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Published as doi: 10.1096/fj.06-5995fje.
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(The FASEB Journal. 2006;20:2378-2380.)
© 2006 FASEB

N-linked glycosylation of IL-13R{alpha}2 is essential for optimal IL-13 inhibitory activity

Mitomu Kioi, Saraswathy Seetharam and Raj K. Puri1

Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA

1Correspondence: Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Food and Drug Administration, Center for Biologics Evaluation and Research, NIH Bldg. 29B, Rm. 2NN20, 29 Lincoln Dr., Bethesda, MD 20892 USA. E-mail: raj.puri{at}fda.hhs.gov

ABSTRACT

A high-affinity receptor for interleukin (IL)-13 (interleukin-13R{alpha} 2) is over-expressed in disease-related fibroblasts and neoplastic cells and is involved in cancer, allergic, and inflammatory diseases. The extracellular domain of IL-13R{alpha}2 (ECD{alpha}2) could be cleaved, which serves as a decoy receptor. We have expressed and purified ECD{alpha}2 in both Escherichia coli (E. coli) and mammalian systems as a soluble fragment and studied its biological activities. Although both products of ECD{alpha}2 showed IL-13 inhibitory activities, mammalian cell-derived ECD{alpha}2 appeared to be superior compared with purified protein from E. coli. When expressed in E. coli, ECD{alpha}2 appeared to be a monomer of 42 but a 60 kDa protein when purified from mammalian cells due to heavy glycosylation. The purified glycosylated ECD{alpha}2 efficiently inhibited IL-13-induced STAT6 phosphorylation in immune and Hodgkin’s lymphoma cell lines, IL-13 binding, and cytotoxicity of IL-13 cytotoxin in various cancer cell lines. The improved potency of mammalian cell-derived ECD{alpha}2 was shown over ECD{alpha}2/Fc fusion protein. The N-linked glycosylation of ECD{alpha}2 was found to be essential for optimal IL-13 inhibitory activity as deglycosylation by PNGase F showed lower activity. ECD{alpha}2 did not inhibit IL-4-induced STAT6 phosphorylation, indicating that inhibitory effects of ECD{alpha}2 are receptor specific. These results indicate that glycosylated ECD{alpha}2 can serve as a potent inhibitor of IL-13 in a variety of conditions in which IL-13 is a key mediator, e.g., pulmonary, allergic, fibrotic, and neoplastic diseases.—Kioi, M., Seetharam, S., Puri, R. K. N-linked glycosylation of IL-13R{alpha}2 is essential for optimal IL-13 inhibitory activity.


Key Words: Type I cytokine receptor • ECD • allergic and inflammatory diseases • cancer

IN RECENT YEARS, INTERLEUKIN-13 (IL-13) has acquired prominent status due to its central role in a variety of inflammatory, allergic, and neoplastic diseases (1 2 3) . Many of these effects are known to be mediated by IL-4, also a Th2-derived cytokine. However, studies conducted using various analytical tools such as knockout and transgenic mice have demonstrated that although IL-13 and IL-4 have some overlapping functions, IL-13 has important distinct roles from IL-4. For example, IL-13 regulates mucus secretion, eosinophilic inflammation, and airway hyperresponsiveness, which are hallmarks of allergic asthma (4 5 6) . Given these important findings, much attention has been directed toward IL-13 and efforts to block its effects.

Both IL-13 and IL-4 bind to specific plasma membrane receptors on a variety of cell types. Both receptor systems share two chains, IL-4R{alpha} and IL-13R{alpha}1, with each other and mediate signal transduction (7 8 9) . IL-13 binds to primary receptor IL-13R{alpha}1 chain with low affinity (9 , 10) . However, with IL-4R{alpha} chain, IL-13R{alpha}1 forms a high affinity receptor complex that binds to both IL-13 and IL-4. Because of this property both cytokines share many biological activities (3) . IL-13 also binds to a second receptor IL-13R{alpha}2 but with high affinity (11 , 12) . Unlike the IL-13R{alpha}1 chain, IL-13R{alpha}2 does not seem to form a complex with any other chain. Because IL-13R{alpha}2 does not bind IL-4, distinct activities of IL-13 are thought to be mediated by IL-13R{alpha}2.

Both IL-13 and IL-4 can activate two signaling cascades through shared receptor chains. These cascades include phosphatidylinositol 3-kinase (PI3K) and Janus kinase-STAT6 pathway. Although IL-13R{alpha}2 internalizes after binding to IL-13, this chain has not been shown to mediate signaling through janus-activated kinase (JAK)-STAT pathway (13 , 14) . This is because the cytoplasmic region of IL-13R{alpha}2 is short (17 amino acids) and does not contain an obvious signaling motif or JAK-STAT binding sequence (12) . However, we have previously demonstrated that overexpression of IL-13R{alpha}2 chain inhibits IL-13-induced STAT6 phosphorylation and activation (14) . Similarly, Rahaman et al. (15) reported that expression of IL-13R{alpha}2 inhibits IL-4-induced signaling due to physical contact between each other. Another study by Rahaman et al. (16) demonstrated that IL-13R{alpha}2 is involved in IL-4-mediated STAT3 activation, which contributes to the pathogenesis of cancer. Most recently, we have demonstrated that IL-13R{alpha}2 signals through the activating protein (AP)-1 pathway (14) . Taken together, IL-13R{alpha}2 is emerging as an important player in signal transduction.

