Structural and Functional Properties of IL-4δ2, an Alternative Splice

Structural and Functional Properties of IL-4δ2, an Alternative Splice Variant of Human IL-4 Anatoly M. Vasiliev,† Raisa N. Vasilenko,† Nataly L. Kulikova,† Sergey M. Andreev,‡ Irina O. Chikileva,† Galina Yu. Puchkova,† Igor V. Kosarev,† Anna V. Khodyakova,† Valentin S. Khlebnikov,† Leonid R. Ptitsyn,† Grygory Ya. Shcherbakov,† Vladimir N. Uversky,*,†,§,| Lawrence M. DuBuske,⊥ and Vyacheslav M. Abramov† Institute of Immunological Engineering, 142380 Lyubuchany, Moscow Region, Russia, State Scientific Center, Institute of Immunology of Russian Ministry of Health, Kaschirskoe Schosse, 24/2, 115478 Moscow, Russia, Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia, Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, and Immunology Research Institute of New England, 955 South Street, Fitchburg, Massachusetts 01420 Received November 21, 2002; Revised Manuscript Received March 28, 2003

Structural and functional properties of recombinant IL-4δ2, a naturally occurring splice variant of human IL-4 with a deletion of the loop region 22-37, have been analyzed. IL-4δ2 has R-helical structure and most likely preserves the “up-up-down-down” topology typical of the four-helix-bundle cytokines. IL-4δ2 interacts specifically with the R chain of IL-4R and competes effectively with IL-4 for the common binding sites. Thus, IL-4δ2 may act as a regulator of the cytokine net, being the natural antagonist of IL-4. Keywords: interleukin 4 • interleukine receptor • splice variant • cytokine • four-helix-bundle cytokine

Interleukin-4 (IL-4)1 is a pleiotropic cytokine produced mainly by T-helper lymphocytes type 2 (TH2) that is involved in the regulation of different biological processes.1,2 The important role of IL-4 is the induction of immunoglobulin class switch in the B-cells expressing IgM into IgG4 and IgE,3 and the upregulation of the expression of the IgE low affinity receptor (CD23) on mast cells and B cells.4 For these reasons, IL-4 constitutes the basis of the allergic response and, therefore, it is considered to be a prime target for the design of anti-allergic drugs. In this connection, development of the effective artificial IL-4 antagonists is considered as a perspective direction. The design of the two helix coiled coil peptide mimetics IL4, which are able to recognize and bind its high affinity receptor IL-4R R-chain (IL-4RR), has been described.5 In this study, the leucine-zipper domain of the yeast transcription factor GCN4 was used as a scaffold into which the putative binding epitope of IL-4 for IL-4RR was transferred in a stepwise manner, using computer-aided molecular modeling. The resulting molecules bind IL-4RR with affinities ranging from 2 mM to 5 µM, * To whom correspondence should be addressed. Vladimir N. Uversky, Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064. Tel: (831) 459-2915. Fax: (831) 459-2935. E-mail: [email protected]. † Institute of Immunological Engineering. ‡ State Scientific Center, Institute of Immunology of Russian Ministry of Health. § Institute for Biological Instrumentation, Russian Academy of Sciences. | Department of Chemistry and Biochemistry, University of California, Santa Cruz. ⊥ Immunology Research Institute of New England. 10.1021/pr025586y CCC: $25.00

 2003 American Chemical Society

depending on the fraction of the IL-4 binding site incorporated and on their stability.5 Mutations in the C-terminal region of IL-4 produce mutants that bind to the IL-4RR with high affinity, but do not induce cellular responses.5 A murine IL-4 mutant with C118 deletion (IL-4R antagonist) inhibited IL-4-induced STAT6 phosphorylation, as well as IL-4-induced IgE production in vitro. Furthermore, the administration of murine IL-4R antagonist during allergen challenge inhibited specifically the development of allergic airway eosinophilia and airway hyperresponsiveness (AHR) in mice previously sensitized with allergen. The inhibitory effect on airway eosinophilia and AHR was associated with reduced levels of IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid. These observations demonstrate the therapeutic potential of IL-4 mutant protein receptor antagonists that inhibit IL-4 in the treatment of allergic asthma.6 Studies on the regulation of IL-4 activity have been also focused on the analysis of promoters, enhancers, and negative regulatory elements within the IL-4 gene.7-11 A potential mechanism for the regulation of human IL-4 activity was described, in which mRNA is alternatively spliced from the IL-4 gene.12,13 Alternative splicing of mRNA encoded by a single gene is a regulated mechanism14 that controls gene expression.15 Alternative splicing of mRNA generates structural and functional diversity of proteins.16,17 Protein variants formed by alternative splicing may be differentially expressed in certain tissues,18,19 during different stages of development18,20,21 and in different states of cell activation.22 For example, human IL-4δ2, an IL-4 splice variant that omits IL-4 exon 2 coding for the 22Journal of Proteome Research 2003, 2, 273-281

