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International Immunology, Vol. 18, No. 1, pp. 19–29 doi:10.1093/intimm/dxh338 ª The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: [email protected] 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 References 1 Keegan, A. D. and Paul, W. E. 1992. Multichain immune recognition receptors: similarities in structure and signaling pathways. Immunol. Today 13:63. 2 Cheever, A. W., Williams, M. E., Wynn, T. A. et al. 1994. Anti-IL-4 treatment of Schistosoma mansoni-infected mice inhibits development of T cells and non-B, non-T cells expressing Th2 cytokines while decreasing egg-induced hepatic fibrosis. J. Immunol. 153:753. 3 Gessner, A., Schroppel, K., Will, A., Enssle, K. H., Lauffer, L. and Rollinghoff, M. 1994. 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