G Model
MIMM-3213; No. of Pages 10
ARTICLE IN PRESS
Molecular Immunology xxx (2009) xxx–xxx
Contents lists available at ScienceDirect
Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm
IgE-binding properties and selectivity of peptide mimics
of the FcRI binding site
Annamaria Sandomenico a , Simona M. Monti a , Daniela Marasco a,b ,
Nina Dathan a , Rosanna Palumbo a , Michele Saviano a , Menotti Ruvo a,∗
a
b
Istituto di Biostrutture e Bioimmagini, CNR, via Mezzocannone 16, 80134 Napoli, Italy
Dipartimento delle Scienze Biologiche, Università di Napoli Federico II, via Mezzocannone 16, 80134 Napoli, Italy
a r t i c l e
i n f o
Article history:
Received 19 June 2009
Accepted 26 July 2009
Available online xxx
Keywords:
IgE
FcRI
Peptide mimics
SPR
ELISA
a b s t r a c t
FcRI␣ found on the surface of mast cells and basophiles mediates allergic diseases, anaphylaxis and
asthma through binding of IgE. Disrupting this interaction with anti-IgE mAbs has proven an efficient
approach to control these diseases. The crystallographic structure of the complex formed between the
IgE-Fc and FcRI␣ extracellular domain has shown that recognition is mediated by residues in the second
Ig-like domain of the receptor (D2) and in the loop connecting the D1 and D2 domains. In an attempt
to obtain specific IgE antagonists, we have designed and prepared a polypeptide named IgE-Trap that
partially reproduces the IgE receptor-binding sites and binds with micromolar affinity to soluble IgE. The
polypeptide contains loops C′ –E [residues 129–134] and F–G [residues 151–161] from the D2 domain
joined by a linker, and loop B–C [residues 110–113]. Peptide binding to IgE has been assessed by SPR
analyses and the data fit with a biphasic model of interaction, in agreement with the two-site mechanism reported for the native receptor. The polypeptide binds to immobilized IgE in a dose-dependent
manner with a KD estimated to be around 6 M, while it does not recognize IgG nor IgA. Polypeptide
sub-domains involved in IgE binding have also been defined, showing that loop C′ –E connected to loop
B–C, but also the isolated loop B–C alone suffice to bind immunoglobulins E with high selectively though
with reduced affinity compared to IgE-Trap. ELISA and cytometric assays on RBL2H3 cells demonstrate
that the interacting peptides are able to displace the binding of IgE to receptor, confirming affinity and
specificity of these ligands and suggesting a potential application as modulators of disorders associated
with inappropriate IgE production.
© 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The human high affinity IgE receptor, hFcRI, found on the
surface of mast cells and basophiles, mediates allergic diseases,
anaphylaxis and asthma through its binding to IgE (Kinet, 1989;
Metzger et al., 1989). hFcRI contains four distinct polypeptide
Abbreviations: BSA, bovine serum albumin; DIEA, di-isopropylethylamine; DMF,
dimethylformamide; DCM, dichloromethne; DTT, di-thiothreitol; DMSO, dimethylsulphoxide; Fmoc, fluorenylmethoxycarbonyl; FPLC, fast purification liquid
chromatography; FITC, fluorescein isothiocyanate; HBTU, 1-H-benzotriazolium,1[bis(dimethylamino)methylene]-hexafluorophosphate(1-),3-oxide;
HOBt,
Nhydroxybenzotriazole; HPLC, high performance liquid chromatography; IPTG,
isopropyl -d-thiogalactoside; LC–MS, liquid chromatography–mass spectrometry; PBS, phosphate buffered saline; PMSF, phenyl-methyl-sulphonyl-fluoride;
SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; 4-VP,
4-vynyl-pyridine; TIS, tri-isopropylsilane; TFA, trifluoroacetic acid; EDC, 1-ethyl3-[3-dimethylaminopropyl]carbodiimide
hydrochloride;
NHS,
N-hydroxysuccinimide.
∗ Corresponding author. Tel.: +39 081 2536644; fax: +39 081 2534574.
E-mail address: menotti.ruvo@unina.it (M. Ruvo).
chains: an extracellular ␣-chain, a -chain and a dimer of ␥-chains.
The extracellular portion of the ␣-chain binds with high affinity
to the Fc region of IgE (KD = 10−9 M) (Cook et al., 1997), whereas
the - and ␥-chains are responsible for down-stream signal
propagation through phosphorylation of their intracytoplasmatic
immune-receptor tyrosine-based activation motifs (ITAM) (Kinet,
1999; Kochan et al., 1988; Kraft and Kinet, 2007). The ␣-chain
is N-glycosylated at seven sites, but sugars are not intrinsically
necessary for proper folding or for binding to immunoglobulins
(Garman et al., 2000). Instead they are deemed to be necessary
for mediating the correct interaction between the ␣-chain and the
ER folding machinery in the endoplasmic reticulum (Letourneur
et al., 1995) and to stabilize the receptor’s secondary and tertiary
structures (Garman et al., 1998). Receptor cross-linking through
allergen/antibody interactions leads to the activation of an intracellular signal cascade mediated mainly by the SRC family kinase
Lyn and Fyn as well as Syc kinases (Scharenberg et al., 1995; Turner
and Kinet, 1999). In mast cells, this signal cascade leads to degranulation and the release of histamine, cytokines and other mediators
of the allergic response. The pharmacological therapy for allergy
0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2009.07.025
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
G Model
MIMM-3213; No. of Pages 10
ARTICLE IN PRESS
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
2
and asthma makes use of drugs that block histamine release, of
bronco-dilatators and corticosteroids that essentially reduce symptoms and the concomitant inflammatory reaction. However, given
the increasing incidence of allergic diseases, that nowadays affect
more than 20% of the western world population (Cookson, 1999;
Meltzer and Grant, 1999), the search for non-symptomatic drugs
that block the early events of allergic response is becoming a very
important field of investigation. The recent introduction on the
market of an anti-IgE antibody (Corren et al., 2009; MacGlashan,
2009) has provided the proof of concept that blocking FcRI signalling is a successful therapeutic strategy and that preventing IgE
binding to the receptor or at least modulating its binding kinetics, is a useful way for counteracting these disorders. Based on this
background knowledge, several studies now focus on the development of potent and selective IgE receptor antagonists, with several
groups having already developed small peptides targeting the IgEFcRI interaction derived from screening phage display libraries or
designed on the basis of known receptor agonists (Buku et al., 2001,
2003, 2004, 2005, 2008; McDonnell et al., 1996, 1997; Nakamura
et al., 2001, 2002; Rossi et al., 2008; Stamos et al., 2004). However,
because of the high affinity and selectivity of the IgE receptor complex, developing effective antagonists still remains a challenging
task. The crystallographic structure of the FcRI␣/FcIgE complex
has elucidated the molecular mechanism and the stoichiometry
of this interaction (Garman et al., 2000, 2001; McDonnell et al.,
2001). The reported structure shows that the receptor is comprised
of two Ig-like domains, D1 and D2, that form an acute angle like an
inverse V and that IgE recognition is mediated by a cluster of aromatic residues localized on D2 and on the linker connecting D1
and D2. The D1 domain, instead, seems to contribute to the high
affinity by stabilizing D2 and the linker conformation (Basu et al.,
1993). Remarkably, sugar residues present on both the ligand and
the receptor, also have only a stabilizing role and do not actively
participate in the interaction (Garman et al., 2000, 2001).