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research articles 37 fragment, is preferentially expressed in the thymus and airways.23 IL-4δ2 inhibited IL-4-induced T-cell proliferation and was found to be specifically bound to the cell lines expressing IL-4R. Here, the unlabeled IL-4 inhibited the labeled IL-4δ2 binding to the receptors.24 In B-cells, IL-4δ2 blocks IL-4induced IgE synthesis and CD23 expression. IL-4δ2 mRNA was found in peripheral blood and CD3+ -T-cells.25 These results show that an alternative splicing may be used by nature to create an antagonist of human IL-4 that is preferentially expressed in certain tissues. Structurally, IL-4 belongs to the group of short-chain fourhelix-bundle cytokines.26 This protein family shows the typical “up-up-down-down” topology of R-helices, which makes long connecting loops necessary between helices running in the same direction. IL-4 contains three disulfide bridges, Cys3Cys127, Cys24-Cys65, and Cys46-Cys99, and no free cysteines. The N- and C-termini of the protein are connected by the Cys3Cys127 disulfide bond. Cys24, being located near the C-terminal end of helix A, forms a disulfide bond with Cys65, whereas disulfide bridge between Cys46 and Cys99 connects helix B to the CD loop. It has been established that in IL-4 residues 2830 and 106-108 form a two-stranded antiparallel β-sheet, which is expected to be absent in IL-4δ2 due to the loss of the 22-37 fragment. IL-4, as many other cytokines, evokes a cellular response by promoting the formation of a heterodimeric receptor complex in the plasma membrane.27-29 The high-affinity binding of human IL-4 to its cellular receptor30 is mediated nearly exclusively by the receptor R chain.31 The affinity is only marginally increased if both the R chain and γc, the second functional receptor chain, are present.27 Antagonistic IL-4 variants retain the high-affinity binding to the R chain but are deficient in γ chain binding.32-34 The structure of receptorunbound human IL-4 has been determined in crystal and in solution.35-38 The understanding of molecular recognition between IL-4 and IL-4RR has greatly been increased by the X-ray analysis of the complex between IL-4 and the extracellular part of the IL-4RR chain (IL-4BP).39 Numerous mutants of the human IL-4 protein have been generated and characterized to identify residues important for receptor binding.32-34,40 From these studies, it was deduced that the highest energetic contributions upon binding to the receptor R chain come from Glu9 and Arg88, with a lesser contribution from Arg85. This suggested that contrary to the human growth hormon, the main binding epitops of IL-4 are located within the helices A and C, rather than within the helix D. Structural and biological properties of IL-4δ2 are still purely understood. However, a theoretical model of IL-4δ2 has been suggested according to which the deletion of the loop region 22-37 leads to the transformation of IL-4 with an “up-updown-down” structural pattern to the structure with a “downup-down-down” structural pattern.41 In the present paper, the structural properties of IL-4δ2 were studied by CD, FTIR, chemical modification, and radioligand analysis. It has been shown that IL-4δ2 may be IL-4 antagonist and agonist as well. Molecular model of IL-4δ2 has been elaborated, in which protein preservs the “up-up-down-down” topology typical of the four-helix-bundle cytokines.

Materials and Methods Materials. Production of Recombinant Proteins. Recombinant IL-4 and IL-4δ2 have been isolated from E. coli as described.42,43 274

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Protein Concentration. The protein concentration has been determined spectrophotometrically using the calculated values for the excinction coefficients (0.54 and 0.6 for IL-4 and IL4δ2, respectively). Human Thymocytes. Human Thymocytes have been isolated from the thymus fragments removed during the surgeries, related to the planned treatment of children with the innate heart disease (Cardiological Center, Moscow, Russia). Cells have been cultivated at 37 °C in the presence of 5% CO2, in RPMI1640 medium containing 5-10% fetal calf serum. Cell Line. Human thymic epithelial cell line VTEC2.HS transformed with virus SV-40 was supplied by the National Scientific CentresThe Institute of Immunology, Ministry of Public Health Service of Russia, Moscow. The cells were cultured in reach RPMI-1640 medium as a suspensioned line.44

