©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding Interactions of Human Interleukin 5 with Its Receptor Subunit
LARGE SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS (*)

Kyung Johanson (1), Edward Appelbaum (2), Michael Doyle (3), Preston Hensley (3), Baoguang Zhao (3), Sherin S. Abdel-Meguid (3), Peter Young (4), Richard Cook (4), Steven Carr (5), Rosalie Matico (1), Donna Cusimano (2), Edward Dul (2), Monica Angelichio (2), Ian Brooks (3), Evon Winborne (3), Peter McDonnell (4), Thomas Morton (4), Donald Bennett (4), Theodore Sokoloski (6), Dean McNulty (3), Martin Rosenberg (7), Irwin Chaiken (4)(§)

From the (1) Departments of Protein Biochemistry, (2) Gene Expression Sciences, (3) Macromolecular Sciences, (4) Molecular Immunology, (5) Physical and Structural Chemistry, and (6) Pharmaceutical Technologies and the (7) Biopharmaceuticals Division, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human interleukin 5 (hIL5) and soluble forms of its receptor subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Å resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor subunit (shIL5R). Additionally, the relative abundance of ligand and receptor in the hIL5shIL5R complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 n M, respectively, for the binding of hIL5 to shIL5R and to a chimeric form of the receptor containing shIL5R fused to the immunoglobulin Fc domain (shIL5R-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kvalues from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R in Drosophila membranes was found to be 6 n M, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5receptor complex and signal transduction.


INTRODUCTION

Interleukin 5 (IL5)() is a disulfide-linked, glycosylated dimeric protein which plays a prominent role in the maturation, proliferation, and activation of eosinophils (Basten and Beeson, 1970; Metcalf et al., 1974; Warren and Sanderson, 1985; Sanderson et al., 1985; McKenzie and Sanderson, 1992; Campbell et al., 1987; Clutterbuck et al., 1987; Lopez et al., 1988). IL5's central role in the control of eosinophilia has suggested that it is a major cause of tissue damage in asthma and other eosinophil-related disorders (Hamid et al., 1991; Bentley et al., 1992; Corrigan and Kay, 1992).

Interleukin 5 exerts its biological effects via cell surface receptor proteins (Chihara et al., 1990; Lopez et al., 1990; Plaetinck et al., 1990; Migita et al., 1991). The human IL5 receptor is composed of two types of subunits, denoted and (Tavernier et al.,1991). The cloned cDNA for the subunit (Murata et al., 1992) encodes a glycoprotein of 420 amino acids with an amino-terminal hydrophobic region (signal sequence, 20 amino acid residues), a glycosylated extracellular domain (324 residues), a transmembrane domain (21 residues), and a cytoplasmic domain (55 residues). The subunit expressed in a soluble form (the extracellular domain) can bind to IL5 without the chain and is IL5-specific (Tavernier et al., 1991; Murata et al., 1992; Devos et al., 1993). In contrast, the chain is identical to the chains of GM-CSF and IL3 receptors (Tavernier et al., 1991; Lopez et al., 1990) and appears to be needed for signal transduction.

The ligand binding of human IL5 receptor chain (hIL5R) is of high affinity, with a Kof 0.6-1 n M reported for the subunit expressed in various heterologous cells (Tavernier et al., 1991, 1992; Takaki et al., 1993). The above studies showed that the affinity for subunit alone was lower by a factor of 2-4-fold than that for cells transfected with both and subunits. Direct interaction of the subunit with IL5 has been suggested by cross-linking (Devos et al., 1991; Tavernier et al., 1991, 1992; Takaki et al., 1993) and as such could contribute energetically to the IL5 receptor affinity.

The stoichiometry of IL5 binding to subunit has been suggested to be 1:1 (one subunit/IL5 dimer). This was based on both cross-linking data as well as from quantitating the abundance of each component in a complex isolated by immunoaffinity capture followed by gel filtration chromatography (Devos et al., 1993). A 1:1-sized cross-linked IL5-receptor chain complex also has been observed indirectly (Mita et al., 1988; Migita et al., 1991; Devos et al., 1991; Tavernier et al., 1992). The above 1:1 stoichiometry is unexpected, since the IL5 dimer has two 4-helix bundle domains. Each of these domains resembles the 4-helix bundle domain of monomeric growth factor proteins such as growth hormone (Abdel-Meguid et al., 1987; de Vos et al., 1992) and IL4 (Redfield et al., 1991), and each of the latter can bind at least one molecule of receptor/molecule ligand. Each IL5 monomeric domain might therefore be expected to bind at least one receptor molecule. Interestingly, the molecular weight of the cross-linked product of IL5 with chain also suggests a 1:1 stoichiometry (Devos et al., 1991; Tavernier et al., 1992). The structural basis for the 1:1 stoichiometry of IL5 and its and receptor subunits is as yet undetermined. The above methods used to define 1:1 stoichiometry, namely cross-linking and chromatographic isolation of complexes, likely would not detect weakly associating multimeric complexes. Nonetheless, if correct, 1:1 stoichiometry must be accounted for in explaining signal transduction.