In this study, we have attempted to obtain IgE binders derived
from the receptor-binding sites thus having a neutralizing activity against receptor activation. Peptides have been prepared by
solid phase synthesis and tested by both SPR and ELISA techniques,
showing that they bind to human and mouse IgE. Despite their affinity in the micromolar range, binding appears highly selective, as
they do not cross-react with IgG and IgA and also block binding of
human IgE to the human D2 domain and the binding of mouse IgE
to rat receptors on cell membranes.
Fig. 1. Schematic representation of the IgE-Trap structure. The loop C′ –E contains
residues from the IgE-binding site 1. Loop B–C contains residues from the region
110–113 and a tryptophan from the linker region (Trp87). A linker joins this last
region with the loop F–G containing residues 151–161. Loop B–C and loop F–G
reproduce the IgE–binding site 2. In the cyclic variant (IgE-Trapox ), a disulfide bond
bridges Cys151 to a cysteine in position 162 replacing the native serine. In the linear
variant (IgE-Trapred ) the disulfide bond is in the reduced form.
2. Experimental procedures
acids and resins, activation and deprotection reagents) were from
Novabiochem (Laufelfingen, Switzerland) and InBios (Pozzuoli,
Italy). Solvents for peptide synthesis and HPLC analyses were
from Romil (Dublin, Ireland); reversed phase columns for peptide
analysis and the LC–MS system were from ThermoFisher (Milan,
Italy). Human IgE were from Chemicon International, while mouse
IgE were from Sigma–Aldrich. Non-aggregated polyclonal human
IgG and IgA were a kind gift of Dr. A. Verdoliva of Tecnogen SpA
(Caserta, Italy). Solid phase peptide syntheses were performed
on a fully automated multichannel peptide synthesizer Syro I
(Multisynthech, Germany). Preparative RP-HPLC were carried out
on a Shimadzu LC-8A, equipped with a SPD-M10 AV detector
and with a Phenomenex C18 Jupiter column (50 mm × 22 mm ID;
10 m). LC–MS analyses were carried out on an LCQ DECA XP
Ion Trap mass spectrometer equipped with an OPTON ESI source,
operating at 4.2 kV needle voltage and 320 ◦ C with a complete Surveyor HPLC system, comprised of MS pump, an autosampler and a
photo diode array (PDA). Narrow bore 50 mm × 2 mm C18 BioBasic
LC–MS columns were used for these analyses. All ELISA assays
to screen the synthetic peptides, were carried out using a fully
automated system (Hamilton Robotics, Milano, Italy) comprising a
liquid handler, a robotic arm, a washer and an automated Synergy
4 multi-wavelength reader (BIOTEK Instruments, Inc., Highland
Park, VT, USA). Dulbecco’s modified eagle’s medium (DMEM) and
fetal bovine serum (FBS) were from BioWhittaker (Verseviers,
Belgium); l-glutamine was from Gibco (Milano, Italy). FITC-avidin
was from Santa Cruz Biotechnology (CA, USA).
2.1. Materials
2.2. Peptide synthesis
Oligonucleotides were synthesized by Sigma-Genosys
(Sigma–Aldrich, Milano, Italy). pETM vectors were from EMBL
(Heidelberg, Germany). Pfu DNA polymerase was from Stratagene
(Milano, Italy). Restriction enzymes were from New England
Biolabs (Milano, Italy). All molecular biology kits were from Qiagen
(Milano, Italy). Escherichia coli bacterial strains were from Novagen
(Milano, Italy). IPTG was from Inalco (Milano, Italy). Reagents for
bacterial medium were from Beckton-Dickinson (Milano, Italy).
All reagents for SDS-PAGE, columns and ÄKTA FPLC were from
GE Healthcare (Milano, Italy). Sigma-fast o-phenylenediamine
dihydrochloride Tablet Sets were from Sigma–Aldrich (Steinheim,
Germany). The BIAcore 3000 SPR system for Real Time kinetic
analysis and related reagents were from GE Healthcare (Milano,
Italy). All other reagents and chemicals were commercially available by Sigma–Aldrich or Fluka (Steinheim, Germany). The hFcRI
cDNA was kindly provided by Prof. Pucillo from the University
of Udine. Reagents for peptide synthesis (Fmoc-protected amino
Peptides were prepared by solid phase synthesis as C-terminally
amidated and N-terminally acetylated derivatives following standard Fmoc chemistry protocols (Fields and Noble, 1990). A
Rink-amide MBHA resin (substitution 0.53 mmol/g) and amino acid
derivatives with standard protections were used in all syntheses.
A lysine with a 4-methyl-trityl (Mtt) protection was used to link
the side chain of Lys133 to the C-terminus of Trp113 (see Fig. 1).
A -alanine was instead used to connect Trp110 and Trp87. A
linker comprising a succinic acid and an ethylenediamine moiety was introduced between Trp87 and the C-terminus of loop
B–C (Garman et al., 2000, 2001). The synthesis was performed
at room temperature (RT) starting from His134 and completing,
in a first step, the loop C′ –E (129–134). After acetylation using
acetic anhydride in DMF (0.5 M, 5% DIEA, 20 min at room temperature), selective deprotection of Lys (Mtt) was achieved by
repeated treatments with a dichloromethane (DCM)/trifluoroacetic
acid (TFA)/tri-isopropylsilane (TIS) (94:1:5, v/v/v) mixture for
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
G Model
MIMM-3213; No. of Pages 10
ARTICLE IN PRESS
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
Table 1
Names, sequences and corresponding and MW of peptides used in this study.
Peptide
Sequence
MW
IgE-Trap
See Fig. 1
3474.20
Loop (C′ –E + B–C)
Loop C′ –E
Loop B–C
Loop F–G
Ac-Y-W-Y-E-K-H-NH2
Ac-G-W-A-W-R-N-W-NH2
Ac-C-T-G-K-V-W-Q-L-D-Y-E-C-NH2
1909.12
1009.12
1016.14
1484.70
2 min at RT. Trp87, -Ala, Trp110, Arg111, Asn112 and Trp113
were incorporated under canonical conditions of peptide synthesis
(HBTU/HOBt/DIEA pre-activation, 5-fold excess of Fmoc-protected
amino acids). Succinic acid was incorporated by treatment with
a 10-fold excess of succinic anhydride (0.5 M in DMF containing
1 M DIEA). Ethylenediamine was then introduced after on resin
pre-activation of the carboxylate group with conventional reagents
(HBTU/HOBt/DIEA) and reaction with a 2-fold excess of reagent in
DMF at RT for 20 min. Loop F–G was subsequently assembled using
classical conditions of peptide synthesis. Cleavage of peptides from
the solid support was performed by treatment with a TFA/TIS/water
(90:5:5, v/v/v) mixture for 90 min at room temperature, affording the crude peptide after precipitation in cold di-ethylether. The
cyclic peptide (named IgE-Trapox ) was obtained by spontaneous
intramolecular oxidation of cysteines in 100 mM carbonate buffer
pH 8.0 for 16 h at RT at the concentration of 10−5 M. Cyclization
progression was monitored by RP-HPLC. The solution was finally
acidified and lyophilized. Products were purified to homogeneity
by RP-HPLC using a C18 Jupiter column (50 mm × 2.2 mm) applying a linear gradient of 0.1% TFA in acetonitrile from 5% to 70%
over 20 min (flow rates of 20 mL/min). Peptide purity and integrity
were confirmed by LC–MS mass measurements using a Surveyor
LC system coupled to an LCQ Deca XP mass spectrometer. Characterizations were conducted under standard conditions of peptide
analysis. The shorter polypeptides were similarly prepared. Final
peptides are reported in Table 1, with names, sequences and molecular weights. The corresponding reduced precursor of IgE-Trap,
IgE-Trapred , was also purified and characterized.