To clarify the role of IL-13R{alpha}2, we have expressed and purified a soluble form of IL-13R{alpha}2 (ECD{alpha}2) in E. coli and mammalian expression systems. The effects of both E. coli and mammalian cell-derived ECD{alpha}2 were tested in various systems including its impact on blocking the cytotoxicity of IL-13 cytotoxin in tumor cells.

MATERIALS AND METHODS

Cell culture and reagents
Human THP-1, TF-1, U251MG, and monkey COS-7 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA), and HEK 293FT cell line was purchased from Invitrogen (Carlsbad, CA). PM-RCC cell line was established as described previously (17) . L591 and L1236 cell lines were kindly provided from Dr. Daniel Re of Universität Klinik Köln, Germany. All cell lines were maintained and cultured as described previously (18) . Recombinant human IL-13 and IL-4 were purchased from PeproTech (Rocky Hill, NJ). Recombinant IL13-PE38 was produced and purified in our laboratory (19) . Recombinant IL-13R{alpha}2 in the extracellular domain fused to the Fc protein (ECD{alpha}2/Fc) was purchased from R&D Systems (Minneapolis, MN).

Construction of plasmids
In all His-tagged constructs for ECD{alpha}2 expression, His-tag was incorporated at the carboxyl terminus of the ECD{alpha}2 sequence. The maltose-binding protein (MBP)/ECD{alpha}2 fusion protein construct was generated by cloning the ECD of IL-13R{alpha}2 into the pMAL-p2E vector (New England Biolabs, Ipswich, MA) downstream of the malE gene encoding MBP. ECD{alpha}2 was amplified by standard polymerase chain reaction (PCR) methods with the following set of primers (sense: 5'-CCCGGTACCGGACACCGAGATAAAAGTTAACCC-3'; antisense: 5'-CCCAAGCTTCTAACGTAGCAAAGTTTCTTCGATAGG-3') for insertion of a KpnI site at 5'-terminal, HindIII at 3'-terminal, and cloning into the pMAL-2pE vector. The Nus/ECD{alpha}2 fusion protein construct was generated by cloning the ECD{alpha}2 into the pET44a vector (Novagen, Madison, WI), downstream of NusA gene. ECD{alpha}2 was amplified with a set of primers (sense: 5'-TCCCCCGGGCGACACCGAGATAAAAGTTAACCC-3'; antisense: 5'-CCCAAGCTTCTAACGTAGCAAAGTTTCTTCGATAGG-3') for insertion of a NdeI site at 5'-terminal and HindIII at 3'-terminal and cloning into the pET44a vector. The E. coli ECD{alpha}2 construct (ECD{alpha}2-EC) insert was prepared by cloning into the SalI and XhoI restriction sites of the pET24a expression vector using the primers 5'-TGTACTTCAGAGCTCGACACCGAGATAAAAGTTAAC-3', 5'-TGGTAGCCACTCGAGTAGCAAAGTTTTCTTCGA-3'. cDNA of CAKi-1 cells served as a template for ECD{alpha}2 amplification in E. coli.

For ECD{alpha}2 expression in mammalian cells using the pFB-Neo retroviral vector (Stratagene, La Jolla, CA), restriction enzyme sites were inserted by PCR using sense primer 5'-ACGCGTCGACCATGGCTTTCGTTTGC-3' and antisense primer 5'-TTTTCCTTTTGCGGCCGCTCAATGGTGATGGTGATG-3' to incorporate SalI restriction site at 5'-terminal and NotI at 3'-terminal. For ECD{alpha}2 expression in mammalian cells using plasmid vector with transient transfection, restriction enzyme site was inserted by PCR using sense primer 5'-CCGGTACCATGGCTTTCGTTTGCTTGGC-3' and antisense primer 5'-GCTCTAGAACGTAGCAAAGTTTTCTTCGATAGG-3' to incorporate KpnI restriction site at 5'-terminal and XbaI at 3'-terminal using CAKi-1 cDNA as a template. After the PCR products were digested, ECD{alpha}2 sequence was inserted into pCDNA6/myc-His vector for expression in 293FT cells (Invitrogen). The sequences of all constructs were verified by ABI Prism 310 genetic analyzer (Perkin-Elmer, Wellesley, MA).

Protein expression and purification in E. coli
The ECD{alpha}2-EC plasmid was transformed into E. coli BL21 cells. The bacterial cultures were induced with 0.25 mM IPTG at optical density (OD)600 = 3.0 for 6 h at 37°C. Proteins were obtained as inclusion bodies that being washed, were solubilized with 100 mM Tris buffer containing 6 M guanidine-hydrochloride and 10 mM imidazole and applied onto a Ni-NTA superflow culumn (Qiagen, Valencia, CA). After being washed with decreasing concentrations of urea-containing buffers (6–0 M) to renature the proteins, ECD{alpha}2 was eluted with 200 mM imidazole-containing TBS buffer (pH 8.3).