Methods Circular Dichroism (CD). CD spectra were recorded on a Jasco J-715 spectropolarimeter using a cell with path length of 0.1 cm. Protein concentration was 0.26 mg/mL. The results were expressed as molar ellipticity, [θ] (deg cm2 dmol-1), assuming mean amino acid residue weight (MRW) of 117.4. The molar ellipticity was determined as [θ] ) (θ × 100MRW)/ (cl), where c is the protein concentration in mg/mL, l is the light path length in centimeters, and θ is the measured ellipticity in degrees. The instrument was calibrated with Jasco standard nonhydroscopic ammonium (+)-10-camphorsulfonate, assuming [θ]290.5 ) 7910 deg cm2 dmol-1.45 R-Helical content has been estimated as described earlier.46 FTIR Spectra. The FTIR spectra were collected on a Nicolet 800SX FTIR spectrophotometer equipped with an MCT detector. The IRE (72 × 10 × 6 mm, 45° germanium trapezoid) was held in a modified SPECAL out-of-compartment ATR apparatus. The hydrated thin-films were prepared as described previously.47,48 Typically, 1024 interferograms were co-added at 4 cm-1 resolution. Data analysis was performed with GRAMS32 (Galactic Industries). Secondary structure content was determined from curve fitting to spectra deconvoluted using second derivatives and Fourier self-deconvolution to identify component band position. Electrophoresis. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli.49 The gel was stained with Coomassie Brilliant Blue R250 or subjected to immunoblotting. Chemical Modification of Proteins. The number of free sulfhydril groups in a protein have been determined using an Ellman reaction, i.e., absorbance of 2-nitro-5-benzoate at 412 nm have been measured. (The molar extinction coefficient of this reagent was taken to be 13 600.)50 Hydrolysis of the peptide bond in the vicinity of the methionine carboxyl group has been accomplished using the cyanogen bromide-cleavage.51 To this end, the lyophilized IL4δ2 sample was dissolved in 150 µL 70% formic acid, then 20 µL of freshly prepared cyanogen bromide solution (50 mg/mL in 70% formic acid) were added. The reaction mixture was incubated in dark at room temperature for 18 h, then 10-fold diluted by water and lyophilized. Products were analyzed using reducing and nonreducing SDS-PAGE with subsequent Nterminal analysis.52 Quantitative cleavage of peptide bonds adjacent to tryptophanyl residues have been accomplished using the cyanogen bromide-cleavage in heptafluorobutyric acid.53 The lyophilized IL-4δ2 sample was dissolved in a mixture of 380 µL 90% formic

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IL-4δ2 Structure and Function

Figure 1. Primary and secondary structure of human IL-4 (A) and its natural splice vartiant IL-4δ2 (B). Cylinders represent R-helical regions, whereas arrows represent extended β-sheets. The numbering of amino acid residues in IL-4δ2 was kept identical to IL-4. Positioning of secondary structure segments within the IL-4 sequence is based on the crystallographic 3D-structure of the protein.35-40 In IL-4δ2, residues 22-37 of IL-4 are omitted. This missing fragment contains short R-helix H2 (residues 23-26) and short β-strand B1 (residues 27-29). IL-4δ2 lacks Cys26, which is forming a disulfide bridge with Cys65 in IL-4.

acid and 500 µL heptafluorobutyric acid, then 120 µL of freshly prepared cyanogen bromide solution (50 mg/mL in 90% formic acid) were added. The reaction mexture was incubated in dark at room temperature for 24 h. Unreacted reagent and solvents were evaporated in N2-stream. The remainder was diluted 10fold by water and lyophilized. Products were analyzed using reducing and nonreducing SDS-PAGE with subsequent Nterminal analysis. The cleavage of peptide bonds adjacent to histidine residues have been accomplished using the succinimide bromatecleavage.54 Radiolabeling of Proteins. Radiolabeling of proteins was carried out using Iodogen technique.55 The specific activity of 125I-labeled IL-4δ2 and IL-4 was 0.1 mCi/µg protein. Binding Assay of 125I-Labeled Proteins to the Cells. To study the reception of proteins VTEC2.H/S cells were cultivated in 6-well plates (Nunc) for 72 h. The cells were collected and washed 3× with culture medium, reaching concentrations up to 107 cells per ml. Different doses of 125I-labeled proteins were inserted into the cell culture and incubated for 1 h at 4 °C. The volume of the incubated mixture was of 300 µL. Following the incubation, 50 µL of the cellular mixture was layered on 250 µL of dibutylphthalat-bis2-ethylhexyl-phthalat (v/v) mixture and centrifuged for 2 min at 14 000 g. Supernatant radioactivity was measured using 1275MINI GAMMA (LKB, WALLAC). For the determination of nonspecific binding of 125Ilabeled proteins, a 1000-fold surplus of unlabeled proteins was