2.3. Cloning, expression and purification of the FcRI extracellular
domain (ECD)
The hFcRI ECD D2 domain residues 84–170 were appropriately
amplified by PCR from the full length h␣FcRI cDNA (sequence
code NM 002001 from PubMed) and cloning into pETMA11 vector.
100 ng of FcRI-D2-pETMA11 was transformed into BL21(DE3)trxB
E. coli competent cells and the recombinant protein expressed as
an N-terminal polyhistidine tagged protein at 22 ◦ C in the presence
of 0.2 mM IPTG. After incubation for 16 h, the bacterial culture was
centrifuged and the pellet was re-suspended in 10 mL of 30 mM
Tris–HCl, 1 mM PMSF, 0,1% Triton X-100, 1 g/mL lysozime, 3 M
urea, 1 mM DTT pH 8.0 (lysis buffer). After incubation for 30 min
at room temperature, cells were sonicated on ice for 10 min (10′ s
on, 10 s off) and centrifuged for 30 min at 12,000 rpm at 4 ◦ C.
Total cell protein fraction and soluble fraction (supernatant of cell
lysate) were analyzed by SDS-PAGE. Proteins were detected in
gels by Coomassie brilliant blue R-250 staining or transferred onto
a nitrocellulose membrane for Western blot analysis using HRPanti-polyhistidine. The protein was purified on a Nichel affinity
(Ni-NTA) column, eluting with increasing concentrations of imidazole. Purified protein fractions were dialyzed over night at 4 ◦ C
against 30 mM Tris–HCl, 150 mM NaCl, 1 mM DTT 2 M urea pH 8.0,
then folding was induced by rapid dilution in the same buffer and
slowly decreasing the urea concentration by performing subse-
3
quent dialysis against 30 mM Tris–HCl, 150 mM NaCl, 1 mM DTT
pH 8.0. The last step of dialysis was carried out in 30 mM Tris–HCl,
150 mM NaCl, 5 mM reduced glutathione, 0.5 mM oxidized glutathione pH 8.0 buffer over night at 4 ◦ C. Any precipitate formed
during the refolding procedure was removed by centrifugation at
12,000 rpm for 30 min at 4 ◦ C. The recovered material was analyzed
by 15% SDS-PAGE under reducing and non-reducing conditions and
by LC–MS.
2.4. Circular dichroism spectroscopy
CD spectra were obtained at room temperature on a Jasco J715 dichrograph, calibrated at 290 nm with an aqueous solution
of d(+)-10-camphor sulphonic acid, using 0.1 mm quartz cuvettes.
Spectra of the FcRI-D2 domain were acquired in 20 mM phosphate
buffer pH 8.0 with C = 8.0 × 10−6 M. Spectra were recorded using a
0.1-mm path length quartz cuvette. Data were collected at 0.2-nm
intervals with a 20 nm/min scan speed, a 2 nm bandwidth, and a 16 s
response, within the spectral range of 260–190 nm. The recorded
spectra were then signal-averaged over at least three scans, and the
baseline was corrected by subtracting the spectrum of the buffer.
Spectra were then transformed in molar ellipticity.
2.5. Binding and competition assays
Real time binding assays were performed on a Biacore 3000 Surface Plasmon Resonance (SPR) instrument (GE healthcare, Milan,
Italy). Human IgE, human IgG and human IgA immobilization were
achieved on CM5 Biacore sensor chips using EDC/NHS chemistry
at pH 5.0 in 10 mM acetate buffer (flow rate 5 L/min) according to the manufacturer’s instructions (Johnsosson et al., 1991).
Residual reactive groups were deactivated by treatment with 1 M
ethanolamine hydrochloride, pH 8.5. Reference channels were
prepared for each biosensor by activating with EDC/NHS and deactivating with ethanolamine. All binding assays were carried out in
HBS buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, pH 7.4) at
a flow rate of 30 L/min. Analyte injections of 140 L (IgE-Trap),
90 L (loop C′ –E + B–C) or 130 L (loop B–C) were performed at the
indicated concentrations. Data were manipulated to obtain kinetic
and thermodynamic parameters using the BIA evaluation analysis
package (version 4.1, GE Healthcare, Milano, Italy). Data fitting was
carried out using monophasic or biphasic models and evaluating
the goodness of fitting by the analysis of residuals (Cook et al., 1997;
Henry et al., 1997). Non-specific binding was subtracted from the
specific binding prior to analysis. Binding to human IgG and human
IgA was performed at 30 M for IgE-Trap and loop (C′ –E + B–C) and
at 60 M for loop B–C.
To perform ELISA assays, 96-well microtiter plates (Nunc, Milan,
Italy) were coated with human IgE at fixed concentrations (2 and
5 g/mL) in PBS buffer (10 mM Na2 HPO4 , 2 mM KH2 PO4 , 137 mM
NaCl, 2.7 mM KCl pH 7.4) for 16 h at 4 ◦ C. Some wells were filled
with buffer alone and were used as blank. After coating, plates
were washed three times with PBS-T buffer (PBS containing 0.04%
Tween-20) and blocked with 250 L PBS containing 1% BSA, for
2 h at 37 ◦ C. After washing, solutions at increasing concentrations
of D2 between 0.50 and 25 M in PBS or Bis–Tris 25 mM pH 6.5
were added to coated wells and incubated 1 h at 37 ◦ C. After washing again with PBS-T, 100 L of anti-His-HRP conjugated antibody
(Santa Cruz Biotechnology, CA, USA) diluted 1:1000 with PBS was
added. After 1 h incubation at 37 ◦ C, plates were washed again with
PBS-T and 100 L of chromogenic substrate, o-phenylendiamine
0.4 mg/mL in 50 mM sodium–phosphate–citrate buffer pH 5.0 containing 0.4 mg/mL hydrogen peroxide, were added and the reaction
was stopped by adding 50 L of 2.5 M H2 SO4 in each well. The
absorbance was measured at 490 nm.
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
G Model
MIMM-3213; No. of Pages 10
ARTICLE IN PRESS
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
4
For the ELISA competition assays, human IgE, diluted in PBS1×,
were coated overnight on microtiter plates at 2.0 g/mL (10 nM).
