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N-linked glycosylation of IL-13R2 is essential for optimal IL-13 inhibitory activity

Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, 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. E-mail: raj.puri{at}fda.hhs.gov

SPECIFIC AIMS

As interleukin-13 (IL-13) is a central mediator of allergic, pulmonary, parasitic and neoplastic diseases, the aim of this study was to develop a novel inhibitor of human IL-13. The soluble form of IL-13R2 (ECD2) binds IL-13 with high affinity and may serve as a potent inhibitor of IL-13. As IL-13R2 has 12 cysteine residues, it is difficult to express in Escherichia coli (E. coli); therefore, the other aim of this study was determine whether glycosylation of soluble protein might have an essential role in its biological activities.

PRINCIPAL FINDINGS

1. Recombinant protein expression and purification
Recombinant extracellular domain of IL-13R2 (ECD2) was expressed in Escherichia coli and purified from inclusion bodies (ECD2-EC) or expressed in mammalian 293FT cells transfected with ECD2 expression vector and purified from culture media (ECD2-His6). Visual inspection of bands suggested purity of both proteins to be >99%.

2. ECD2 blocks proliferative activity of IL-13
We determined effect of ECD2 on IL-13-induced proliferation of TF-1 cell line. Both ECD2-EC and ECD2-His6 inhibited the mitogenic activity of IL-13 and IL-13-induced protein synthesis in a concentration-dependent manner. ECD2-His6 appeared to be better in blocking the activity of IL-13-induced proliferation compared with ECD2-EC.

3. ECD2 specifically inhibits IL-13-mediated signal transduction in immune, fibroblast, and Hodgkin’s lymphoma cell lines
STAT6 phosphorylation is an early cellular response induced by IL-13 or IL-4 and is responsible for the biological effect mediated by these cytokines. We therefore tested whether ECD2 could inhibit the IL-4 and IL-13-induced signaling. THP-1 human monocytic cell, COS-7 fibroblast cell, and Hodgkin’s lymphoma (HL) Reed-Sternberg (L591 and L1236) cell lines were used for this purpose. ECD2-His6 inhibited IL-13-induced STAT6 phosphorylation in a dose-dependent manner in all four cell lines (Fig. 1 ). An equal concentration of ECD2-His6 caused 50% inhibition of IL-13-induced STAT6 phosphorylation. In sharp contrast, IL-4-induced STAT6 phosphorylation was not attenuated by ECD2-His6.

4. N-linked glycosylation of ECD2 is essential for optimal inhibition of IL-13-induced STAT6 phosphorylation
To determine whether better inhibitory activity of ECD2-His6 over the ECD2-EC was due to posttranslational glycosylation of ECD2-His6, we examined the effect of glycosylated and deglycosylated ECD2-His6 on IL-13-induced STAT6 phosphorylation in THP-1 cells. ECD2-His6 preincubated with N-glycosidase PNGase F caused only partial inhibition of STAT6 phosphorylation, whereas equal concentration of glycosylated ECD2-His6 completely blocked STAT6 phosphorylation (Fig. 1) .

5. ECD2 can neutralize the cytotoxicity mediated by IL-13 cytotoxin
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. IL13-PE38 mediates cytotoxicity primarily through binding to IL-13R2 chain and followed by receptor internalization. To determine whether ECD2 could inhibit IL13-PE38-induced cytotoxicity, IL-13R2-positive U251 and PM-RCC cell lines were incubated with IL13-PE38 in the absence and presence of ECD2. ECD2-His6 blocked the cytotoxicity of IL13-PE38 in a concentration-dependent manner in both U251 and PM-RCC cell lines. Commercially available ECD2/Fc fusion protein and IL-13 also blocked IL13-PE38-induced cytotoxicity; however, ECD2-His6 appeared to be superior compared with ECD2/Fc and IL-13.

6. ECD2 competes for the binding of radiolabeled IL-13
To elucidate the mechanism involved in superior neutralizing activity of ECD2-His6 in IL-13 cytotoxin-mediated cytotoxicity, binding studies were performed using PM-RCC and U251 cell lines. ECD2-His6, ECD2/Fc, and IL-13 inhibited the binding of radiolabeled IL-13 on both cell lines (not shown). However, ECD2-His6 showed superior blocking activity of radiolabeled IL-13 binding on PM-RCC and U251 cells. The EC₅₀ (concentration causing 50% inhibition of ¹²⁵I-IL-13 binding) of ECD2-His6, ECD2/Fc, and IL-13 in PM-RCC was 0.3, 2.2, and 9 nM, respectively, while it was 0.08, 0.5, and 2 nM, respectively, in U251 cells. Thus, ECD2-His6 appeared to show 6- to 7-fold better binding avidity compared with ECD2/Fc to both cell lines.