used. The results were expressed as the mean cpm (specific binding) in which nonspecific binding level was subtracted. Cell Culturing for Proliferation Research. As it had been described earlier,56 the thymocytes were cultured on flat 96well microplates (Nunc) at the concentration of 5 × 10-5 cells per well in 200 µL of RPMI-1640 medium containing 2 nM L-glutamin, 10% FCS and 100 U/ml penicillin and streptomycin (all reagents were from ICN). Concanavalin A (ConA 0.5-1 µg/ mL, Sigma) or phytohemagglutinin (PHA 0.5-1 µg/mL, ICN) were added directly into the wells for cell culture initiation. Tritium labeled thymidine was added into the cultures in 60 h after the mitogen addition. 72 h after the culture initiation, the cells were washed and transferred onto the glass filter paper and counted using fluid scintillation counter. All of the cultures were thrice repeated. The results were presented as a mean impulse number per minute ( SD (standard deviation). Three-Dimensional Modeling. Molecular model of IL-4δ2 was constructed using a homology modeling (threading) performed with the SWISS-MODEL Protein Modeling Server program package and the Swiss-Pdb Viewer 3.757,58. The package 3D-PSSM was used to predict the elements of secondary structure of IL-4d2.59

Results Structural Characterization of IL-4 and IL-4δ2. Primary Structure Analysis. Figure 1 represents the distribution of secondary structure elements within the amino acid sequences Journal of Proteome Research • Vol. 2, No. 3, 2003 275

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Figure 3. Secondary structure analysis of human IL-4 (A) and IL-4δ2 (B) by FTIR. Figure represent FTIR spectra of proteins (bold lines) and their curve fit spectra (thin dotted lines). Figure 2. Far-UV CD (A) and amide I FTIR (B) spectra of human IL-4 (solid lines) and its natural splice vartiant IL-4δ2 (dotted lines). Measurements were carried out at pH 7.5 and 25 °C.

Table 1. Secondary Structure Content of Human IL-4 and Its Natural Splice Variant IL-4δ2 by FTIR human IL-4

of human IL-4 and its splice variant IL-4δ2. The numbering of amino acid residues in IL-4δ2 was kept identical to IL-4. Note that the positioning of secondary structure segments within the IL-4 sequence is based on the crystallographic threedimensional structure of the protein.35-40 Figure 1A shows that IL-4 is a highly helical protein. In fact, according to X-ray crystallographic data, the helicity of this protein is as high as 62%. In IL-4δ2, the second exon (residues 22-37) of IL-4 is omitted by natural alternative splicing, with exons 1, 3, and 4 joined in an open reading frame. Figure 1A shows that in IL-4 the missing fragment contains short R-helix H2 (residues 2326), short β-strand B1 (residues 27-29), with the remaining residues being involved into the formation long loop. Importantly, IL-4δ2 lacks Cys26, which is forming a disulfide bridge with Cys65 in IL-4 (see Figure 1). We assumed that both size and distribution of the remaining R-helical segments in IL-4δ2 are identical to those in full-length protein (see Figure 1B). Thus, the release of the 22-37 fragment should result in a measurable increase in protein helicity (from 62% to 67%). These assumptions were tested experimentally. Secondary Structure Analysis by CD and FTIR. Figure 2 represents far-UV CD and FTIR spectra measured for IL-4 (solid lines) and IL-4δ2 (dotted lines). Far-UV CD and FTIR spectra of both proteins are typical of higly helical polypeptides. In fact, far-UV CD analysis revealed that full length IL-4 and its splice variant IL-4δ2 possessed 60% and 63% R-helical structure, respectively. This conclusion was further confirmed by the 276

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structural assignment

wavenumber (cm-1)

%

wavenumber (cm-1)

%

turn turn R-helix β-sheet β-sheet/side chains

1681.5 1669.0 1654.1 1636.1 1622.0

8.4 15.3 60.4 10.5 5.4

1682.9 1671.1 1654.1 1635.2 1620.9

7.3 11.0 64.5 12.2 5.0

analysis of FTIR spectra. The curve-fit spectra for IL-4 and IL4δ2 are compared in Figure 3, and the results of secondary structure analysis are summarized in Table 1. The CD and FTIR data are consistent with the important conclusion that both proteins belong to the family of R-helical proteins (containing more than 60% R-helices) and have very close (almost identical) secondary structure composition. Moreover, in agreement with a hypothesis on preservation of R-helical segments, both farUV CD and FTIR data showed that IL-4δ2 possessed higher helical content. Disulfide Structure Analysis by Chemical Modifications. Clarification of Cys127 Status. According to the crystal structure, IL-4 is stabilized by a net of the disulfide bridges, Cys3-Cys127, Cys24-Cys65, and Cys46-Cys99.35-40 In agreement with this observation, using the method of chemical modification, we have established that IL-4 does not have any free SH-group. On the other hand, our data show that IL-4δ2 contains 5 cysteine residues, with four of them involved into the formation of two disulfide bridges and one free SH-group. The question then arose on the disulfide structure of IL-4δ2. The theoretical model