Recombinant receptor concentration was maintained at 10 M and
mixed with competitor peptides at increasing concentrations as
indicated in the legend of Fig. 8. The subsequent steps were carried
out as previously described. Data points were in triplicate. Assays
were performed at least three times obtaining reproducible results.
2.6. IgE biotinylation
Mouse IgE (0.5 mg) were dissolved in 1 mL of PBS, pH 7.4, and
treated with 10 mM of succinimididyl1-6-(biotinamido)hexanoate
(Pierce, Italy) in DMSO. After 1 h of incubation on ice, 1 mL of Tris
50 mM pH 8 was added to deactivate residual active groups. The
biotinylated-IgE were extensively dialyzed 4 ◦ C against PBS pH 7.4.
2.7. Cell culture and flow cytometry
The rat basophilic leukemia (RBL2H3) cell line was maintained
in DMEM supplemented with 10% heat-inactivated FBS, containing
2 mM l-glutamine at 37 ◦ C in a humidified atmosphere at 5% CO2 .
For competitive binding experiments, RBL2H3 cells were readily
detached by gentle scraping with PBS/EDTA 0.05%, centrifuged at
100 × g for 5 min, washed with PBS, 0.2% BSA and re-suspended in
the same buffer to a final density of 5 × 106 cells/mL (Buku et al.,
2001). Increasing concentration of peptide between 0 and 600 M
was pre-incubated with 13 nM biotynilated-IgE at 4 ◦ C for 1 h. The
mix was added to cell suspensions for 45 min followed by the
addition of 10 L FITC-avidin. After 30 min the cells were washed
twice with PBS/BSA and analyzed with a flow cytometer equipped
with a 488-nm argon laser (FACSCalibur, Becton Dickinson). For
each sample, 20,000 events were acquired and analyzed using the
Cell Quest software.
et al. (2000) (PDB code 1F6A). A sketch of the peptide structure is
reported in Fig. 1, where the two receptor-binding sites and loops
involved in the interaction with IgE are evidenced by boxes and
arrows The polypeptide recapitulates both receptor sites which are
shared by three distinct loop regions, named loop C′ –E, loop B–C,
and loop F–G, in agreement with the nomenclature reported in Garman et al. Loop C′ –E (boxed) contains residues 129–134 (YWYEKH),
which comprise most of site 1. Here, Asn133 originally present in
the receptor sequence and not involved in contacts with the IgE-Fc,
was mutated to a lysine in order to exploit the side chain amino
group to join residues 110–113 (WRNW) from loop B–C. The NH2 of lysine was thus connected to the C-terminus of Trp113.
An additional tryptophan, putatively mimicking Trp87, on the Nterminus of Trp110 was linked using a -alanine as spacer. Residues
of loop F–G (Cys151-Ser162, loop 3) were connected to Trp87 by
a linker comprising a succinic acid and an ethylendiamine moiety. Loops B–C and F–G were used to mimic the receptor-binding
site 2. In order to reduce the conformational flexibility of loop
F–G, Ser162 was replaced by an isosteric cysteine and a disulfide
bridge was inserted. To increase the polypeptide’s stability, the
N-terminal and C-terminal ends were acetylated and amidated,
respectively.
3.2. Peptide synthesis
3. Results
Cyclic IgE-Trap, named IgE-Trapox , was obtained with an overall yield of about 5% after cyclization and purification. The linear
precursor (named IgE-Trapred ) was also prepared and purified. The
yield was about 10%. The other peptides (see Table 1) were obtained
in higher yields (ranging from about 20 to 50%). Molecular weights
were consistent with the expected values. Disulfide cyclization of
IgE-Trap was assessed by high resolution mass spectrometry and
confirmed by the lack of reactivity following iodoacetamide treatment (not shown).
3.1. Peptide design
3.3. Peptide binding to IgE
The peptide IgE-Trap was designed on the basis of the crystallographic structure of the IgE-FcRI complex reported by Garman
The capacity of peptides to bind to immobilized human IgE was
determined by Real Time kinetic analysis using the SPR technique.
Fig. 2. (A) Sensorgrams relative to the binding of IgE-Trapox to human IgE immobilized on the surface of a Biacore sensor chip. Concentrations were between 2.5 M and
40 M. Experiments were carried out at a 25 ◦ C, at a constant flow rate of 30 L/min using HBS as running buffer (140 L injected for each experiment). (B) Plot of RUmax from
each single experiment versus concentration (M). Data were fitted by non-linear regression analysis. In (C) and (D), data obtained for IgE-Trapred are reported. Experiments
were performed at concentrations between 2.5 M and 50 M.
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
ARTICLE IN PRESS
G Model
MIMM-3213; No. of Pages 10
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
5
Table 2
Association and dissociation rates and dissociation constants for the active peptides reported in this study. Data for the two IgE-Trap variants and for loop (C′ –E + B–C) are
referred to a fitting with a biphasic model of binding to IgE. The KD for loop (B–C) has been obtained by fitting the data with a monophasic model of association with IgE.
