Download Identification of core functional region of murine IL-4 using

International Immunology, Vol. 18, No. 1, pp. 19–29
doi:10.1093/intimm/dxh338
ª The Japanese Society for Immunology. 2005. All rights reserved.
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Identification of core functional region of murine IL-4
using peptide phage display and molecular modeling
Gang Yao1, Weiyan Chen2, Haibin Luo3, Qunfeng Jiang1, Zongxiang Xia2*, Lei Zang1,
Jianping Zuo3, Xin Wei4, Zhengjun Chen1, Xu Shen3, Chen Dong5 and Bing Sun1,6*
1
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences,
Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China
2
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, China
3
Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203,
China
4
GL Biochem (Shanghai) Ltd, 351 Guo Shoujing Road, Shanghai 201203, China
5
Department of Immunology, MD Anderson Cancer Center, 7455 Fannin, Unit 902, Houston, TX 77030-1903, USA
6
Immunology Division, E-Institutes of Shanghai Universities
Keywords: 11B.11, core functional region of IL-4, IL-4, molecular modeling, peptide phage display
Abstract
Murine IL-4 is a pleiotropic cytokine with undefined core functional region for eliciting downstream
signaling. We used molecular modeling to predict the binding sites recognized by an anti-IL-4neutralizing mAb (11B.11) and peptide phage display to delineate their makeup. The results of these
approaches were confirmed by site-directed mutagenesis analysis. The results suggest that the
amino acid residues spanning from 79 to 86 (QRLFRAFR) on IL-4 are of the major binding site for
11B.11. Furthermore, the functional experiments demonstrate that the residues R80, R83 and R86,
which are located in the helix C of murine IL-4, play a crucial role in binding to the IL-4R a-chain. Taken
together, a new core functional region of murine IL-4 is identified, which provides new insight into the
interaction between IL-4 and IL-4Ra. In addition, the results demonstrate that 11B.11 binds to a core
functional region of murine IL-4, which prevents this cytokine from interacting with its cognate
receptor.
Introduction
IL-4 is a pleiotropic cytokine derived from different cells of
hematopoietic origin, such as T cells, mast cells and basophils
(1). IL-4 is essential for the development of Th2 cells (2–6). As
shown for the first time in a model of murine cutaneous
leishmaniasis, IL-4-mediated induction of Th2 cells in susceptible strains of mice is responsible for severe disease due to
insufficient activation of the leishmanicidal functions of macrophages (7). The fact that transgenic mice overexpressing IL-4
develop allergy-like diseases and autoimmunity is another
example of a pathogenic role of IL-4 (8). More recently, it was
described that IL-4 can direct Th1 differentiation and CD8+
T-cell activation (9, 10).
IL-4 exerts a number of biological activities through two
types of receptors (11). The type I IL-4R is a heterodimer of
IL-4Ra and cc, and type II IL-4R is a heterodimer of IL-4Ra and
IL-13Ra1 (12, 13). Type I IL-4R is preferentially expressed in
hematopoietic cells, whereas type II IL-4R is preferentially
expressed in non-hematopoietic cells. However, in some
hematopoietic cells, including human B cells (14, 15) and
murine macrophages (16), both types of IL-4R are expressed,
and IL-4 can activate cell signaling through type II IL-4R in the
absence of cc.
11B.11 is a rat anti-mouse mAb and IgG1 isotype. It can
neutralize the function of IL-4 (17, 18). Sato et al. (19) found
that administration of soluble IL-4R, like antibody to IL-4
(11B.11), similarly inhibited IgE production in response to antiIgD. All these data indicate that 11B.11 may neutralize the
function of IL-4 by preventing IL-4 from binding to IL-4Ra (20).
However, the precise binding sites on IL-4 recognized by
11B.11 mAb remain unknown.
Our previous work identified the binding sites of trichosanthin (an allergen) to two mAbs (4B5, 2E9) using peptide phage
Received 10 March 2005, accepted 16 September 2005
*Correspondence to: B. Sun; E-mail: [email protected], Zx. Xia; E-mail: [email protected]
Transmitting editor: Dr J. Allison
Advance Access publication 16 December 2005
20
Identifying a core functional region if IL-4
display and molecular modeling analysis (21, 22). In the
current study, the same strategy was used and the binding site
of IL-4 to 11B.11 was predicted and confirmed.
Methods
Reagents and cell line
Random phage peptide libraries (23) expressing circular
nine peptides with random amino acid sequences fused to
pVIII of filamentous bacteriophage fd, vector pC89, wild-type
(WT) phage f1 and bacterial strain DH5aF9 were all kindly
provided by Paolo Monaci of Istituto di Ricerche di Biologia
Molecolare (P. Angeletti SPA, Rome, Italy). Vector pAdEasy-1,
pAdTrack-CMV and bacterial strain BJ5183 were all kindly
provided by LinHua Qin of Shanghai Institutes of Biological
Sciences (Chinese Academy of Sciences).
Recombinant mouse IL-4R/Fc Chimera was purchased
from R&D Systems Inc. (Minneapolis, MN, USA), recombinant
mouse IL-4, 11B.11 and 24G2, was purchased from Endogen
(Woburn, MA, USA) and 4G10 and anti-STAT6 antibody and all
secondary antibodies were purchased from BD PharMingen
(San Diego, CA, USA). All antibodies were used according to
the manufacturer’s instructions.