IL-4δ2 Structure and Function

Figure 4. SDS-PAGE of IL-4 and IL-4δ2 before and after the BrCNcleavage in formic acid. Analysis has been performed under reducing (10 mM β-mercaptoethanol) and nonreducing conditions. Lanes are as follows: 1, 3, 7, and 9sproteins before the creavage; 2 and 8 proteins after the creavage; 1, 2, and 3sin the presence of β-mercaptoethanol; 7, 8, and 9sproteins under nonreducing conditions; 4 and 5smolecular mass markers.

of IL-4δ2 has been earlier elaborated,41 according to which it has been suggested that the deletion of 22-37 fragment might be accompanied by the dramatic structural rearrangements. Particularly, it has been assumed that IL-4δ2 has “down-updown-down” topology, realizing as a result of almost 180° revolution of helix A and stabilizing by a new net of disulfide bridges Cys3-Cys65, Cys46-Cys99, with Cys127 being disengaged. Thus, to check the validity of this model it was necessary to gain information on whether Cys127 is free or is involved into the formation of disufide bond. Fortunately, IL-4δ2 (as well as IL-4) contains the sole site for the cyanogen bromide-cleavage in formic acid, Met120. Thus, the treatment of the protein by BrCN in formic acid should lead to the formation of a short C-terminal peptide, Arg121Glu122-Lys123-Tyr124-Ser125-Lys126-Cys127-Ser128-Ser129, containing Cys127. The existence of this peptide has been confirmed by the N-terminal analysis of the reaction mixture. The products of hydrolysis have been analyzed by SDS-PAGE under the reducing and nonreducing conditions (Figure 4). As BrCN does not affect disulfide bridges, such kind of analysis might give information on Cys127 status. Figure 4 shows that molecular masses of IL-4 and IL-4δ2 treated with BrCN did not change in the absence of the reducing agent (Figure 4, lanes 8). However, the mobilities of both proteins after the cyanogen bromide-cleavage in formic acid were slightly increased in the presence of β-mercaptoethanol (Figure 4, lanes 2). This means that in the absence of reducing agent, the C-treminal fragment 121-129 stayed bound to the main part of the protein throughout a disulfide bond, whereas it might be released in the presence of reducing agent β-mercaptoethanol. Analysis of Cys99 Status. At the next step we have analized the status of Cys99. IL-4δ2 and IL-4 contain the sole site, Trp91, for the BrCN-cleavage in heptafluorobutyric acid. The effective hydrolysis of the protein under these conditions should lead to the appearance of 38 amino acid residues-long C-treminal fragment containing Cys99, Cys127, and Met120. On the other hand, the combined BrCN-cleavage of the protein in formic

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Figure 5. SDS-PAGE of IL-4 and IL-4δ2 before and after the double BrCN-cleavage in formic and heptafluorobutyric acids. Analysis has been performed under reducing (10 mM β-mercaptoethanol) and nonreducing conditions. Lanes are as follows: 1 and 8sprotein before the creavage; 2, 3, 6, and 7sprotein after the creavage; 1, 2, 7, and 8-10 µg of protein; 3 and 6-5 µg of protein IL-4δ2; 1, 2, and 3sprotein in the presence of β-mercaptoethanol; 6, 7, and 8sprotein under nonreducing conditions; 4smolecular mass markers.

and then heptafluorobutyric acids should result in formation of two fragments, 28-mer92-119 and 9-mer121-129, each containing the single cystein residue (Cys99 and Cys127, respectively). Obviously, if Cys99 would not involved into the formation of intramolecular disulfide bond, the mass of the hydrolyzed protein, measured by SDS-PAGE under the nonreducing conditions, would be essentially lower (∼27%) because of the missing fragment of 28 amino acid residues. Figure 5 shows that in SDSPAGE under the nonreducing conditions the electrophoretic mobility of the double hydrolyzed IL-4δ2 does not increase in comparison with the controle (lanes 6 and 7). It is known that the yield of BrCN-cleavage in heptafluorobutyric acid does not usually exceed 20%.53 As a result, SDS-PAGE analysis under the reducing conditions gave two bandsscorresponding to the intact protein and to the 1-90 fragment of IL-4δ2 (Figure 5, lanes 2 and 3). Thus, our data clearly show that Cys99 forms disulfide bond in Il-4δ2. These data also specify, that in IL4d2 there is no disulfide bond Cys99-Cys127. Similar SDS-PAGE patterns have been obsrerved for the products of IL-4 double cleavage by BrCN in formic and heptafluorobutyric acids (Figure 5). Studies on the Cys65 Status. At the final stage of the chemical modification studies, we have analized the status of Cys65. There are five histidine residues in IL-4 and IL-4δ2, His1, His58, His59, His76, and His78. Quantitative cleavage of the IL-4δ2 peptide bonds adjacent to histidine residues by succinimide bromate with the subsequent analysis of the hydrolysis products by SDSPAGE under the nonreducing conditions leaded to the deletion of 58-78 fragment (with the molecular mass of ∼2 kDa), which was accompanied by an essential increase in the protein electrophoretic mobility (Figure 6). These data show that Cys65 did not form disulfide bond in IL-4δ2. Thus, we have established that IL-4δ2 has two disulfide bridges formed by Cys3, Journal of Proteome Research • Vol. 2, No. 3, 2003 277