A
Peptide
Mean 1st
association rate,
Ka1 (M−1 s−1 )
Mean 1st
dissociation
rate, Kd1 (s−1 )
Mean 2nd
association rate,
Ka2 (M−1 s−1 )
Mean 2nd
dissociation
rate, Kd2 (s−1 )
Mean KD1 (M)
Mean KD2 (M)
Global KD (M)
IgE-Trapox
IgE-Trapred
Loop (C′ –E + B–C)
1.63 × 102
3.30 × 104
1.35 × 104
1.06 × 10−2
6.43 × 10−1
8.70 × 10−1
6.14 × 103
2.87 × 102
1.33 × 102
3.69 × 10−1
1.05 × 10−2
2.54 × 10−2
65 (±5) × 10−6
19 (±6) × 10−6
65 (±6) × 10−6
60 (±6) × 10−6
37 (±7) × 10−6
19 (±5) × 10−5
16 (±4) × 10−6
6.2 (±0.7) × 10−6
19 (±4) × 10−6
B
Peptide
Mean association rate, Ka1 (M−1 s−1 )
Mean dissociation rate, Kd1 (s−1 )
KD (M)
Loop (B–C)
1.81 × 102
1.50 × 10−1
8.3 (±0.5) × 10−4
Human IgE, IgG and IgA were efficiently immobilized to the surface of a CM5 sensor at a similar extent (about 15,000 RU). At
first, IgE-Trapox and IgE-Trapred were tested. Sensorgrams relative to IgE-Trapox are reported in Fig. 2A, while thermodynamic
parameters are shown in Table 2. A preliminary analysis of residuals (see Supplementary Fig. S1A–D) suggested that experimental
data were fitted better by a biphasic model of interaction, therefore we hypothesized that two distinct association and dissociation
kinetic constants characterized the binding. This observation was
in agreement with the fact that the peptide contains two distinct receptor-binding sites for which we obtained, incidentally,
two very similar KD s of about 60 M (see Table 2A). Data were
further analyzed to obtain a value for the apparent macroscopic
dissociation constant. For this purpose, RUmax values from each
determination were plotted against polypeptide concentrations
and the KD evaluated by data fitting using a non-linear regression
analysis (Fig. 2B). In agreement with the “two-site binding” hypothesis, the KD extrapolated by this approach was lower than the KD s
for the two distinct sites, namely 16 ± 4.0 M compared to about
60 M (Table 2), also suggesting a cooperative mechanism. In order
to evaluate the contribution to binding provided by the presence
of the disulfide bridge, we also analyzed the reduced polypeptide IgE-Trapred (Fig. 2C). Again, a preliminary analysis of residuals
(not shown) indicated that experimental data were best fitted by
biphasic curves, thus confirming the occurrence of a two-site binding also for this molecule. As reported in Table 2, slightly higher
affinities compared to the cyclic variant were observed for both
sites; this was also reflected by the somewhat lower macroscopic
KD (6.2 ± 0.7 M) determined as reported for the cyclic variant
(Fig. 2D). In order to dissect the contribution to IgE recognition by
the different IgE-Trap regions and to further confirm the occurrence
of multiple and cooperative interaction sites, we then analyzed
the binding properties of synthetic peptides corresponding to different receptor loops. Peptides, together with sequences and the
corresponding elements of secondary structure in the native receptor, are reported in Table 1 and Fig. 1. Sensorgrams relative to IgE
binding by the peptide named loop (C′ –E + B–C), at concentrations
ranging between 2.5 and 50 M, are reported in Fig. 3A. The peptide bound in a dose-dependent fashion to the functionalized chip
and, in agreement with the fact that residues from two distinct
binding sites are present within this sequence, experimental data
were again fitted better by biphasic curves (see Supplementary
Fig. S2A–D). Importantly, the first KD was very close to that determined for the whole IgE-Trapox (see Table 2), suggesting that a
site with a higher affinity is present on both peptides. The affinity for the second site was fairly lower (190 M), while the global
apparent KD determined for this peptide was 19 ± 4.0 M (Fig. 3B
and Table 2), very similar to that determined for the IgE-Trapox
variant. This result, based on the preliminary assumption that two
sites were present, suggested that the contribution to binding of
loop F–G, either in the cyclic or reduced form, was negligible.
In order to confirm this hypothesis, we then analyzed the binding properties of the isolated cyclic and reduced synthetic loop
F–G, which indeed showed no recognition (not shown). To further dissect the contribution to binding from loops C′ –E and B–C
and confirm the occurrence of two sites in this peptide too, we
investigated their separate effects using the corresponding synthetic peptides. Unexpectedly, loop C′ –E was unable to bind alone
to IgE (not shown), while loop B–C, assayed at concentrations up
to 75 M (at higher concentrations it was insoluble), bound very
weakly (KD = 8.3 ± 0.5 × 10−4 M, Fig. 4A and Table 2). Furthermore,
although a dose–response trend was observed, the dependence of
RUmax versus concentration was essentially linear and not saturable
in the concentration range investigated (Fig. 4B), indicating a prevalence of less specific and very weak interactions. Importantly, in
this case, experimental data were fitted better by a monophasic
Fig. 3. (A) Sensorgrams relative to the binding of loop (C′ –E + B–C) to immobilized
IgE. Concentrations were between 2.5 M and 50 M. Experiments were carried
out at 25 ◦ C, at a constant flow rate of 30 L/min using HBS as running buffer (90 L
injected for each experiment). (B) Plot of RUmax from each single experiment versus
concentration (M). Data were fitted by non-linear regression analysis.
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
G Model
MIMM-3213; No. of Pages 10
6
ARTICLE IN PRESS
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
Fig. 4. (A) Sensorgrams relative to the binding of loop B–C to immobilized IgE. Concentrations were between 20 M and 75 M. Experiments were carried out at 25 ◦ C,
at a constant flow rate of 30 L/min using HBS as running buffer (130 L injected
for each experiment). (B) Plot of RUmax from each single experiment versus concentration (M). Data could not be fitted by non-linear regression analysis in the
explored concentration range.
association model (Fig. S3A–D), in agreement with the assumption
that this short peptide can mimic only one site. Collectively, the
data support the hypothesis that, most contribution to the binding
properties of IgE-Trap is provided by residues belonging to loops
C′ –E and B–C. Furthermore, despite the low affinity compared to the
native receptor, a strong cooperativity occurs between these loops
when they are covalently interconnected, while they appear poorly
or essentially inactive when are assayed separately. To further characterize the binding properties of these peptides, we performed
comparative assays with IgG and IgA, which are the most abundant
immunoglobulins and the most similar in primary sequence to IgE.
As shown in Fig. 5A and C, the peptides IgE-Trapox , loop (C′ –E + B–C)
and loop B–C, did not bind immunoglobulins from other classes,
suggesting a very high specificity of these molecules for IgE.
3.4. Preparation of the recombinant D2 domain of the FcRI
receptor
It has been reported that binding of the receptor to IgE is mediated by the D2 domain and by residues located on the linker
between D1 and D2. D1, instead, favours an optimal receptor conformation, but does not actively participate in receptor recognition
(Vangelista et al., 2002). Glycosylation is also deemed to only
slightly affect affinity, being involved in the stabilization of the protein structure and not in direct contacts with IgE (Garman et al.,
2000). To carry out binding and competition experiments with the
bioactive peptides and to further confirm the specificity of recognition, we designed and prepared a D2 variant starting from Phe84
and including Ile170 (the carboxy-terminal tail involved in membrane anchoring was excluded, Fig. 6A) The protein, expressed in
E. coli, also bear at its N-terminus a polyhistidine tag that facilitated its purification using a Nichel-immobilized affinity resin
Fig. 5. (A) Comparative sensorgrams for the binding of IgE-Trap to immobilized IgE,
IgG and IgA. The peptide only binds to IgE. The peptide was analyzed at a concentration of 30 M with a constant flow rate of 30 L/min. (B) The same comparative
analysis performed with loop (C′ –E + B–C) at 30 M and with loop B–C at 60 M. (C)
Experiments were carried out at 25 ◦ C, at a constant flow rate of 30 L/min using
HBS as running buffer.
(Fig. 6B). A yield of 0.5 mg of folded protein from 100 mL of bacterial
cell culture was obtained in typical preparations. The final material was more than 90% pure as estimated by SDS-PAGE (Fig. 6B)
and LC–MS analysis (not shown). The experimental molecular
weight was in agreement with the theoretical value (MWExper./Calc. :
13,253/13253.2 amu). Protein refolding was assessed by CD analysis (Fig. 6C); the protein spectrum showed canonical -sheet
bands (minimum at 215 nm and a maximum at about 195 nm), as
expected on the basis of the domain structure (Garman et al., 1999,
2000, 2001). The presence of the disulfide bridge was confirmed
by LC–MS analysis of a 4-VP-treated sample, which was indeed not
affected by the alkylation reaction (not shown).