The cell line for a rat IgG1 mAb (11B.11) against murine IL-4
was kindly provided by W. E. Paul (17, 18). CTL44 cell line was
generously provided by P. Erb (24). EL-4, 293 and L02 cell
lines were obtained from the cell bank of Type Culture
Collection of Chinese Academy of Sciences.
Epitope mapping of mAb 11B.11
Random phage peptide libraries (23) expressing circular nine
peptides with random amino acid sequences were panned
against mAb 11B.11; binding phages were selected from the
library according to immunoscreening method as previously
described (22). Single clones displaying relevant sequences
were tested for binding to 11B.11 by ELISA as described
below (22).
ELISAs
The ability of antibody binding to an antigen was studied by
ELISA as described elsewhere (22). In brief, to study the
capacity of IL-4 or phage-displayed peptides to bind 11B.11,
96-well plates were coated with 11B.11 (2 lg mlÿ1). Then 100 ll
(500 U) of IL-4, phage solution or cultured supernatant in
PBS/1% BSA/0.05% Tween 20 was added for 1 h at 37C and
developed with HRP-conjugated second antibody. For competition ELISA, 96-well plates were coated with 11B.11 at 2 lg
mlÿ1. Serial dilutions in PBS/1% BSA/0.05% Tween 20 of the
selected positive phage clones were incubated with different
concentrations of competing recombinant murine IL-4 (rmIL-4)
overnight at 4C and transferred thereafter for incubation at
37C for 1 h. After extensive washing, the HRP-conjugated
secondary antibody (rabbit anti-phage IgG, Sigma) was
added. For detecting sera antibodies, 50 ll (1000 U) of
rmIL-4 was coated to 96-well plates, and the diluted sera in
PBS/1% BSA/0.05% Tween 20 were added for 1 h incubation at
37C. The plates were washed prior to adding peroxidaseconjugated goat anti-mouse Igs. For detecting the cultured
supernatant, 50 ll (250 U) of rmIL-4, WT IL-4, mutant IL-4 and
control supernatant (from cells infected by control adenovirus)
was added to a 96-well plate coated by 11B.11 or 24G2 mAb
and anti-IL-4 polyclonal antibody as a second antibody to
determine IL-4 production. At last, plates were washed and
developed by adding 100 ll substrate TMB-H2O2 and
incubated at 37C in dark for 15 min. Optical density was
measured using ELISA reader (Multiskan MK3, Thermo Co.)
at 450 nm.
Immunization of mice with selected phage peptide
The selected positive phage clones were amplified in DH5aF9
and purified by PEG/NaCl precipitation. Then the phage clone
was re-suspended in 1 3 PBS at a concentration of 5 3 1012
phage particles mlÿ1 (21). Male C57BL/6 mice of 8–12 weeks
of age were immunized by intraperitoneal injection with 250 ll
of positive phage clones and control WT phage f1. The mice
were immunized three times on days 0, 28 and 56. The sera
were collected on day 10 after the third immunization. The
reactivity of sera antibodies to rmIL-4 was determined using
1:300 dilution of sera in the ELISA.
Cloning of the 11B.11-variable regions by PCR
Total mRNA was prepared from 11B.11 hybridoma cell and
first-strand cDNA was synthesized by using random primers
as described elsewhere (25, 26). The variable heavy and light
chains of 11B.11 were amplified after cDNA synthesis by PCR
using the following specific primers: VH sense primer,
CAGGTCCAACTGCAGGAG, and antisense primer, TGA
GGA GAC GGT GAC CGT GGT CCC TTG GCC CCA; VL
sense primer, GAC ATT CAG CTG ACC CAG TCT CCA, and
antisense primer, ACG TTT GAT CTC CAG CTT GGT CCC. The
amplified DNA was cloned into the T vectors (Promega Co.,
Madison, WI, USA) and sequenced.
Molecular modeling
The sequence-based homologue search and sequence
alignment were carried out using the program Antheprot (27)
and Swiss-Model, a web-based tool for automated comparative protein modeling (28, 29). The initial models were manually
built using the graphics software TURBO-FRODO (30) for
mouse IL-4 and 11B.11, the former based on the crystal
structure of recombinant human IL-4 (PDB entry 1RCB) (31),
the VH domain of 11B.11 was built based on the Fab fragment
of mouse anti-human FAS mAb HFE7A (PDB entry 1IQW) (32)
and the VL and other domains of 11B.11 were from mouse
IgG1 mAb FabE8 (PDB entry 1QBL) (33). The manual docking
between mouse IL-4 and complementarity-determining regions (CDRs) of 11B.11 was carried out using TURBO-FRODO
to build a set of starting models of the complex.
The program CNS (34) was used for molecular modeling:
500 cycles of energy minimization were followed by Cartesian
molecular dynamics simulation (integration time 0.0005 ps,
1000 steps, 298 K), and 500 cycles of further energy
minimization gave the final models of mouse IL-4 and 11B.11
Fab. The interaction energy between IL-4 and 11B.11 Fabs
was calculated using program X-PLOR, and the buried
surface area of IL-4 in the complex was calculated using the
program AREAIMOL (in DIFFERENCE mode) of CCP4 SUITE
Identifying a core functional region if IL-4
(Collaborative Computational Project No. 4, 1994). Three final
models were selected with the criteria of low interaction
energy, good complementary surface contacts and reasonable intermolecular interactions.