Protein expression and purification in mammalian cells
Retrovirus-mediated expression of ECD{alpha}2 was performed in the ViraPort Retroviral Gene Expression System following the manufacturer’s instructions with minor modifications. To obtain retrovirus-containing supernatants, 293T cells were cotransfected with pVPack-VSV-G, pVPack-GP, and pFB-Neo-ECD{alpha}2. After 48 h of transfection, the medium containing retroviruses was collected, filtered, treated by diethylaminoethyl (DEAE)-Dextran (1 µg/ml), and transferred onto Hela cells. Infected cells were selected with G418 (1000 µg/ml) for 7 days. The plasmid for expression of ECD{alpha}2 (pcDNA6-ECD{alpha}2) was transfected into 293FT cells using FuGene 6 (Roche, Indianapolis, IN) and cultured for 24 h. After the culture medium was changed to serum-free Dulbecco’s modified Eagle’s medium (DMEM) medium, the cells were incubated an additional 48 h. Supernatant was harvested, centrifuged, and loaded onto ProBond His-tagged protein purification column (Invitrogen). The purity at each step was verified by SDS-PAGE and Western blotting. The purity (>99%) of the final recombinant protein (ECD{alpha}2-His6) was verified by SDS-PAGE.

Western blot analysis
To determine the phosphorylation of STAT6, 2 x 106 cells were incubated with various concentrations (0–100 ng/ml) of IL-4 or IL-13 in the presence or absence of glycosylated ECD{alpha}2-His6 (12.5 ng/ml) for 15 min at 37°C. The reaction was quenched with 10 ml of cold PBS containing 1 mM sodium vanadate, 25 mM NaF, 10 mM sodium pyrophosphate, and 1 mM EDTA (pH 8.0). The cells were lysed with 1% Nonidet P-40, 300 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 µg/ml of leupeptin, 10 µg/ml of aprotinin, 1 mM PMSF, 1 mM sodium vanadate, 25 mM NaF, 10 mM sodium pyrophosphate, and 1 mM EDTA. The blotted membrane was blocked with PBS containing 5% skim milk at room temperature for 1 h, and then the membrane was incubated with antiphosphorylated STAT6 polyclonal antibody (Ab; Cell Signaling Technology, Beverly, MA) overnight at 4°C. Immunoreactive signal was visualized by enhanced chemiluminescence (ECL) system following the manufacturer’s instructions. After being stripped, the membrane was incubated with anti-STAT6 Ab (s-20, Santa Cruz Biotechnology, Santa Cruz, CA) for detection of STAT6.

To determine the presence of ECD{alpha}2, conditioned media were dialyzed against phosphate buffer, cold-acetone precipitated, and dissolved in SDS sample buffer. The blotted membrane was reacted with anti-IL-13R{alpha}2 monoclonal Ab (R&D Systems).

Deglycosylation of ECD{alpha}2
Deglycosylation of ECD{alpha}2 was performed using E-DEGLY kit (Sigma, St. Louis, MO) according to manufacturer’s instructions. Briefly, purified ECD{alpha}2-His6 was incubated with or without PNGase F or O-Glycosidase under normal or denaturing conditions at 37°C overnight. After cold-acetone precipitation, samples were analyzed by Western blotting. To determine the effect of deglycosylation of ECD{alpha}2 on inhibition of STAT6 phosphorylation, ECD{alpha}2-His6 was preincubated with or without PNGaseF at 37°C overnight. THP-1 cells were incubated with IL-13 (50 ng/ml) in the presence of ECD{alpha}2-His6 (50 or 500 ng/ml) for 15 min.

Cell proliferation assay
The biological activity of ECD{alpha}2 was determined by cell proliferation assays (20) . Briefly, TF-1 cells (1x104) were incubated with different concentrations (0–100 ng/ml) of IL-13 in the presence or absence of ECD{alpha}2-EC, ECD{alpha}2/Fc, or ECD{alpha}2-His6 for 48 h at 37°C and pulsed with 0.5 µCi of [3H]thymidine (NEN Research Products, Boston, MA) for an additional 12 h.

Protein synthesis assay
TF-1 (1x104) cells were incubated with different concentrations of human IL-13 in the presence or absence of ECD{alpha}2 for 52 h at 37°C in 10% dialyzed FBS-containing leucine-free medium. Next, 1 µCi of [3H]leucine was added to each well, and cells were incubated for an additional 8 h. Cells were harvested, and radioactivity incorporation was measured by a ß plate counter (Wallac, Gaithersburg, MD).

Cytotoxicity assay
The in vitro cytotoxic activity of IL13-PE38 was measured by the inhibition of protein synthesis (19) . Briefly, cells (1x104) were cultured in leucine-free medium with varying concentrations of IL13-PE38 for 22 h at 37°C. One µCi of [3H]leucine (NEN Research Products, Boston, MA) was added to each well and incubated for an additional 4 h. All assays were performed in quadruplicate, and the concentration of IL13-PE38 at which 50% inhibition of protein synthesis occurred was calculated (IC50).

Radio-receptor binding assay
Recombinant human IL-13 was labeled with 125I (Amersham, Piscataway, NJ) by the IODO-GEN iodination reagent (Pierce, Rockford, IL) according to the manufacturer’s instructions. The IL-13 equilibrium binding studies were performed using the previously described method (20) . Briefly, 1 x 106 cells in 100 µl of binding buffer (RPMI 1640 containing 0.2% human serum albumin and 10 mM HEPES) were incubated for 2 h with 200 pM 125I-interleukin-13 (specific activity, 20.5 µCi/µg) with or without various concentrations (0.78 pM to 200 nM) of unlabeled IL-13, ECD{alpha}2/Fc, or ECD{alpha}2-His6 at 4°C. Cell-bound 125I-interleukin-13 was separated from unbound by centrifugation through a cushion of phthalate oils. Radioactivities in pelleted cells were counted by a gamma counter.