3.5. Binding assays between IgE and the recombinant D2 domain
The recombinant receptor domain was at first tested for binding with IgE. An ELISA assay was thus set up in order to use it
in subsequent competition experiments with the synthetic peptides. The assay was performed by coating human IgE at 25 nM
(5 g/mL) and the recombinant receptor in solution at concentra-
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
G Model
MIMM-3213; No. of Pages 10
ARTICLE IN PRESS
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
7
Fig. 6. (A) Sequence of the recombinant D2 domain of FcRI. Residues used to design the peptides are reported in red. Residues corresponding to the linker region are in italic
and boxed. Cysteines involved in the disulfide bridge are underlined. Residues coming from the pETMA11 vector containing the polyhistidine linker are reported in the first
line of the sequence. (B) SDS-PAGE characterization of the recombinant protein in the presence and absence of -mercaptoethanol. The reduced form of the protein migrates
slightly slower. (C) CD spectrum of the protein in 20 mM phosphate buffer, pH 8.0. The polypeptide, analyzed at a concentration of 8.0 M shows the typical spectrum of
-sheet rich structures. Molecular weight markers are reported on the gel left margin. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of the article.)
tions ranging between 50 nM and 20 M. As shown in Fig. 7A, the
receptor bound to immunoglobulins in a dose-dependent manner, but showed a clear two-step curve with a first saturation
point at about 500 nM and a second saturation point at concentrations higher than about 10 M (Fig. 7B and C). Fitting of data
by non-linear regression over the two concentration ranges, provided the respective KD s of 0.11 ± 0.04 M and 4.1 ± 0.1 M. This
behaviour, reproducibly observed over at least three independent
experiments, is indicative of two independent sites, but with much
lower affinities compared to the full length receptor and to previous
data (Cook et al., 1997; Henry et al., 1997). However, given the lack
of the adjacent D1 domain and the presence of the polyhistidine tag
at the N-terminus where the linker between D1 and D2 is located, it
is likely that residues Ser85, Asp86 and Trp87, which contribute to
the receptor-binding site 2 (Garman et al., 2000), are not properly
disposed or are partially inaccessible, thereby causing a reduced
affinity. This hypothesis is consistent with the large difference of
KD values determined for the two distinct sites. ELISA competition assays using the recombinant D2 domain and IgE were then
carried out in order to confirm that peptides bind the immunoglobulins on the same sites as the receptor. In this experiment, 10 nM
human IgE (2.0 g/mL) were coated on the plates and 5.0 M D2
was used in solution, since, at this receptor concentration, both
binding sites are saturated. For the dose-dependent competition
assay, we explored peptide:receptor molar ratios between 1 and
100. Competitor concentrations therefore ranged between 5.0 M
and 500 M. Results reported in Fig. 8A and C, show that all peptides efficiently block the interaction, confirming that peptides
actually mimic the receptor-binding sites. Noticeably, IC50 values
are consistent with the relative binding affinities determined by
SPR. Indeed, IgE-Trapox displayed an IC50 of 36.0 ± 0.1 M, loop
(C′ –E + B–C) an IC50 of 103.0 ± 0.2 M, whereas loop B–C alone
showed an IC50 of only 379.0 ± 0.2 M.
3.6. Inhibition of biotinylated-IgE binding to RBL2H3 cells by loop
(C′ –E + B–C)
The competitive binding experiments was performed with
increasing concentration of loop (C′ –E + B–C) at a fixed concentra-
tion of 13 nM biotinylated-IgE. As shown in Fig. 9, the peptide at
60 M reduced the binding of biotinylated-IgE to 60% of the control
value. The positive control was defined as the fluorescence intensity of 13 nM biotinylated-IgE bound to the cells. The results are
expressed as percentage fluorescence intensity of the sample minus
the fluorescence of the cells incubated with FITC-avidin alone. In the
same assay 100-fold excess of non-labelled-IgE was used to verify
the specificity of IgE binding to FcRI receptor on RBL2H3. The addition of a competition mix containing 1 mM of a scrambled peptide
provided a proof of the specificity of loop (C′ –E + B–C) for binding
to IgE (not shown).
4. Discussion
Therapeutic protocols for allergy are still largely based on
symptomatic drugs which are directed at reducing the degree of
inflammation, vasodilation, and congestion (Jardieu, 1995). More
recently, new strategies targeting the IgE-FcRI molecular complex that triggers the allergic cascade, have been undertaken. These
efforts have culminated so far in the development of the antiIgE monoclonal antibody named Omalizumab which has proven
to be of broad clinical efficacy in reducing the severity of several allergic diseases (Ames et al., 2004; Corren et al., 2009;
MacGlashan, 2009). With the aim of identifying smaller antagonists
capable of binding to either the immunoglobulin or the receptor,
several techniques have been employed, including combinatorial
approaches (Nakamura et al., 2001, 2002; Rossi et al., 2008), the
use of truncated IgE-Fc or receptor loops (Rigby et al., 2000b), or
by modifying known receptor agonists (Buku et al., 2003). Most of
these molecules disrupt IgE binding and despite their low affinity
(McDonnell et al., 1996; Rossi et al., 2008), have proved effective
in preventing activation of cells or even in in vivo assay (Rossi et
al., 2008). Elucidation at the atomic level of the interaction interface between IgE and the FcRI has open the way to the design
of new, effective drugs blocking receptor signalling and thus to
new opportunities for therapeutic intervention at the molecular
level (Garman et al., 2000). However, the presence of two distinct interaction sites and the convex nature of the receptor make
the design of receptor-binding peptides quite difficult. In addi-
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
G Model
MIMM-3213; No. of Pages 10
8
ARTICLE IN PRESS
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
Fig. 7. (A) ELISA binding assay between coated human IgE and the soluble recombinant D2 domain of human FcRI fused with an N-terminal polyhistidine tag
(His6x-D2). The binding curve shows a clear two-step dependence from protein
concentration with a first saturation point for values higher than about 500 nM
and a second saturation point for concentrations higher than 10 M. In (B) and (C),
the two steps are highlighted by spitting the plot between 0.01 nM and 2.0 M (B)
and between 2.0 M and 50 M (C). Experiments were in quadruplicate and were
repeated at least three times. Data ± SD are reported.
tion, receptor-binding compounds can lead to unwanted agonistic
activities. Instead, blocking the circulating IgE by soluble factors
is a validated approach with a proven clinical efficacy (Corren et
al., 2009; MacGlashan, 2009). On this background, starting from
the receptor structure we have designed small polypeptides that
mimic the IgE-binding interface and thereby specifically interact
with IgE. Despite their relatively low binding capacity (low micromolar range) compared to the full length receptor (KD = 10−9 M),
these peptides show a cooperative two-site mechanism of binding,
as suggested by the lower value (2–3 times) of the macroscopic KD
compared to the single-site KD s and by a preliminary analysis of
residuals for the fitting of experimental binding curves. The occurrence of a cooperative mechanism, which implies the existence of
at least two interacting sites, is also suggested by the lack of activity
shown by the isolated loops C′ –E and F–G, which, instead, synergically contribute to binding when covalently connected with other
Fig. 8. Competitive binding assays between the recombinant D2 domain and the
synthetic bioactive peptides to immobilized human IgE. (A) Competition assay with
IgE-Trapred . The experiment was performed at concentrations ranging between
10−7 M and 10−3 M. The IC50 was 36.0 ± 0.1 M. (B) The same experiment carried
out with loop (C′ –E + B–C) at concentrations ranging between 1 M and 500 M. The
estimated IC50 was 103.0 ± 0.2 M. (C) Competition assay with the peptide corresponding to loop B–C. Concentrations of the competing peptide were between 3 M
and 750 M. The estimated IC50 was 379.0 ± 0.2 M. For all experiments human IgE
were coated at 10 nM while the recombinant receptor domain was maintained at
10 M. Data on the vertical axis are reported as B/B0 × 100, where B is the absorbance
at a given concentration of competitor and B0 is the absorbance from wells without
competitors.