21
were infected with AdIL-4, AdmuIL-4 or AdCtrl, using 1 h exposure to 30 ll of the adenovirus (1 3 108 plaque-forming units
mlÿ1). One hour after infection, the L02 cells were washed and
cultured in 3 ml of RPMI medium in 6-cm bacteriologic petri
dishes for 24 h, then the supernatant of the cell culture was used.
Binding measurements
BIAcore 3000 (BIAcore AB, Sweden) instrument was used to
study the interactions between synthetic peptide79–86, IL-4
and 11B.11. 11B.11 or IL-4Ra was covalently and directly
coupled to the dextran matrix of CM5 sensor chip surface at
the level of 6000 RU (resonance unit). Another flow cell was
activated, blocked and then used for reference. All the signals
were obtained by subtracting the signals of reference flow
cell. The analyses were injected to the surface at the flow
rate of 20 ll minÿ1, then dissociated in the system running
buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA,
0.1% P20). After each injection, the flow cell was regenerated
by 20 mM NaOH to remove the sticked material. The kinetic
rate constants, kon and koff, were evaluated using the BIAcore
evaluation software supplied by the manufacturer. Kd values
were calculated from kon and koff rate constants (Kd = kon/koff).
Site-directed mutagenesis
Site-directed mutagenesis based on PCR was performed as
previously described (21, 22). Briefly, mutagenic primers were
designed according to the experiments. For the binding site
mutation, the primers MF1 and MR1 were used. The second
set of primer FKpnI and RXhoI was used to amplify the wholelength mIL-4 gene. The primers were described as follows: MF
sense primer (59-CTG CAG GCA CTC TTT GCG GCT TTT GCA
TGC CTG-39) and MR antisense primer (59-CAG GCA TGC
AAA AGC CGC AAA GAG TGC CTG CAG-39), FKpnI sense
primer (59-GGGGTACC-ATG GGT CTC AAC CCC C-39) and
RXhoI antisense primer (59-CCGCTCGAG- AAG TTAAAG CAT
GGT GGC-39). The mutations were confirmed by restriction
enzyme digestion and sequence analysis.
Cloning and expression of murine IL-4 and its mutant
8
Total mRNA was prepared from 5 3 10 EL-4 cells and the fulllength mIL-4 gene was cloned. The adenoviral vectors for IL-4
gene expression were constructed by use of the AdEasy
System (35). In brief, the full-length IL-4 gene was first cloned
into the KpnI–XhoI site of a shuttle vector (pAdTrack-CMV)
that contained cytomegalovirus promoters and a green fluorescent protein gene. The resultant plasmid was linearized by
digesting with a restriction endonuclease and was subsequently co-transformed into Escherichia coli BJ5183 cells
with an adenoviral backbone plasmid, pAdEasy-1. Recombinants were selected for kanamycin resistance, and recombination was confirmed by restriction endonuclease analysis.
The linearized recombinant plasmid was then transfected
into 293 cells (E1-transformed human embryonic kidney cells).
The IL-4 recombinant adenovirus (AdIL-4) and its mutant
(AdmuIL-4) were generated and verified by PCR analysis of
the DNA samples prepared from the cultured cells. The control
recombinant adenovirus AdCtrl, which carries a green fluorescent protein gene regulated by the cytomegalovirus promoter, was constructed with pAdTrack and pAdEasy-1 and
used as a control in these experiments. L02 cells (5 3 106)
T-cell proliferation assay
IL-4-induced cell proliferation was determined by tritiated
thymidine ([3H]TdR) incorporation as previously described
(24). The mouse T-cell line CTL44 was cultured in RPMI 1640
plus 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05
mM 2-mercaptoethanol, 100 U mlÿ1 penicillin, 100 lg mlÿ1
streptomycin and 100 U mlÿ1 murine IL-4 for 1 week. The cells
were harvested, washed to remove all residual ILs and plated
in 96-well flat-bottom plates at 5000 cells per 50 ll per well
in triplicate in CM. The amount of IL-4 in the cultured
supernatants obtained from L02 cell transferred by AdIL-4,
AdmuIL-4 and AdCtrl was quantified by ELISA with IL-4
antibody, then the supernatants containing equal IL-4 were
added at the beginning of the CTL44 culture. The plates were
incubated for 48 h and [3H]TdR (0.6 lCi per well) was added
for the last 6 h. The results are expressed as mean counts per
minute 6 SD for triplicate wells.
Immunoprecipitation and western blotting analysis
CTL44 cells were incubated at 37C in serum-free medium for
12 h and stimulated under various conditions for 30 min. Total
cellular lysate of 107 cells was prepared as described previously (24, 36) and separated by electrophoresis on 7.5%
SDS-PAGE. After electrotransfer to the PVDF membrane, blots
were probed with anti-STAT6 antibody. Blots were stripped
with the phosphate buffer containing 2% SDS and 0.1 M
2-mercaptoethanol and reprobed with anti-phosphotyrosine
antibody, 4G10.
Reproducibility and data presentation
Experiments were repeated at least twice, and usually three
times. Figures show data compiled from several experiments,
or from a representative experiment, as specified. Results
represent the mean 6 SD, where applicable. Statistical significance of differences was analyzed using the independent
Student’s t test (37, 38). Values of P < 0.05 were considered
significant. The results of statistical analyses are given in the
figure legends.