Detection of ECD{alpha}2-bound IL-13
Ninety six well immunoplates were coated with IL-13R{alpha}2 monoclonal Ab (4 µg/ml), washed and blocked with 5% milk in PBS. ECD{alpha}2-His6 (1 µg/ml) was preincubated with or without 0.5 U of PNGase F for overnight and mixed with serially diluted IL-13 (0–400 ng/ml) for 30 min at room temperature. ECD{alpha}2-His6/interleukin-13 solutions were then added. After 1 h incubation, biotinylated anti-human IL-13 Ab was added for 1 h at room temperature. After washing, HRP-conjugated streptavidin and TMB (3,3',5,5'tetra-methylbenzidine) peroxidase substrate was added to detect IL-13.

Statistical analysis
The statistical significance of data was calculated by Student’s t test. All statistical tests were two sided.

RESULTS

Expression and purification of ECD{alpha}2
We transformed BL21 cells with pET24a expression vector carrying the ECD{alpha}2 gene (Fig. 1 ). The expression and purification of ECD{alpha}2 from inclusion bodies were performed. As shown in Fig. 2 A, expression of ECD{alpha}2-EC in BL21 cells when induced by IPTG was very efficient. Highly purified protein was obtained after refolding and running through Ni-NTA affinity column. Refolding of ECD{alpha}2-EC was attained by rapid dilution of guanidine hydrochloride. However, when stepwise dilution was performed in Ni-NTA column after protein had bound to Ni-NTA beads, an efficient refolding was achieved. SDS-PAGE analysis showed its purity and a molecular mass of 42 kDa, indicating that this protein was expressed and purified as monomer. The overall yield of highly purified ECD{alpha}2-EC (>99%) was 1–2 mg from 1 liter of bacterial culture. As shown in Fig. 2B , the mobility of ECD{alpha}2-EC in the nonreducing condition was faster compared with the reducing condition in SDS-PAGE analysis. This different mobility indicates the refolding of ECD{alpha}2-EC.


Figure 1
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  		Figure 1. Schematic chart for the expression and purification of IL-13R{alpha}2 extracellular domain (ECD{alpha}2). To construct the expression vector for fusion protein, ECD of IL-13R{alpha}2 DNA was inserted into the following: A) pMAL-p2E vector down-stream from malE gene, which encodes maltose-binding protein (MBP); B) into pET44a vector, down-stream from NusA gene, which encodes NusA protein; or C) ECD{alpha}2-His tag insert cloned in pET24a expression vector for expression in E. coli. For expression in mammalian cells, 293T cells were cotransfected with retroviral vectors (pVPack-VSV-G, pVPack-GP, and pFB-Neo-ECD{alpha}2). After 48 h, the retrovirus-containing supernatant was collected, filtered, treated by DEAE-Dextran (1 µg/ml), and transferred to Hela cells. Transduced cells were selected with G418 (1000 µg/ml) for 7 days. For transient transfection, the plasmid for expression of ECD{alpha}2 (pcDNA6-ECD{alpha}2) was transfected into 293FT cells using FuGene 6 and cultured for 24 h. After culture medium was changed to serum-free DMEM medium, a further 48 h ofincubation was performed. Further purification of protein was performed as described in Materials and Methods.


Figure 2
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  		Figure 2. Expression, purification, and deglycosylation of ECD{alpha}2. SDS-PAGE analysis in 4–20% nonreducing gel was performed for ECD{alpha}2-EC (A and B) and ECD{alpha}2-His6 (D). A, lane 1) uninduced bacterial cell lysate; lane 2, post-IPTG bacterial cell lysate; lane 3, 2 µg purified ECD{alpha}2-EC. B, lane 1) nonreduced form; lane 2, reduced form; lane 3, molecular mass marker. C) Western blot analysis of ECD{alpha} 2 expression in supernatant of retrovirus-infected Hela cells. Lane 1, conditioned medium (CM) after first infection; lane 2, after second infection; lane 3, after third infection; lane 4, after fourth infection. D, lane 1) nontransfected 293FT cell conditioned medium; lane 2, pCDNA6-myc/His-ECD{alpha}2-transfected 293FT cell CM; lane 3, 2 µg of purified ECD{alpha}2-His6. E) ECD{alpha}2-His6 was incubated with control (phosphate buffer), PNGase F, or O-glycosidase under non- or denaturing conditions. Samples were analyzed by Western blotting.

To obtain posttranslationally modified protein, we expressed ECD{alpha}2 in Hela cells by retroviral transduction. As shown in Fig. 2C , expression of ECD{alpha}2 was confirmed from supernatant of cell culture. To obtain high levels of ECD{alpha}2 expression, retrovirus infection into the cells was repeated two to four times. The maximum expression level of ECD{alpha}2 was observed after third infection of ECD{alpha}2 gene-containing retrovirus. The expression level of ECD{alpha}2 in third infection when cells were cultured under serum free condition for 48 h was {approx}0.5 µg/ml. We next expressed and purified ECD{alpha}2-His6 in 293FT cells by transient transfection. As shown in Fig. 2D, a highly efficient expression of ECD{alpha}2 was obtained in culture medium. The yield of final product (ECD{alpha}2-His6) was 200 µg from 200 ml culture. SDS-PAGE analysis showed that molecular mass of ECD{alpha}2-His6 was {approx}58 kDa under nonreducing condition.