CONCLUSIONS AND SIGNIFICANCE

The IL-13R2 deletion mutein of cytoplasmic and transmembrane domain (ECD2) was expressed in E. coli and mammalian cells and purified from inclusion bodies and culture media, respectively. ECD2 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. We have optimized the expression and purification of IL-13R2 extracellular domain. In addition, as the E. coli system lacks the machinery for posttranslation modification, we also expressed ECD2 in mammalian cells, which allowed investigation of biological activity of ECD2 produced in prokaryotic and mammalian systems.

We demonstrate that ECD2 (ECD2-His6) inhibits 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 (Fig. 2 ). It is of interest to note that ECD2-His6 produced in mammalian cells demonstrated higher activity compared with E. coli-derived ECD2-EC in these assays. The molecular mass of mammalian cell-derived ECD2 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. As ECD2 molecule has four potential glycosylation sites, we determined whether ECD2 is glycosylated and this glycosylation modulates its biological activity. Our results indicate that ECD2-His6 is N-linked glycosylated, and this conclusion was drawn from blocking N-linked glycosylation before expression. This was accomplished by expression of ECD2 by pcDNA6-ECD2-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 ECD2. Based on this observation, it was concluded that N-linked glycosylation is critical for the expression and stabilization of ECD2. In addition, deglycosylated ECD2-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 ECD2 to IL-13. This is consistent with known observation that N-linked glycosylation affects protein folding and trafficking in other cytokine receptors. Furthermore, it is suggested that the discrepant activities between E. coli-derived ECD2 (ECD2-EC) and mammalian cell-derived ECD2 (ECD2-His6) are contributed to N-linked glycosylation of ECD2.

The detailed mechanism by which IL-13R2 regulates IL-13 activities is not completely understood. It has been suggested that IL-13R2 on the cell surface sequesters IL-13 from IL-13R1/interleukin-4R complex. As IL-13R2 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-13R1. In addition, IL-13R2 internalizes after binding to IL-13, decreasing availability of IL-13 on the cell surface and thus suppressing IL-13 biological activities. Since ECD2 inhibited the mitogenic activity of IL-13 and protein synthesis in TF-1 cells expressing IL-13R1 and IL-4R chains, these results indicate that ECD2 blocks the IL-13 binding to IL-13R1/interleukin-4R complex, as well. Based on these studies, we predict that IL-13R2 interacts with -helix D or close to -helix D of IL-13 and causes steric hindrance in physical interaction between IL-13 and IL-13R1, as -helix D is thought to interact with IL-13R1 subunit.

Since IL-13 is an autocrine growth factor for HL cells and ECD2-His6 blocked the IL-13-induced and constitutive STAT6 phosphorylation in HL cells, it is possible that ECD2-His6 may have a significant role in the therapy of HL. Taken together, ECD2-His6 may be 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.

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of ECD2 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, or 2000 ng/ml of ECD2-EC (A) or 0, 25, 50, or 100 ng/ml ECD2-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 ECD2-His6, respectively. D) Band intensity was measured using National Institutes of Health-Image 1.67 to determine the inhibitory effect of ECD2-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 ECD2-His6 that was or was not deglycosylated by PNGase F. Cells were lysed, and Western blotting analysis was performed.

View larger version (26K): [in this window] [in a new window]   Figure 2. Schematic diagram of the experimental findings. As glycosylated ECD2 binds to IL-13 with high affinity, it effectively blocks IL-13-induced STAT6 phosphorylation, cell proliferation, protein synthesis, and IL-13 cytotoxin-induced cytotoxicity. N-linked glycosylation is critical for optimal inhibitory effect as deglycosylated ECD2 demonstrated little effect on IL-13-induced STAT6 phosphorylation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5995fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5995fjev1 20/13/2378    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kioi, M. Articles by Puri, R. K. Search for Related Content PubMed PubMed Citation Articles by Kioi, M. Articles by Puri, R. K.