Results
Peptide phage display reveals the binding sites
recognized by 11B.11 mAb
Our previous work demonstrated that the peptide phage
display identifies a binding site between a given mAb and its
corresponding antigen (21, 22). In this study, the binding sites
for 11B.11 mAb interaction with murine IL-4 were analyzed by
peptide phage display. After three rounds of affinity selection
and immunoscreening, the positive phage clones binding
to 11B.11 mAb were selected and amplified. The binding
activities of the clones to 11B.11 mAb were further confirmed
22
Identifying a core functional region if IL-4
by ELISA and the sequences of the clones were analyzed by
sequence analysis (Table 1). The results showed that 23
selected positive clones had relatively high binding affinities
for 11B.11 mAb. According to their sequences, all clones were
divided into four groups. It was interesting to observe that
17/23 clones showed an identical peptide sequence
(RELRYLRRA) (we termed it as C6 clone), while the other six
clones had three different sequences. Although four groups
had different sequences, all of them were able to bind to
11B.11 as determined by ELISA.
It is reasonable to assume that the positive clones have
peptide sequences similar to the epitopes of IL-4. In this case,
they could effectively compete with IL-4 for binding to 11B.11
mAb. To address this question, a competitive ELISA was
performed by using IL-4 as a competitor. The results showed
that the C6 clone had a higher specific binding affinity to
11B.11 mAb than any of the other clones (data not shown). The
C6 clone effectively competed with IL-4 for binding to 11B.11
mAb in a dose-dependent manner, but the control clone (WT
phage f1) did not (Fig. 1A). The data suggest that the peptide
sequence of C6 clones may mimic an epitope of IL-4.
To further confirm whether the peptide sequence of C6
clone was able to mimic the epitope of IL-4 in vivo, the crossreactivity between C6 clone-immunized sera from C57BL/6
mice and IL-4 protein was measured by ELISA. The data
showed that the immunized sera from the C6 clone were able
Table 1. 11B.11-binding peptides selected from the phagedisplayed random peptide library
Sequence
No. of selected clones
ELISA (OD units)
RELRYLRRAa
PRPLRGVYM
PRSLRHLAN
LLEKLRMRA
17
3
2
1
0.68
0.57
0.53
0.41
6
6
6
6
0.03
0.04
0.03
0.01
Twenty-three of all phage clones that had relative high binding
activities to 11B.11 were selected. Their sequences were analyzed
and the binding activities to 11B.11 were determined by ELISA.
a
The selected clone appeared with high frequency.
Fig. 1. The epitopes recognized by 11B.11 mAb are predicted by
analysis of peptide phage display. (A) The affinity of 11B.11 binding to
IL-4 or phage-displayed peptide was detected by competition ELISA.
11B.11 was used to coat a multi-well plate, C6 clone in three dilution
(106 for diamonds, 107 for squares and 108 for triangles) and control
phage clone of f1 (108 for circles) were incubated overnight at 4C with
a different concentration of IL-4. The anti-phage antibody served as
the secondary antibody. (B) The binding activity of IL-4 to sera from
phage C6 clone-immunized mice. The multi-well plate was coated with
IL-4 and the reaction with sera from phage C6 clone and WT f1immunized mice was detected. The sera from naive mice (preimmunization; pre-imm) and 1 3 PBS (the nature of blank, ‘Blank’ bar)
served as a control. (C) 11B.11 lost the binding activity to heatinactivated IL-4 (de-rmIL-4). The different 50-ll concentrations [5 3
103 U mlÿ1 (black bar) and 5 3 104 U mlÿ1 (white bar)] of recombinant
murine IL-4 (rmIL-4) and denatured IL-4 (de-rmIL-4) were used to coat
the multi-well ELISA plate, then 11B.11 mAb was used to detect their
binding activities and 1 3 PBS (the nature of blank, ‘Blank’ bar) served
as a control. Asterisk denotes statistically significant difference from
control (P < 0.05).
to bind to IL-4 protein (Fig. 1B). The results strongly suggest
that the peptide sequence of C6 clone (RELRYLRRA) mimics
an epitope of IL-4 and this epitope was related to binding sites
of 11B.11 mAb.
Identifying a core functional region if IL-4
Since both linear and conformational epitopes play an
important role in acting on B cells to secrete antibodies,
denatured rmIL-4 was selected as a coating antigen and its
binding activity to 11B.11 mAb was determined by ELISA. The
data showed that 11B.11 had a lower reactivity with denatured
rmIL-4 as compared with native rmIL-4 (Fig. 1C), indicating
that the binding sites of 11B.11 on IL-4 were conformational
epitopes.
Prediction of three-dimensional structures of murine IL-4
and Fab fragment of 11B.11 mAb by molecular modeling
In order to predict the epitopes recognized by 11B.11, it is
necessary to use the molecular modeling method to predict
the three-dimensional (3D) structures of IL-4 and Fab fragment
of 11B.11. Initially, two mAbs (FabE8 and HFE7A) were
selected as templates and their crystal structures were
obtained from the PDB database (32, 33). The 11B.11-variable
regions were cloned from the cell line of 11B.11 and its 3D
structure was constructed, as described in Methods. The
model of the VH of 11B.11 Fab is similar to HFE7A (1IQW_H)
since they share 75% identity and there is no insertion or
deletion in the H chain, as shown in Fig. 2(A). The sequence
alignment showed that the VL domain of FabE8 (1QBL_L) is
75% identical with that of 11B.11, and there is no insertion or
deletion, as shown in Fig. 2(B). To obtain the modeled
structure of murine IL-4, the available crystal structure of
23
human IL-4 was selected as a template (31). The amino acid
sequence alignment of IL-4 from mouse and human shows
43% sequence identity (data not shown). The mouse IL-4
model contains a four a-helix bundle and a two-stranded antiparallel b-sheet, which is very similar to the molecular structure
of human IL-4 determined by X-ray structure analysis (31).