Deglycosylation of ECD{alpha}2 produced in 293FT cells
To investigate whether the molecular mass discrepancy between ECD{alpha}2-EC purified from E. coli BL21 cells (42 kDa) and ECD{alpha}2-His6 (58 kDa) is due to glycosylation, ECD{alpha}2-His6 was incubated with PNGase F or O-glycosidase, and Western blot analysis was performed. As shown in Fig. 2E , the molecular mass of ECD{alpha}2-His6 returned to 42 kDa after incubation with PNGase F under the denaturing condition. In contrast, O-glycosidase incubation did not affect the molecular mass of ECD{alpha}2-His6, indicating that glycosylation of ECD{alpha} 2-His6 is N linked.

ECD{alpha}2 inhibits TF-1 proliferation and protein synthesis induced by IL-13
It is known that TF-1 erythroleukemia cells proliferate and synthesize protein in response to IL-13 (21 , 22) . We have previously demonstrated that IL-13E13K, an antagonist of IL-13, can inhibit IL-13-induced proliferation and protein synthesis of TF-1 cells (23) . We therefore determined the effect of ECD{alpha}2 on IL-13-induced proliferation of TF-1 cells. Both ECD{alpha}2 produced in E. coli and 293FT cells inhibited the mitogenic activity of IL-13 (Fig. 3 A, B) and IL-13-induced protein synthesis (Fig. 3C ). This inhibition of IL-13-induced protein synthesis appeared to be concentration dependent (Fig. 3D ). Interestingly, 293FT-derived ECD{alpha}2-His6 was better in blocking the effect of IL-13-induced proliferation compared withECD{alpha}2 derived from E. coli (Fig. 3A, B ).


Figure 3
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  		Figure 3. Inhibition of IL-13-induced proliferation and protein synthesis in TF-1 cells by ECD{alpha}2 proteins. A) TF-1 cells were cultured with various concentrations of ECD{alpha}2-EC (square) or IL-13 (0–100 ng/ml) for 48 h in the presence (triangle) or absence (rhombus) of 1 µg/ml ECD{alpha}2-EC. B) TF-1 cells were incubated with various concentrations of IL-13 (0–100 ng/ml) in the absence (circle), presence of ECD{alpha} 2/Fc (triangle) or ECD{alpha}2-His6 (square) 100 ng/ml. C) Cells were cultured with various concentrations of IL-13 (0–100 ng/ml) in the presence or absence of 100 ng/ml ECD{alpha} 2-His6. D) Cells were cultured with or without 10 ng/ml IL-13 in the presence or absence of 12.5 or 25 ng/ml ECD{alpha}2-His6. Data are presented as the percentage of count without IL-13 stimulation (as 100%) and average of quadruplicate samples. Error bars are SD within a data set. Experiments were repeated 2–3 times.

ECD{alpha}2 specifically inhibits IL-13-mediated signal transduction
IL-13 has been shown to activate two intracellular signaling pathways: PI3K pathway that promotes cell growth in some cell types and JAK-STAT pathway (1 , 2) . After phosphorylation of JAKs, STAT6 is phosphorylated and activated when stimulated by IL-13 or IL-4, which in turn induces the expression of responsive genes (3) . We therefore tested whether ECD{alpha}2 could inhibit the IL-13- and IL-4-induced signaling. THP-1 human monocytic cell and COS-7 fibroblast cell lines were used for this purpose, since both cell lines have been shown to express IL-13R{alpha}1 and IL-4R{alpha} chains (20) . ECD{alpha}2-EC inhibited IL-13-induced STAT6 phosphorylation in a dose-dependent manner in both cell lines (Fig. 4 A). As shown in Fig. 4B , ECD{alpha}2-His6 also inhibited IL-13-induced STAT6 phosphorylation. However, the inhibitory effect of ECD{alpha}2-His6 on STAT6 phosphorylation was {approx}10-fold higher compared with ECD{alpha}2-EC. In sharp contrast, IL-4-induced STAT6 phosphorylation was not inhibited by ECD{alpha}2-His6 even though the concentration of ECD{alpha}2-His6 used in this assay was eight times higher than in IL-13-induced STAT6 phosphorylation assay (Fig. 4C ). An equal concentration (12.5 ng/ml) of ECD{alpha}2-His6 caused {approx}50% inhibition of IL-13-induced STAT6 phosphorylation (Fig. 4D ). This inhibitory efficiency is similar to inhibition of IL-13-induced TF-1 proliferation by ECD{alpha}2-His6 (Fig. 3B ).


Figure 4
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  		Figure 4. Effect of ECD{alpha}2 on IL-13- or IL-4-induced STAT6 phosphorylation in THP-1 and COS-7 cell lines. Cells were incubated with 50 ng/ml IL-13 or IL-4 in the presence of 0, 500, and 2000 ng/ml of ECD{alpha}2-EC (A) or 0, 25, 50, and 100 ng/ml ECD{alpha}2-His6 (B) for 15 min. C) Cells were incubated with various concentrations (0–100 ng/ml) of IL-13 or IL-4 with 12.5 or 100 ng/ml ECD{alpha}2-His6, respectively. D) Band intensity was measured using National Institutes of Health-Image 1.67 to determine the inhibitory effect of ECD{alpha}2-His6 on IL-13-induced STAT6 phosphorylation. E) Cells were incubated with 50 ng/ml IL-13 in the presence of 0, 50, or 500 ng/ml ECD{alpha}2-His6 that was or was not deglycosylated by PNGase F. Cells were lysed, and Western blotting analysis was performed.

