; WT, wild type; FACScan,
fluorescence-activated cell scanner.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|