Regional IL-4/11B.11-binding prediction
After construction of the IL-4 and 11B.11 structure models, the
potential binding regions between them were predicted by
molecular modeling analysis. Based on the high affinity of C6
clone (RELRYLRRA) to 11B.11, its sequence served as
a candidate binding site in murine IL-4 and it was taken as
a reference when the complex structure of IL-4/11B.11 was
predicted by molecular modeling. Three models were selected as the predicted mouse IL-4/11B.11 complex models
(Fig. 3A–C). In each of the three models, the helix C is bound in
the antigen-binding pocket of 11B.11 and makes contacts with
the CDRs, and the orientations of this helix in the three models
differ from each other. The buried surface area due to the
binding in each complex indicates the surface complementarity, which is the basis of one of the mechanisms of antigen–
antibody recognition (39). The calculated values of the buried
surface areas of each complex between IL-4 and 11B.11 are in
the order of model I (1131) > model II (1025) > model III (948).
The interaction energy (kcal molÿ1) of IL-4 with 11B.11 Fab in
each complex has the following rank order: model I (ÿ388) <
model III (ÿ279) < model II (ÿ251). These data show that
model I is the most likely model for interaction of mouse IL-4
with 11B.11.
The segment R80–R86 of a-helix C contains three positively
charged arginines, R80, R83 and R86, and they can form salt
bridges with the negatively charged residues of 11B.11 Fab,
such as D50 in VH, D28 in VL and D93 in VL, which are shown in
Table 2. The intermolecular interactions involve R83 and R86 in
model I, R80 and R83 in model II and all the three arginines in
model III, as shown in Fig. 3A, B and C, respectively. By
comparing 11B.11 binding sites deduced by phage peptide
library (RELRYLRRA) and by modeling analysis (RLFRAFR),
a common motif of RXXRXXR has been identified. Furthermore, the three models suggest that IL-4 residues Arg80(R),
Arg83(R) and Arg86(R) had long and positively charged side
chains, which could form salt bridge interactions with
negatively charged residues of 11B.11. These three amino
acids are likely to play a crucial role in determining the binding
activity to 11B.11. In summary, using the two techniques in
combination, we predicted that the binding region of IL-4 to
11B.11 is located at amino acid residues 80 RLFRAFR 86.
11B.11 mAb binds to synthetic peptide79–86
Fig. 2. Amino acid sequences of heavy and light chain variable
regions (VH and VL) of antibody 11B.11. The identical residues are
indicated by asterisks. The amino acid residues and CDR designation
are marked based on Kabat’s observation (56). The sequence data
have been submitted to the GenBank databases under accession
numbers AY633654 and AY633655. (A) VH aligned with that of the
apoptosis-inducing mouse anti-human FAS mAb HFE7A (1IQW_H).
(B) VL aligned with that of mouse IgG1 mAb FabE8 (1QBL_L).
To directly confirm whether the predicted binding site
recognized by 11B.11 mAb is correct, initially, we synthesized
the peptide79–86 (79 QRLFRAFR 86 which was termed as QR8)
from IL-4. It was reasonable to assume that if the predicted
binding site was capable of binding to 11B.11 mAb, the
synthetic QR8 should have some affinity to 11B.11 mAb. To
address this question, 11B.11 mAb was linked to BIAcore
CM5 sensor chips, and the affinity of QR8 to the chips was
measured by the BIAcore. The data showed that QR8 had
24
Identifying a core functional region if IL-4
Fig. 3. Stereo diagrams of the mouse IL-4–11B.11 complex models. The main chain of IL-4, 11B.11 VL and VH are shown in pink, green and light
blue, respectively. The side chains of R56, K64, R80, R83 and R86 in IL-4 are shown in dark blue and those of the aspartic acids in 11B.11
interacting with them are shown in red. These diagrams were prepared using the program SETOR. (A) Model I; (B) model II; (C) model III.
Table 2. The salt bridge interactions of R80, R83 and R86 of
mouse IL-4 with CDR of 11B.11
predicted region (i.e. 79 QRLFRAFR 86) for binding of IL-4 to
11B.11 mAb is correct.
Model I
The component of IL-4 corresponding to 11B.11 mAb
plays a crucial role in IL-4 function
Model II
Model III
Residue 1 Residue 2 Residue 1 Residue 2 Residue 1 Residue 2
in IL-4
in 11B.11 in IL-4
in 11B.11 in IL-4
in 11B.11
(CDR)
(CDR)
(CDR)
R83
R86
D50 (H2)
D93 (L3)
R80
R83
D93 (L3)
D28 (L1)
R80
R83
R86
D50 (H2)
D93 (L3)
D28 (L1)
a relatively higher affinity to 11B.11 mAb as compared with
control peptide (S3: YKGYQPIDVVRDLPSGFNTL) (Fig. 4).