Although the inhibitory effect of ECD{alpha}2-His6 on IL-13-induced TF-1 proliferation was better compared with ECD{alpha}2-EC (Fig. 3A, B ), it was not clear whether this was due to posttranslational glycosylation of ECD{alpha}2-His6 or simply different folding of ECD{alpha}2 derived from E. coli. To differentiate between these two possibilities, we examined the effect of glycosylated and deglycosylated ECD{alpha}2-His6 on IL-13-induced STAT6 phosphorylation in THP-1 cells. IL-13 induced phosphorylation of STAT6; however, in the presence of 500 ng/ml ECD{alpha}2-His6, complete inhibition of IL-13-induced STAT6 phosphorylation was observed (Fig. 4E , lane 4). An equal concentration of ECD{alpha}2-His6 preincubated with PNGase F caused only partial inhibition of STAT6 pohosphorylation (Fig. 4E , lane 8). The existence of PNGase F itself did not affect the inhibitory effect of ECD{alpha}2-His6 on IL-13-induced STAT6 phosphorylation (Fig. 4E , lane 6). These results indicate that glycosylation of ECD{alpha}2 is essential for its optimum inhibitory activity on IL-13-induced STAT6 phosphorylation.

ECD{alpha}2 inhibits IL-13-induced and constitutive STAT6 phosphorylation in Hodgkin’s lymphoma cell lines
We examined the effect of ECD{alpha}2 in IL-13-induced STAT6 phosphorylation in Hodgkin’s lymphoma (HL) Reed-Sternberg cell lines (L591 and L1236). These cell lines have been shown to secrete IL-13 and respond to both IL-4 and IL-13. As ECD{alpha}2 inhibited IL-13-induced signaling and proliferation of TF-1 cells, we hypothesized that it would also inhibit IL-13 signaling in HL cells. As shown in Fig. 5 A, both IL-13 and IL-4 induced phosphorylation of STAT6 in L591 cells. IL-13-stimulated phosphorylation was completely blocked by ECD{alpha} 2-His6. In contrast, ECD{alpha}2-His6 did not block IL-4-induced STAT6 phosphorylation. L1236 cells showed constitutive phosphorylation of STAT6, which was blocked by ECD{alpha}2-His6 in a dose-dependent manner (Fig. 5B ).


Figure 5
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  		Figure 5. Effect of ECD{alpha}2 on IL-13-induced STAT6 phosphorylation in Hodgkin’s lymphoma cell lines. A) L591 cells were incubated with 50 ng/ml IL-13 or IL-4 in the presence or absence of 200 ng/ml ECD{alpha}2-His6 for 15 min. B) L1236 cells were incubated with 50 ng/ml IL-13 with various concentrations (0–200 ng/ml) of ECD{alpha}2-His6. Cells were lysed, and Western blot analysis was performed.

Neutralization of IL-13 cytotoxin-mediated cytotoxicity by ECD{alpha}2
We have previously demonstrated that a chimeric fusion protein composed of IL-13 and a truncated form of Pseudomonas exotoxin (IL13-PE38) is highly cytotoxic to IL-13R-positive tumor cells in vitro and in vivo (24 25 26 27) . IL13-PE38 mediates cytotoxicity primarily through binding to IL-13R{alpha}2 chain and followed by receptor internalization (13) . To determine whether ECD{alpha}2 could inhibit IL13-PE38-induced cytotoxicity, IL-13R{alpha}2-positive U251 and PM-RCC cell lines were incubated with IL13-PE38 in the absence and presence of ECD{alpha}2. As shown in Fig 6 A, IL13-PE38 mediated cytotoxicity in a dose-dependent manner, and ECD{alpha} 2-His6 blocked this cytotoxicity in both cell lines. The blocking of cytotoxicity was observed in a concentration-dependent manner in both U251 and PM-RCC cell lines (Fig. 6B ). As expected, commercially available ECD{alpha}2/Fc and IL-13 also blocked IL13-PE38-induced cytotoxicity; however, ECD{alpha}2-His6 appeared to be superior compared with ECD{alpha}2/Fc and IL-13.


Figure 6
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  		Figure 6. Effect of IL-13, ECD{alpha}2/Fc, and ECD{alpha}2-His6 on IL13-PE38-induced cytotoxicity in U251 and PM-RCC cell lines. Ten thousand cells/well were cultured in Leu-free medium containing various concentrations of IL-13 cytotoxin (IL13-PE38) and 100 ng/ml ECD{alpha}2-His6 (A) or 1 ng/ml IL13-PE38 with or without various concentrations of IL-13, ECD{alpha}2/Fc, or ECD{alpha}2-His6 (B) for 20–22 h before the addition of 1 µCi of [3H]Leu. Cells were then incubated for 4 h and harvested, and radioactivity was counted with a ß counter. Data are average of quadruplicate determinations, and error bars are SD within a data set. Experiments were repeated 2 times with similar results.