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Figure 6. SDS-PAGE of IL-4 and IL-4δ2 before and after the succinimide bromate-cleavage. Analysis has been performed under reducing (10 mM β-mercaptoethanol) and nonreducing conditions. Lanes are as follows: 1 and 5sprotein before the creavage; 2 and 4 protein after the creavage; 1 and 2sin the presence of β-mercaptoethanol; 5 and 6sprotein under nonreducing conditions; 3smolecular mass markers.

Cys46, Cys99, and Cys127. Cys65, loosing in IL-4δ2 its natural partner Cys24 due to the deletion of the 22-37 fragment, does not participate in the formation of any intra- or intermolecular disulfide bonds. In other words, our data suggest that the deletion of the 22-37 fragment most likely does not affect the IL-4 topology and IL-4δ2 possesses “up-up-down-down” structure, typical of the other four-helix-bundle cytokines. Thus, 3D model of IL-4δ2 suggested earlier,41 based on the assumption on the formation of a new disulfide bridge Cys3-Cys65 and, thus, disengaging of Cys127, is wrong. Specific Binding of IL-4 and IL-4δ2 to Human Thymocytes and VTEC2.HS. It is known that IL-4 regulatory function as a cytokine is performed via cooperative two-centered specific interaction with heterodimer receptor IL-4R, characterized by high affinity interaction (Kd1) with R chain and some weaker interaction (Kd2) with the receptor γ chain. For the thymocytes (Figure 7A) and the VTEC2.HS cells (Figure 8A), the following values have been obtained: Kd1 ) (0.5 ( 0.09) × 10-10 M, Kd2 ) (2.5 ( 0.2) × 10-9 M and Kd1 ) (0.87 ( 0.4) × 10-10 M, Kd2 ) (4.5 ( 0.9) × 10-9 M, respectively. Importantly, IL-4δ2 is also able to interact with these cells (see Figure 7B and Figure 8B). This binding, being specific, is characterized by Kd ) (2.7 ( 0.3) × 10-10 M (thymocytes) and Kd ) (3.3 ( 0.3) × 10-10 M (VTEC2.HS cells). Figure 9 represents the results of inhibitory studies and illustrates the existence of the effective inhibition of 125I-IL-4 (4 ng) binding by the addition of nonlabeled IL-4 (Figure 9A) or nonlabeled IL-4δ2 (Figure 9B). It can be seen that IL-4δ2 is 278

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Figure 7. Scatchard’s analysis for the specific binding of 125IIL-4 (A) and 125I-IL-4δ2 (B) to human thymocytes. B and F represent bound and free protein.

able to effectively inhibit the IL-4 binding and this inhibitory effect is realized at higher protein concentration than that of nonlabeled IL-4. This indicates that IL-4 and IL-4δ2 binds only to the one mutual site of the heterodimer receptor. Effect of IL-4 and IL-4δ2 on the Proliferative Activity of Human Thymocytes and VTEC2.HS. Figure 10 shows that IL4δ2 is able to suppress the IL-4-dependent proliferation of human thymocytes in a dose-dependent manner. ConAinduced prolifiration of human thymocytes may be essentially stimulated by IL-4 (1 to 10 ng). Addition of 100 ng/mL IL-4δ2 decreased the proliferative activity of thymocytes by 10-15%, whereas in the presence of higher IL-4δ2 (1000 ng/mL) the proliferation is suppreset almost completely. Our results are in a good agreement with the data on IL-4δ2 inhibition of IL4-stimulated proliferation of plasma T-cells.24 At the next step, we have ahalyzed the effect of IL-4 and IL4δ2 on the VTEC2.HS growth. These cells are involved in the process of T-cell differenciation and in the clone selection of the thymus limphoid cells throughout the direct contact or by the secretion of different cytokines. Figure 11 shows that the low IL-4 concentrations (10-5-10-2 ng/mL) stimulated the VTEC2.HS proliferation, whereas this process was inhibited in the presence of higher IL-4 concentrations (10-100 ng/mL). All these effects have been eliminated by the IL-4δ2 addition (100 ng/mL). Thus, IL-4δ2 acts as a natural IL-4 antagonist under the studied conditions. Modeling of IL-4δ2 Three-Dimensional Structure. On the basis of the structural and functional data reprted in this paper, the 3D molecular model of IL-4δ2 has been restored using a structural homology of this protein with IL-4 (Figure 12).