Mutagenesis confirms binding region identity
To further confirm the predicted binding site recognized by
11B.11 mAb, the predicted binding site (QR8) that corresponds to 11B.11 was induced to mutate using site-directed
mutagenesis. The three Arg80, Arg83 and Arg86 in murine
IL-4 were mutated into alanine. DNA-sequencing analysis
verified the DNA sequence of the mutant. To express the
functional IL-4 protein, IL-4 gene was cloned in an adenoviral
vector and the IL-4 protein was expressed in an AdEasy
system. The L02 cells were infected with AdIL-4, AdmuIL-4 or
AdCtrl. After transfection for 24 h, nearly 100% of the cells
expressed the green fluorescent protein. Total mRNA was
isolated from 5 3 106 transfected L02 cells and the cultured
supernatants were harvested for ELISA analysis. The data
showed that the WT IL-4 and mutant IL-4 mRNA expression
were detectable in the transfected L02 cells, in which the
starting mRNA was normalized to equal by detecting the
OD260/280 value. Their synthesized proteins were also determined in the supernatant from the cultured L02 cells
measured by 24G2 mAb (murine IL-4-specific mAb), indicating that WT IL-4 and mutant IL-4 were expressed (Fig. 5). It is
interesting to observe that in a parallel experiment the mutant
IL-4 was unable to bind to 11B.11. The data indicate that the
Since the 11B.11 mAb can neutralize IL-4 bioactivity by
blocking IL-4 interaction with IL-4Ra, there is a possibility that
the identified binding site recognized by 11B.11 mAb plays an
important role in IL-4 bioactivity. Therefore, the mutated IL-4
should consequently lose the activity to stimulate IL-4R. To
address this question, an IL-4-dependent cell line (CTL44)
was selected and the cell proliferation and STAT6 phosphorylation were examined in the IL-4-treated cells. Interestingly,
the expressed WT IL-4 was capable of stimulating CTL44
cell proliferation and triggering STAT6 phosphorylation which
was mediated by IL-4R, but the expressed mutant IL-4 lost
these functions. Blocking with 11B.11 served as a positive
control showing a complete arrest of mitogenic activity upon
exposure to IL-4 (Fig. 6A and B). The results indicate that the
IL-4 region binding to 11B.11 mAb had the ability to interact
with the IL-4R and trigger a cellular signal necessary for cell
proliferation.
The synthetic QR8 was capable of binding to IL-4R a-chain
Based on available information about the interaction between
IL-4 and IL-4R and knowing that the QR8 peptide was identical
to amino acid residues 79–86 in IL-4, it was assumed that QR8
may be able to bind to IL-4R a-chain (IL-4Ra). To obtain
supporting evidence for this assumption, IL-4Ra was linked to
BIAcore CM5 sensor chips and the affinity of QR8 to IL-4Ra
was measured by BIAcore. The affinity of IL-4 for IL-4Ra
served as a positive control in these experiments. The data
show that QR8 was capable of binding to IL-4R a-chain as
compared with the S3 control peptide (Fig. 7A–C).
Discussion
IL-4 plays a crucial role in Th2 cell differentiation. The interaction of IL-4/IL-4R controls the signaling transduction and cell
Identifying a core functional region if IL-4
25
Fig. 5. Murine IL-4 protein expression and detection. IL-4 and mutant
IL-4 proteins are expressed in adenovirus expression system. The
proteins were collected from the supernatant of virus-infected L02
cells (24 h after infection). Fifty microliters (250 U) of rmIL-4, WT IL-4,
mutant IL-4 and cultured medium control (from cells infected by
control adenovirus) was added to a 96-well plate coated with 11B.11
or 24G2 mAb and anti-IL-4 polyclonal antibody as a second antibody
to determinate IL-4 production. Triplicate samples were run for each
supernatant tested. Asterisk denotes statistically significant difference
from control (P < 0.05). The top right data showed that IL-4 mRNA
expression amplified by reverse transcription–PCR from the L02 cells
(5 3 106) which had been transferred by pAdIL-4, pAdmuIL-4 or
pAdCtrl.
Fig. 4. Binding properties of mAb 11B.11 to IL-4 protein and
peptide79–86 (QR8). (A) Binding of IL-4 to 11B.11. Samples of serially
diluted IL-4 (3.5 lM to 6.85 nM) were analyzed on 11B.11 mAb-labeled
sensor surface. The Kd of IL-4 (served as a positive control) was 1.19 3
10ÿ7 (M). (B) Interaction kinetics for QR8 binding to 11B.11. Serial dilutions of QR8 (1.12 mM to 8.82 lM) were analyzed on the same
sensor surfaces. The QR8 was fitting by steady-state fitting model and
its Kd was 6.18 3 10ÿ4 (M). (C) The values of AU/MW of QR8 and
control peptide (S3) binding to 11B.11 were compared at the same
experimental conditions. The top right shows the interaction kinetics
for QR8 and control peptide binding to 11B.11. The peptide (300 lM)
was analyzed on parallel sensor surfaces, and control peptide cannot
bind to IL-4Ra.
activation. 11B.11 mAb is a neutralizing antibody that blocks
the function of IL-4. To identify the binding sites of 11B.11 mAb
on murine IL-4, both peptide phage display and molecular
modeling were used and the core functional region of IL-4 was
localized to amino acid residues 79–86 of IL-4, which is
characterized by an a-helix structure and is capable of binding
to the IL-4R a-chain. The data indicate that the identified core
region of IL-4 is identical to the site that binds to IL-4R a-chain
to trigger IL-4 signaling and to 11B.11 mAb.