peptide regions. The presence of loop C′ –E, containing only residues
from site 1, confers a 100-fold affinity increase to loop B–C, while
it is totally inactive alone. This finding suggests that site 1 on the
IgE is not readily accessible to the peptide, this hypothesis being
corroborated by the occurrence of the slow association kinetics for
one of the two bindings (Ka2 = 1.33 × 102 M−1 s−1 ) (Table 2) and the
concurrent slow dissociation (Kd2 = 2.54 × 10−2 s−1 ). Notably, this
observation is in agreement with previous evidences provided by
Wan and co-workers (Wan et al., 2002) suggesting a poor accessibility of the corresponding site 1 on the IgE and a mechanism of
bending of the immunoglobulin upon receptor engagement.
The presence of the cyclic loop F–G in IgE-Trapox does not greatly
affect the binding of the peptide to IgE, which indeed displays the
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
G Model
MIMM-3213; No. of Pages 10
ARTICLE IN PRESS
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
9
assistance of Dr. Giuseppe Perretta and Mr. Leopoldo Zona is also
gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molimm.2009.07.025.
References
Fig. 9. Flow-cytometric analysis of inhibition of biotinylated-IgE binding to RBL2H3
cells. The number of cells is plotted against the logarithm of fluorescence intensity
of the bound ligand. Biotinylated-IgE were coupled with FITC-avidin and incubated
with cells. Fluorescence intensity was determined by FACS scan analysis in the
absence of competitors and was assumed as the 100% control. Increasing concentrations of peptide loop (C′ –E + B–C) between 0 M and 600 M were incubated with
the cells under the same conditions and fluorescence intensity again determined.
Data shown are the average from at least three independent determinations.
same global KD both with and without it. Conversely, leaving this
loop in the reduced form, slightly enhances the affinity (about 3fold), suggesting that, while the more rigid cyclic moiety is not
capable of adopting a conformation suitable for binding, the linear
peptide does and contributes to slightly increase its global affinity.
The soluble and functional D2 domain of human FcRI has also
been prepared as recombinant protein and utilized to confirm the
ability of these peptides to reproduce the binding sites. The protein
also shows a two-site mechanism of interaction, but despite the
presence of the linker region (residues 84–105), binding affinities
are still low compared to the full length receptor. Indeed, two independent sites are observed with KD s of about 100 nM and 4 M. The
IgE-binding peptides block the interaction between human IgE and
D2 with IC50 values consistent with the relative binding affinities,
thus confirming that recognition occurs on the expected regions.
Competition has been demonstrated also on rat cells expressing the
receptor using the loop (C′ –E + B–C). Remarkably, while IgE-Trap
or the smaller loop (C′ –E + B–C) described here, shows a reduced
affinity compared to receptor-binding peptides reported in previous studies (Buku et al., 2008; Nakamura et al., 2001, 2002), they
show IC50 for IgE displacement from cell-bound receptors, in the
same order of magnitude as the more active peptides (about 70 M
compared to 20 M reported for the MCD analogues). The peptides
we report here also have affinities higher or similar to IgE-binding
compounds with a proven in vivo activity (Rigby et al., 2000a; Rossi
et al., 2008). In addition, the peptides show a very high selectivity
for IgE, being incapable of binding IgG and IgA. This feature is of
particular interest for in vivo applications, as unspecific binding to
IgG, highly abundant in blood and in other tissues, would subtract
the molecule from its target. Similarly, IgA are the most abundant
immunoglobulins in external secretions (saliva, mucus, gastric fluids, sweat, and tears), therefore the lack of binding will ensure no
subtraction in case of oral administration. Finally, given the small
molecular size, they are also not expected to be immunogenic and
could be used in in vivo experiments at high dosages without modifications, therefore they have a potential as modulators of disorders
associated with inappropriate IgE production.
Acknowledgements
This project was supported by the projects FIRB2003, No
RBNE03PX83 005 to M.R. and FIRB No RBRN07BMCT. The technical
Ames, S.A., Gleeson, C.D., Kirkpatrick, P., 2004. Omalizumab. Nat. Rev. Drug Discov.
3, 199–200.
Basu, M., Hakimi, J., Dharm, E., Kondas, J.A., Tsien, W.H., Pilson, R.S., Lin, P., Gilfillan,
A., Haring, P., Braswell, E.H., et al., 1993. Purification and characterization of
human recombinant IgE-Fc fragments that bind to the human high affinity IgE
receptor. J. Biol. Chem. 268, 13118–13127.
Buku, A., Condie, B.A., Price, J.A., Mezei, M., 2005. [Ala12]MCD peptide: a lead peptide
to inhibitors of immunoglobulin E binding to mast cell receptors. J. Pept. Res. 66,
132–137.
Buku, A., Keselman, I., Lupyan, D., Mezei, M., Price, J.A., 2008. Effective mast cell
degranulating peptide inhibitors of the IgE/Fc epsilonRI receptor interaction.
Chem. Biol. Drug Des. 72, 133–139.
Buku, A., Mendlowitz, M., Condie, B.A., Price, J.A., 2003. Histamine-releasing activity
and binding to the FcepsilonRI alpha human mast cell receptor subunit of mast
cell degranulating peptide analogues with alanine substitutions. J. Med. Chem.
46, 3008–3012.
Buku, A., Mendlowitz, M., Condie, B.A., Price, J.A., 2004. Partial alanine scan of mast
cell degranulating peptide (MCD): importance of the histidine- and arginine
residues. J. Pept. Sci. 10, 313–317.
Buku, A., Price, J.A., Mendlowitz, M., Masur, S., 2001. Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding. Peptides 22,
1993–1998.
Cook, J.P., Henry, A.J., McDonnell, J.M., Owens, R.J., Sutton, B.J., Gould, H.J., 1997. Identification of contact residues in the IgE binding site of human FcepsilonRIalpha.
Biochemistry 36, 15579–15588.
Cookson, W., 1999. The alliance of gene and environmental in asthma and allergy.
Nature 402, 5–11.
Corren, J., Casale, T.B., Lanier, B., Buhl, R., Holgate, S., Jimenez, P., 2009. Safety and
tolerability of omalizumab. Clin. Exp. Allergy.
Fields, G.B., Noble, R.L., 1990. Solid phase peptide synthesis utilizing 9fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35, 161–214.