IL-4δ2 Structure and Function

Figure 8. Scatchard’s analysis for the specific binding of 125IIL-4 (A) and 125I-IL-4δ2 (B) to VTEC2.HS cells. B and F represent bound and free protein.

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Figure 9. Inhibition of the specific binding of 125I-IL-4 (4 ng) to attached of TEC2.HS by the nonlabeled IL-4 (A) and nonlabeled IL-4δ2 (B).

Modeling was performed with the SWISS-MODEL protein modeling server program package and the Swiss-Pdb Viewer 3.7.57,58 In our model, IL-4δ2 has a virtually identical structure to the IL-4 and preserves the “up-up-down-down” topology typical for the four-helix-bundle cytokines. Importantly, we have established that the position of the functionally important amino acid residues Glu9 and Arg72, which are responsible for the IL-4R R-chain binding, are preserved and they are located in the antiparallel helices A and C for both in the IL-4δ2 and IL-4.39,40

Discussion IL-4 is a principal regulatory cytokine in the immune response and is a crucial determinant for allergy and asthma.1,2,60 Increased IL-4 production leads to allergic disorders development.61-63 One of the mechanisms, restricting the activity of IL-4 in the organism, is an alternative splicing of IL-4 gene resulting in the elimination of the second exone, encoding for the amino acid sequence fragment 22-37,23-25 thus, giving rise to IL-4δ2, an alternative splice form of IL-4. The missing fragment 22-37 corresponds to the considerable part of a loop connecting R-helixes A and B. Moreover, Cys24, involved into the formation of S-S bridge with Cys65 in IL-4, is in the eliminated amino acid sequence. The understanding of the IL-4δ2 structure might be considerable helped via the comparative analysis of the disulfide frames in IL-4δ2 and IL-4, where there are three disulfide bridges Cys3-Cys127, Cys24-Cys65, Cys46-Cys99. Earlier, IL-4δ2

Figure 10. IL-4δ2 supresses the IL-4-induced proliferation of human thymocytes. Controls were as follows: 1sintact thymocytes; 2sthymocytes in the presence of ConA (0.8 µg/mL).

model has been proposed with a new net of disulfide bridges Cys3-Cys65 and Cys46-Cys99, and with Cys127 being free.41 However, our experiments based on the cleavage of IL-4δ2 with BrCN at Met120 with the subsequent SDS-PAGE electrophoresis under the reducing and nonreducing conditions demonstrated that Cys127 is not free, being involved into the formation of a disulfide bridge. Furthermore, a consequent cleavage of IL-4δ2 with BrCN at Met120 in formic acid and at Trp91 in heptafluorobutyric acid with the subsequent SDS-PAGE under the reducing and nonreducing conditions demonstrated that Cys99 is not free either and is also involved in the formation of a Journal of Proteome Research • Vol. 2, No. 3, 2003 279

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Figure 11. Effect of IL-4δ2 on the IL-4-stimulated proliferation of TEC2.HS. Controls were as follows: 1sintact cells; 2scells in the presence of IL-4δ2.

Figure 12. Hypothetical 3D-structure of IL-4δ2. Amino acid residues important for IL-4R R-chain binding are located on the antiparallel helices A and C: Glu9 (E9) and Arg72 (R72).

disulfide bridge, but not with Cys127. Importantly, the succinimide bromate cleavage of IL-4δ2 at histidine residues with the subsequent analysis of the hydrolysis products by SDS-PAGE under the nonreducing conditions leaded to the deletion of fragment 58-78, containing Cys65. This suggests that Cys65 is not form a disulfide bound in IL-4δ2 as it had been suggested earlier.41 Thus, similar to IL-4, IL-4δ2 has two disulfide bridges formed by Cys3, Cys46, Cys99, and Cys127. In addition, IL-4δ2 possesses free Cys65 that is not involved in formation of any intra- or intermolecular disulfide bonds. The CD and FTIR spectra demonstrated that IL-4δ2 and IL-4 have very close (almost identical) secondary structure arrangement. Although, in agreement with a hypothesis on the preservation of R-helical segments, both far-UV and FTIR data demonstrated that IL-4δ2 possessed a slightly higher helical content. The obtained experimental data allowed the reconstruction of IL-4δ2 3D-structure using the computer modeling (see Figure 12). In the proposed model, IL-4δ2 preserved the “up-updown-down” topology typical for the four-helix-bundle cytokines. Furhtermore, IL-4δ2 structure like that of IL-4 preserves spatial arrangement of the functional amino acid residues on the helices A and C. It is known that even insignificant changes 280