In general, a neutralizing mAb often binds to conformational
epitopes of the antigen (39–41). Since 11B.11 can completely
block IL-4 function, the sites that bind 11B.11 to IL-4 were
proposed to be crucial for IL-4 function. Therefore, it is
possible that the interaction of 11B.11/IL-4 will help to
elucidate the mechanism by which IL-4 stimulates IL-4R.
Initially, the 11B.11 mAb served as a probe and its binding
sites on IL-4 were predicted by peptide phage display. Many
peptide sequences were deduced based on selected positive
phage clones. It was interesting to observe that one peptide
sequence (RELRYLRRA) had relatively high affinity to 11B.11
mAb. The serial synthetic peptides or truncated fragments
from a giving antigen are routinely used to define the binding
sites recognized by an mAb. However, these methods are
believed to impact the structure of the antigen and there is low
efficiency of screening. The phage-displayed peptide libraries
overcome such limitations. In addition, a displayed peptide on
the phage clone could be directly used to immunize mice,
evaluate its antigenicity and mimic the target protein.
Since 11B.11 mAb binds to conformational epitopes,
molecular modeling was used to locate its binding regions
on the 3D structure of IL-4 molecule. Based on the molecular
modeling results, we propose three possible models, as
described above, in which two or three arginines in the
segment Q79–R86 of a-helix C are involved in the interactions
of mouse IL-4 with 11B.11 Fab, and the sequence of this
segment is QRLFRAFR. On the other hand, the peptide
identified by peptide phage display has a sequence of
RELRYLRRA. These two sequences share a similar RXXRXXR
motif. The three predicted models further show that substitution of two phenylalanine residues in the segment Q79–
R86 of 11B.11 Fab by two leucines of QR8 would not
26
Identifying a core functional region if IL-4
Fig. 6. Mutant IL-4 lost the functional bioactivity. (A) The mutant IL-4 lost the capability to stimulate CTL44 cell line proliferation. Fifty microliters
(250 U) of rmIL-4 and cultured supernatants was added to CTL44 (104 per well), and CTL44 cells were cultured for 48 h and pulsed with [3H]TdR
for the last 6 h. Then the [3H]TdR uptake was determined by the counts per minute value as data presented. Asterisk denotes statistically
significant difference from control (P < 0.05). (B) The mutant IL-4 lost the capability to trigger the phosphorylation of STAT6 in the cells.
significantly modify the lengths and sizes of the two side
chains. In contrast, when residues L81 and A84 of 11B.11 Fab
were replaced by glutamic acid and tyrosine of QR8,
respectively, the two side chains would change their size
and property. It was interesting to note that this mutated
peptide segment would still be capable of recognizing 11B.11
Fab, since the side chains of residues L81 and A84 point to the
other side of a-helix C and are not involved in the interactions
with 11B.11. Furthermore, residues R80, R83 and R86 are
located in helix C, and K64 (another basic residue involved in
the interactions with 11B.11 in model I) is in the BC loop, which
is consistent with the results of a study on immunochemical
mapping of domains in human IL-4 recognized by neutralizing
mAbs, wherein helix C and BC loop comprise the antibodyrecognized region of human IL-4 (42). Therefore, it is reasonable to predict that QR8 peptide (79 QRELRYLR 86) in murine
IL-4 might be a binding site for 11B.11 mAb on IL-4.
In order to confirm whether the predicted binding site was
correct, firstly, the peptide79–86 (QR8; 79 QRELRYLR 86) from
murine IL-4 was synthesized, and its binding activity with
11B.11 mAb was analyzed by surface plasmon resonance
assay. The results showed that QR8 was able to bind to
11B.11. Notably, its affinity was lower compared with IL-4.
There are two possible explanations for this result. One is that
the synthetic single peptide could not maintain its 3D structure
like the whole IL-4 molecule or as the peptides displayed on
the phage surface. The other is that amino acid residues such
as R56 and K64 or others in IL-4 could also comprise binding
sites recognized by 11B.11. Site-directed mutagenesis was
performed to further confirm the prediction of QR8 region on
the surface of IL-4 protein. It is accepted that alanine is
a suitable amino acid for substitution in mutagenesis studies.
When both buried and exposed positions of amino acid
residues in proteins were conserved (43, 44), the alanine
substitution in a given protein generally does not affect overall
protein folding (45, 46). When the arginines in positions 80, 83
and 86 of IL-4 were mutated to alanine, the expressed WT IL-4
and mutant IL-4 bound equally well to an IL-4-specific mAb
(24G2) and polyclonal antibody (data not shown), indicating
that the expressed WT IL-4 and mutant IL-4 had a similar
structure. As expected, WT IL-4 maintained its binding activity
with 11B.11 mAb, but the mutant IL-4 did not. The data
suggest that by mutating the IL-4 protein within the 11B.11
(QR8) binding site, its specific binding reactivity to 11B.11 is
eliminated. The results confirm that the predicted 11B.11
binding site (QR8) in the 3D structure of IL-4 protein is indeed
correct, and indicate that the region of IL-4 protein from
residues 79 to 86 is recognized by 11B.11 mAb.