ECD{alpha}2 competes for the binding of radiolabeled IL-13
To elucidate the mechanism involved in superior neutralizing activity of ECD{alpha}2-His6 in IL-13 cytotoxin-mediated cytotoxicity, binding studies were performed using PM-RCC and U251 cell lines. As shown in Fig. 7 , ECD{alpha}2-His6, ECD{alpha}2/Fc, and IL-13 inhibited the binding of radiolabeled IL-13 on both cell lines. However, ECD{alpha}2-His6 showed superior blocking activity of radiolabeled IL-13 binding on PM-RCC and U251 cells. The EC50 (concentration causing 50% inhibition of 125I-interleukin-13 binding) of ECD{alpha}2-His6, ECD{alpha}2/Fc, and IL-13 in PM-RCC was 0.3, 2.2, and 9 nM, respectively, while it was 0.08, 0.5, and {approx}2 nM, respectively, in U251 cells. Thus, ECD{alpha}2-His6 appeared to show 6- to 7-fold better binding avidity compared with ECD{alpha}2/Fc to both cell lines.


Figure 7
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  		Figure 7. Competition for the binding of 125I-IL-13 by IL-13, ECD{alpha}2/Fc, and ECD{alpha}2-His6. PM-RCC or U251 cells (1x106) were incubated with 200 pM 125I-IL-13 with various concentrations of unlabeled ECD{alpha}2/Fc, ECD{alpha}2-His6, or IL-13. Data are percentage of cell-bound 125I-IL-13 without competitor, and error bars are SD of duplicated determinations. Experiments were repeated 2 times with similar results.

Glycosylated ECD{alpha}2 demonstrates better binding to IL-13
We determined whether the higher inhibitory effect of ECD{alpha}2-His6 on IL-13-induced STAT6 phosphorylation compared with deglycosylated form was due to better binding to IL-13. To do this, we established a sandwich ELISA that detects only IL-13R{alpha}2-bound IL-13. As shown in Fig. 8 , ECD{alpha}2-His6 demonstrated dramatically higher (>10-fold) binding avidity to IL-13 compared with deglycosylated form. However, at very high and saturating concentration of IL-13 (400 ng/ml), no difference in binding to ECD{alpha}2 was observed.


Figure 8
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  		Figure 8. Binding of IL-13 to glycosylated and deglycosylated ECD{alpha}2-His6.ECD{alpha}2-bound IL-13 was determined by sandwich ELISA. ECD{alpha}2-His6 (1 µg/ml) was preincubated with or without 0.5 U of PNGase F for overnight and mixed with serially diluted IL-13 (0–400 ng/ml) for 30 min at room temperature. Experiments were repeated two times with similar results.

DISCUSSION

The extracellular domain of IL-13R{alpha}2 chain may provide therapeutic benefit in various pathological conditions including neoplasia, pulmonary, and inflammatory diseases. The availability of natural and correctly folded ECD{alpha}2 would be extremely useful for treatment of these conditions.

We have optimized the expression and purification of IL-13R{alpha}2 extracellular domain in E. coli and 293FT mammalian cells. ECD{alpha}2 contains 12 cysteine residues representing 3.8% of total amino acid content with at least four residues predicted to form intramolecular disulfide-bonds. Because of such cysteine residues, it is often difficult to denature and accurately refold a protein, which leads to low yield and less than optimal biological activity. To overcome this limitation, we developed an expression system in which E. coli was transformed with constructs encoding fusion proteins comprised of ECD{alpha}2 and MBP or Nus A to enhance the expression and solubility of fusion proteins (Fig. 1) . Although both expression constructs resulted in good expression of fusion proteins, both proteins could not be recovered in soluble form (data not shown). We were, however, able to express ECD{alpha}2 in E. coli in inclusion bodies and recover the protein by denaturation in guanidine hydrochloride and refolding in urea on a Ni-NTA affinity column to obtain highly purified His-tagged ECD{alpha}2. As E. coli system lacks the machinery for posttranslation modification such as glycosylation, we also expressed ECD{alpha}2 in mammalian cells, which allowed investigation of biological activity of ECD{alpha}2 produced in prokaryotic and mammalian systems.

ECD{alpha}2 (ECD{alpha}2-His6) inhibited the biological activity of IL-13 as measured by several parameters including IL-13-induced TF-1 cell proliferation and protein synthesis, IL-13 binding to IL-13R-expressing cells, and IL-13-induced STAT6 phosphorylation. Interestingly, ECD{alpha}2-His6 produced in mammalian cells demonstrated higher activity compared with E. coli-derived ECD{alpha}2-EC in these assays. The molecular mass of mammalian cell-derived ECD{alpha}2 was much larger than the E. coli material. This is similar to other type I cytokine receptor subunits, the molecular mass of which is far greater than the theoretical masses calculated from the primary amino acid sequences alone. In many cases, this is due to glycosylation (28 , 29) . As ECD{alpha}2 molecule has four potential glycosylation sites, we determined whether ECD{alpha}2 is glycosylated and this glycosylation modulates its biological activity. Our results indicate that ECD{alpha}2-His6 is glycosylated, and this glycosylation is N linked. This conclusion was drawn from blocking N-linked glycosylation before expression. This was accomplished by expression of ECD{alpha}2 by pcDNA6-ECD{alpha}2-tranfected 293FT cells grown in the presence of tunicamycin, an inhibitor of glycosylation. However, protein expression was very low, and purification did not yield measurable amounts of ECD{alpha}2. Based on this observation, it was concluded that N-linked glycosylation is critical for the expression and stabilization of ECD{alpha}2. Next, we examined the effect of glycosylation on biological activity of ECD{alpha}2 by enzyme-mediated deglycosylation. Interestingly, deglycosylated ECD{alpha}2-His6 showed lower activity in blocking IL-13-induced STAT6 phosphorylation in THP-1 cells compared with glycosylated protein. These results suggest that the N-linked glycosylation is required to maintain the integrity or binding of ECD{alpha}2 to IL-13. This is consistent with known observation that N-linked glycosylation affects protein folding and trafficking in other cytokine receptors (30 31 32) .