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in the mutual arrangement of A and C helices might result in the considerable loss of the ability of IL-4 mutants to interact with IL-4RR.31,34,39,40 IL-4 receptor is a heterodimer, consiting of R and γ chains. It is known that the responses mediated by IL-4 are generated by the heterodimerization of transmembrane receptor chains in a strictly sequential manner. In the first step, IL-4 binds to the extracellular part of IL-4RR. This primary event is followed by the recruitment of the receptor γ chain with lower IL-4 affinity. This low-affinity binding receptor could be the common γ chain, yielding the so-called type I receptor complex, or the IL-13Ra1 chain,64 and resulting in the formation of a type II receptor complex. Besides, γ chain is also a part of the receptor complexes for IL-2, IL-7, IL-9, and IL-15,65 whereas IL-13Ra1 might be also recognized by IL-13. Finally, the crosslinking of receptor ectodomains by the ligand might lead to the association of tyrosine kinases of the Jak family to the cytoplasmic parts of the receptors and, thus, to the activation of the Jak/Stat pathway.66 In our experiments, the Scatchard graph desribing the IL-4 interaction with IL-4R of human thymocytes possesses the nonlinear concave character, indicating the presence of two binding sites. High affinity site is characterized by Kd1 ) (0.50 ( 0.09) × 10-10 M and according to34 is located on the IL-4RR. Low affinity site is characterized by Kd2 ) (2.5 ( 0.02) × 10-9 M and according to64-66 is located on γ-chain of IL-4R. Same results have been obtained in the experiments with VTEC2.HS cells. While evaluating the validity of the obtained dissociation constants, it is necessary to take into consideration the fact that the IL-4 interaction with receptor represents several consecutive processes. In fact, IL-4 interaction with IL-4RR orients the cytokine in the space and produces a positive effect on its subsequent interaction with the second binding center on IL-4Rγ. Furthermore, after IL-4 binding to the receptor R and γ chains, the interaction of IL-4 with IL-4RR might be additionally intensified. Contrarily to IL-4, the Scatchard graphs describing interaction of the IL-4δ2 with IL-4R on thymocytes and VTEC2.HS cells were linear, indicating the existence of only one IL-4δ2 binding site (with the Kd of (2.7 ( 0.3) × 10-10 M and (3.3 ( 0.3) × 10-10 M for thymocytes and VTEC2.HS cells respectively). According to our experimental data and based on the suggested 3D-structural model, IL-4δ2 might preserve the active center responsible for the interaction with IL-4RR. Consequently, the Kd values characterizing interaction of IL-4δ2 with IL-4R might represent the actual Kd1 values for the IL-4-IL-4R complex. Thus, our data suggest that IL-4δ2 interacts only with R chain of IL-4R, whereas binding of IL-4δ2 to γ chain of IL-4R does not occur. This migh have some regulatory implication, as IL4δ2 capability to compete with IL-4 only for IL-4RR on the immunocompetent cells should weaken the inhibition effects manifested by IL-4δ2 and make the process more fluent. In fact, our data on the IL-4δ2 biological properties as IL-4 antagonist testified in favor of this mechanism. Thus, because IL-4 plays an important role in the development of allergic inflammatory responses, and since IL-4δ2 acts as a natural IL-4 antagonist and prevents polarization of immune response of Th2-type, the detection of the mutual levels of IL-4 and IL-4δ2 is needed for the effective development of individual schedules for the treatment of allergic and infectious diseases.67-70 Besides, IL-4δ2 should be considered as a base for construction of anti-allergic drugs of a new generation.

research articles

IL-4δ2 Structure and Function

Acknowledgment. This research was supported by grants from the International Science and Technology Center (ISTC), Moscow, Russia. Abbreviations. IL-4, interleukin 4; IL-4δ2, splice variant of human IL-4; IL-4R, IL-4 receptor; IL-4RR, alpha-chain of IL-4 receptor; IL-4Rγ, γ-chain of IL-4 receptor; UV, ultra violet; CD, circular dichroism; FTIR, Fourier transform infrared spectroscopy; AHR, airway hyperresponsiveness.

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