Garman, S.C., Kinet, J.P., Jardetzky, T.S., 1998. Crystal structure of the human highaffinity IgE receptor. Cell 95, 951–961.
Garman, S.C., Kinet, J.P., Jardetzky, T.S., 1999. The crystal structure of the human
high-affinity IgE receptor (Fc epsilon RI alpha). Annu. Rev. Immunol. 17, 973–
976.
Garman, S.C., Sechi, S., Kinet, J.P., Jardetzky, T.S., 2001. The analysis of the human
high affinity IgE receptor Fc epsilon Ri alpha from multiple crystal forms. J. Mol.
Biol. 311, 1049–1062.
Garman, S.C., Wurzburg, B.A., Tarchevskaya, S.S., Kinet, J.P., Jardetzky, T.S., 2000.
Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc
epsilonRI alpha. Nature 406, 259–266.
Henry, A.J., Cook, J.P., McDonnell, J.M., Mackay, G.A., Shi, J., Sutton, B.J., Gould,
H.J., 1997. Participation of the N-terminal region of Cepsilon3 in the binding of human IgE to its high-affinity receptor FcepsilonRI. Biochemistry 36,
15568–15578.
Jardieu, P., 1995. Anti-IgE therapy. Curr. Opin. Immunol. 7, 779–782.
Johnsosson, B., Lofas, S., Lindquist, G., 1991. Immobilization of protein to a
carboxymethyldextran-modified gold surface for biospecific interaction analysis in SPR sensor. Anal. Biochem. 198, 268–277.
Kinet, J.P., 1989. The high-affinity receptor for IgE. Curr. Opin. Immunol. 2, 499–
505.
Kinet, J.P., 1999. The high-affinity IgE receptor (Fc epsilon RI): from physiology to
pathology. Annu. Rev. Immunol. 17, 931–972.
Kochan, J., Pettine, L.F., Hakimi, J., Kishi, K., Kinet, J.P., 1988. Isolation of the gene
coding for the alpha subunit of the human high affinity IgE receptor. Nucleic
Acids Res. 16, 3584.
Kraft, S., Kinet, J.P., 2007. New developments in FcepsilonRI regulation, function and
inhibition. Nat. Rev. Immunol. 5, 365–378.
Letourneur, O., Sechi, S., Willette-Brown, J., Robertson, M.W., Kinet, J.P., 1995. Glycosylation of human truncated Fc epsilon RI ␣ chain is necessary for efficient
folding in the endoplasmic reticulum. J. Biol. Chem. 270, 8249–8256.
MacGlashan Jr., D., 2009. Therapeutic efficacy of omalizumab. J. Allergy Clin.
Immunol. 123, 114–115.
McDonnell, J.M., Beavil, A.J., Mackay, G.A., Henry, A.J., Cook, J.P., Gould, H.J., Sutton,
B.J., 1997. Structure-based design of peptides that inhibit IgE binding to its highaffinity receptor Fc epsilon RI. Biochem. Soc. Trans. 25, 387–392.
McDonnell, J.M., Beavil, A.J., Mackay, G.A., Jameson, B.A., Korngold, R., Gould,
H.J., Sutton, B.J., 1996. Structure based design and characterization of peptides that inhibit IgE binding to its high-affinity receptor. Nat. Struct. Biol. 3,
419–426.
McDonnell, J.M., Calvert, R., Beavil, R.L., Beavil, A.J., Henry, A.J., Sutton, B.J., Gould,
H.J., Cowburn, D., 2001. The structure of the IgE Cepsilon2 domain and its role
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025
G Model
MIMM-3213; No. of Pages 10
10
ARTICLE IN PRESS
A. Sandomenico et al. / Molecular Immunology xxx (2009) xxx–xxx
in stabilizing the complex with its high-affinity receptor FcepsilonRIalpha. Nat.
Struct. Biol. 8, 437–441.
Meltzer, E.O., Grant, J.A., 1999. Impact of cetirizine on the burden of allergic rhinitis.
Ann. Allergy Asthma Immunol. 83, 455–463.
Metzger, H., Kinet, J.P., Blank, U., Miller, L., Ra, C., 1989. The receptor with high affinity
for IgE. Ciba Found. Symp. 147, 93–101 (Discussion 101–13).
Nakamura, G.R., Reynolds, M.E., Chen, Y.M., Starovasnik, M.A., Lowman, H.B., 2002.
Stable “zeta” peptides that act as potent antagonists of the high-affinity IgE
receptor. Proc. Natl. Acad. Sci. U.S.A. 99, 1303–1308.
Nakamura, G.R., Starovasnik, M.A., Reynolds, M.E., Lowman, H.B., 2001. A novel family of hairpin peptides that inhibit IgE activity by binding to the high-affinity IgE
receptor. Biochemistry 40, 9828–9835.
Rigby, L.J., Epa, V.C., Mackay, G.A., Hulett, M.D., Sutton, B.J., Gould, H.J., Hogarth,
P.M., 2000a. Domain one of the high affinity IgE receptor, FcepsilonRI, regulates binding to IgE through its interface with domain two. J. Biol. Chem. 275,
9664–9672.
Rigby, L.J., Trist, H., Snider, J., Hulett, M.D., Hogarth, P.M., Epa, V.C., 2000b. Monoclonal
antibodies and synthetic peptides define the active site of FcepsilonRI and a
potential receptor antagonist. Allergy 55, 609–619.
Rossi, M., Ruvo, M., Marasco, D., Colombo, M., Cassani, G., Verdoliva, A., 2008. Antiallergic properties of a new all-D synthetic immunoglobulin-binding peptide.
Mol. Immunol. 45, 226–234.
Scharenberg, A.M., Lin, S., Cuenod, B., Yamamura, H., Kinet, J.P., 1995. Reconstitution
of interactions between tyrosine kinases and the high affinity IgE receptor which
are controlled by receptor clustering. Embo J. 14, 3385–3394.
Stamos, J., Eigenbrot, C., Nakamura, G.R., Reynolds, M.E., Yin, J., Lowman, H.B., Fairbrother, W.J., Starovasnik, M.A., 2004. Convergent recognition of the IgE binding
site on the high-affinity IgE receptor. Structure 12, 1289–1301.
Turner, H., Kinet, J.P., 1999. Signalling through the high-affinity IgE receptor Fc
epsilonRI. Nature 402, B24–30.
Vangelista, L., Cesco-Gaspere, M., Lamba, D., Burrone, O., 2002. Efficient folding of
the Fc1RI ␣-chain membrane proximal domain D2 depends on the presence of
the N-terminal domain D1. J. Mol. Biol. 322, 815–825.
Wan, T., Beavil, R.L., Fabiane, S.M., Beavil, A.J., Sohi, M.K., Keown, M., Young,
R.J., Henry, A.J., Owens, R.J., Gould, H.J., Sutton, B.J., 2002. The crystal structure of IgE Fc reveals an asymmetrically bent conformation. Nat. Immunol. 3,
681–686.
Please cite this article in press as: Sandomenico, A., et al., IgE-binding properties and selectivity of peptide mimics of the FcRI binding site. Mol.
Immunol. (2009), doi:10.1016/j.molimm.2009.07.025