It is of interest to note that when we compared the biological activity of ECD{alpha}2-His6 and commercially available ECD{alpha}2/Fc fusion protein for their abilities to block IL-13 signaling, IL-13 binding to cell surface, and neutralization of cytotoxicity of IL-13 cytotoxin, ECD{alpha}2-His6 demonstrated higher potency. The reason for this different activity is not completely clear. It is possible that fusion with Fc portion of IgG causes steric hindrance in binding of ECD{alpha}2 to IL-13. ECD{alpha}2/Fc appeared as a disulfide-linked homodimeric protein in SDS-PAGE analysis under nonreducing condition, while ECD{alpha}2-His6 appeared as a monomer, suggesting that the monomer form may be more biologically active. Taken together, it is clear that heavy glycosylation of ECD{alpha}2-His6 is advantageous in blocking IL-13 activities. This characteristic is extremely valuable, as glycosylated molecules tend to have long serum half-life. Further studies will examine biological activity of this protein in vivo for therapeutic purposes.

The detailed mechanism by which IL-13R{alpha}2 regulates IL-13 activities is not completely understood. It has been suggested that IL-13R{alpha}2 on the cell surface sequesters IL-13 from IL-13R{alpha}1/interleukin-4R{alpha} complex. As IL-13R{alpha}2 has high binding affinity to IL-13 and slow dissociation, this receptor acts as a negative regulator of IL-13 by competing for IL-13 binding to IL-13R{alpha}1. In addition, IL-13R{alpha}2 internalizes after binding to IL-13 (12) , decreasing availability of IL-13 on the cell surface and thus suppressing IL-13 biological activities. Since ECD{alpha}2 inhibited the mitogenic activity of IL-13 and protein synthesis in TF-1 cells expressing IL-13R{alpha}1 and IL-4R{alpha} chains, these results indicate that ECD{alpha}2 blocks the IL-13 binding to IL-13R{alpha}1/interleukin-4R{alpha} complex, as well. Based on these studies, we predict that IL-13R{alpha}2 interacts with {alpha}-helix D or close to {alpha}-helix D of IL-13 and causes steric hindrance in physical interaction between IL-13 and IL-13R{alpha}1, as {alpha}-helix D is thought to interact with IL-13R{alpha}1 subunit (20 , 23) .

IL-13R{alpha}2 chain has been shown to be over-expressed in various human solid cancer cell lines and tissues derived from glioma, head, and neck cancer, AIDS-associated Kaposi’s sarcoma, and renal cell carcinoma (24 25 26 27) . Although the significance of its expression is not clear, the expression of this receptor is up-regulated by TNF-{alpha}, IFN-{gamma} in fibroblasts, and adrenomedulin in cancer cells (33 34 35) . IL-13R{alpha}2 is naturally over-expressed in fibroblasts derived from various pathological conditions such as pneumonia, infection, and pulmonary and liver fibrosis (36 37 38) . In the murine system, the extracellular domain of IL-13R{alpha}2 has been shown to be secreted in plasma and urine of mice (39) . In humans, the ECD of IL-13R{alpha}2 chain has been shown to be elevated in serum of patients with schistosomiasis mansoni infection (40) . Although the significance of IL-13R{alpha}2 cleavage is not clear, ECD{alpha}2 has been shown to be a dominant negative decoy receptor for IL-13. Whether ECD of IL-13R{alpha}2 acts as a carrier protein to deliver IL-13 to target organs or it acts to quench the IL-13 signaling to maintain the homeostasis in the body is not completely clear. Availability of ECD{alpha}2 offers opportunities to examine these possibilities.

Recent studies have identified a possible role of ECD IL-13R{alpha}2 in the attenuation of various diseases. It is reported that administration of soluble IL-13R{alpha}2-Fc attenuated IL-13-induced airway hyperresponsiveness in murine models of allergic inflammation (41) . Since IL-13 is an autocrine growth factor for HL cells and ECD{alpha}2-His6 blocked the IL-13-induced and constitutive STAT6 phosphorylation in HL cells, it is possible that ECD{alpha}2-His6 may have a significant role in the therapy of HL. Taken together, ECD{alpha}2-His6 may be a useful for therapy of pathological conditions in which IL-13 plays a major role such as asthma, fibrosis, infection, and cancer. Further studies should be performed to examine its biological activities in vivo.

ACKNOWLEDGMENTS

We thank Pamela Dover for technical support and Neel Talwar (summer student) for help in purification of proteins, and NCI Fellows Editorial Board for editorial assistance. We are grateful to Dr. Wendy C. Weinberg for review and helpful suggestions. These studies were conducted as part of a collaboration between the Food and Drug Administration (FDA) and NeoPharm, Inc., under Co-operative Research and Development Agreement. This article does not present an official position of the FDA.

Received for publication March 2, 2006. Accepted for publication June 2, 2006.

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