In addition, the crystal structure of the complex of human
IL-4 with its receptor a-chain (IL4-BP) revealed an interface
with three separate clusters of trans-interacting residues (47).
The second cluster contains a central salt pair linking R88
of human IL-4 and D72 of IL4-BP. The R88 of human IL-4
corresponds to R83 of mouse IL-4. From the modeling result, it
was demonstrated that R80, R83 and R86 of mouse IL-4 were
the important amino acid residues involved in the interaction of
mouse IL-4 with the antibody 11B.11.
To determine whether 11B.11 could completely neutralize
the IL-4 function by competitively inhibiting the ability of IL-4 to
bind with IL-4Ra or not, we presume that the binding site
recognized by 11B.11 on IL-4 might be functionally important
for IL-4 and may be related to IL-4Ra. From the functional
experiments, the mutant IL-4 protein lost its function in
inducing the phosphorylation of STAT6 and cell proliferation
in CTL44 cells, but the expressed WT IL-4 protein maintained
these functions. Furthermore, the synthetic peptide QR8 from
IL-4 can directly bind to IL-4Ra as determined by biosensor
assay. These results show that this region in the IL-4 protein is
very important for binding to the receptor a-chain. Generally,
site-directed mutagenesis has led to the discovery of two
regions in the human IL-4 molecule that are important for
interaction with the receptor chains (48). The substitution of E9
or R88 leads to a near-complete loss of binding to IL-4Ra. A
second important region of the human IL-4 molecule is defined
Identifying a core functional region if IL-4
by mutations of R121, Y124 and S125, close to the C terminus
of the protein (20, 49). However, only a few reports have been
published so far concerning murine IL-4 important regions
necessary for binding with the receptor a-chain. Morrison
27
and Leder (50) analyzed the receptor-binding domain of
mouse IL-4 using a solid-phase-binding assay and in vitro
mutagenesis. They reported that the 16 amino-terminal
residues and the 20 carboxyl-terminal residues are required
for species-specific receptor binding. It is important to confirm
that the C-terminal residues bind to the receptor cc (51–54).
Here, we identified another sequence of amino acid residues
(Q79–R86) which is important for binding to the receptor
a-chain. These residues (Q79–R86) could form a cluster with
the 16 amino-terminal residues as in human IL-4 (20, 50) and
bind to the receptor a-chain. It is possible that the neutralizing
mAb, 11B.11, could occupy the Q79–R86 binding site and
then interfere with the contact between IL-4/IL-4R. We have to
emphasize that it cannot be ruled out that 11B.11 may also
bind to other sites on IL-4, since 11B.11 mAb recognizes
conformational epitopes.
In conclusion, screening of phage-displayed random peptide libraries with neutralizing mAb to IL-4, 11B.11, yielded
peptide sequences that mimic the conformational epitopes of
IL-4. When combining molecular modeling analysis, a core
functional region of murine IL-4 was identified. The region was
recognized by 11B.11 and was also involved in binding to
IL-4R a-chain. The identified region will improve our understanding of the mechanisms of IL-4 in triggering IL-4R-linked
signaling. Since a neutralized mAb is valuable in disease
treatment, the techniques used here allow us to identify core
functional regions of a given receptor/ligand, therefore, there
is a potential to design rational mimicking molecules based on
the identified region (55, 56). Defining the functional region of
ligand or receptor recognized by neutralizing mAb will be
essential in identifying novel drug targets.
Acknowledgements
We are grateful to LinHua Qin and Qiong Wang for excellent technical
assistance. We greatly appreciate the critical reading of this paper by
Jian-Ping Ding, Zihe Rao (Chinese Academy of sciences, China) and
Peter Reinach (Columbia University, USA). This work was supported
by the grants of Technology Commission of Shanghai Municipality
03DZ19113, 04DZ19108, 04DZ14902 and 03DZ19119, National
Key Basic Research Program of China 2001CB510006, The National
Natural Science Foundation of China 30228016, 30325018 and
30421005 and grant from E-institutes of Shanghai Universities Immunology Division.
Abbreviations
CDR
[3H]TdR
rmIL-4
WT
complimentarity-determining region
tritiated thymidine
recombinant murine IL-4
wild type
Fig. 7. Binding properties of IL-4 protein and peptide79–86 (QR8) to
IL-4Ra. (A) Binding of IL-4 to IL-4Ra. Samples of serially diluted IL-4
(3.5 lM to 6.85 nM) were analyzed on an IL-4Ra sensor surface. The
Kd value for IL-4 binding to IL-4Ra was 3.24 3 10ÿ7 (M). (B) Interaction
kinetics for QR8 binding to IL-4Ra. Serial dilutions of QR8 (1.12 mM to
8.82 lM) were analyzed on parallel sensor surfaces. The curves of the
synthetic QR8 peptide binding to IL-4Ra were fitted by the steadystate model, its Kd value was 8.77 3 10ÿ4 (M). (C) The value of AU/MW
of QR8 and control peptide (S3) binding to IL-4Ra at the same
concentration; the top right shows the Interaction kinetics for QR8 and
control peptide binding to IL-4Ra. The peptide (300 lM) was analyzed
on parallel sensor surfaces, and control peptide cannot bind to IL-4Ra.
28
Identifying a core functional region if IL-4
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