We have initiated an investigation of the quantitative and structural properties of the interaction of hIL5 with its receptor. In this report, we describe the expression and production of Drosophila-expressed hIL5 and both soluble and membrane-associated forms of its receptor chain. We report the three-dimensional structure of deglycosylated Drosophila-expressed hIL5 and show that it is nearly identical to the protein produced in Escherichia coli (Milburn et al., 1993). We further report the interaction properties of these functionally active proteins. The equilibrium binding parameters of soluble and full-length IL5R chain show that the soluble domain of the receptor accounts for most, if not all, of the binding energy of cell-bound receptor subunit recognition. We provide rigorous confirmatory evidence for a stoichiometry of 1:1 for the hIL5receptor chain complex. Finally, we report new data on the thermodynamics and kinetics of the IL5receptor interaction which argue that conformational change occurs in this process. Together, the data suggest a model for the interaction of hIL5 with its receptor and subunits leading to signal transduction.


EXPERIMENTAL PROCEDURES

Materials For protein purification, Q-Sepharose fast flow, phenyl-Sepharose fast flow high substitution, Superose 12 and 6, and Superdex 75 were from Pharmacia LKB Biotechnol. Hydroxylapatite high resolution medium was from Calbiochem. ABX-plus ion exchange resin was from J. T. Baker Chemical Co. Lentil lectin-agarose and methyl D-mannosylpyranoside were from E-Y Lab (San Mateo, CA). For BIAcore kinetics measurements, the sensor chips CM5, surfactant P20, and the amine coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl- N`-(3-diethylaminopropyl)carbodiimide (EDC), and ethanolamine hydrochloride all were from Pharmacia Biosensor. Murine IL3 was obtained from Genzyme (Cambridge, MA), and purified murine IL5 (specific activity 4.9 10units/mg) was prepared in a baculovirus expression system using Spodoptera frugiperda 21 cells (Mitchell et al., 1993). The murine pre-B cell line, denoted B13, was obtained courtesy of R. Palacios, Basel Institute of Immunology, Switzerland. Expression of Recombinant IL5, shIL5R, and shIL5R-Fc Human IL5 and various forms of shIL5R were expressed in a Drosophila cell culture system (van der Straten et al., 1989; Angelichio et al., 1991). Each gene to be expressed was cloned between a copper sulfate-inducible metallothionein promoter and an SV40 late polyadenylation site and introduced into Drosophila Schneider 2 (S2) cells by cotransfection with a vector carrying a hygromycin B resistance gene. Hygromycin B-resistant cells were selected as a stable polyclonal population, induced with copper sulfate, and examined for expression by Western blot analysis. Antisera used for Western blots were raised by immunization of rabbits with denatured fusion proteins (expressed in E. coli and gel-purified) carrying hIL5 or hIL5R sequences.

hIL5 Expression

Human IL5 expression was achieved with a vector encoding a fusion of a human tissue plasminogen activator (tPA) secretion signal sequence to amino acids 4-115 of mature hIL5. Cleavage of the signal sequence during secretion leaves four amino acids from tPA fused to the NHterminus of hIL5, as reported for other such fusions (Culp et al., 1991). Western blot analysis of medium from induced cultures revealed a protein with an apparent molecular mass of 15 kDa under reducing conditions and 30 kDa under nonreducing conditions, as expected for the disulfide-linked homodimer. The cell line was expanded to 30 liters to produce hIL5.

shIL5R Expression

To express shIL5R in Drosophila, a cDNA fragment encoding the shIL5 protein precursor (signal sequence followed by mature shIL5R) was cloned from human eosinophil RNA by polymerase chain reaction and inserted into an expression vector (pMTAL) to yield pMTAL-shIL5R, a vector that does not contain the tPA sequence. DNA sequence analysis showed the shIL5R sequence to be identical to that reported by others (Tavernier et al., 1992). Western blot analysis of induced culture carrying this construct revealed a broad band with an apparent molecular mass of 40-43 kDa under both reducing and nonreducing conditions. The cell line was expanded to 4 liters in spinner flasks for production of shIL5R.

shIL5R-Fc Chimera Expression

A fusion of shIL5R to the C2-C3 region of human IgG1 was constructed and then expressed in Drosophila cells as described above for shIL5R. In this construct, the COOH terminus of shIL5R (Argof mature shIL5R) was linked to the NHterminus of the IgG1 hinge region (Glu, Kabat numbering) by the short sequence Ile-Glu-Gly-Arg, a Factor Xa cleavage site, allowing recovery of shIL5R by proteolytic cleavage of the purified chimera. The Fc region also had a mutation of Cysto Ala. Western analysis using anti-shIL5R antiserum showed that this protein (70 kDa under reducing conditions, 140 kDa and hence dimeric under nonreducing conditions) was produced in Drosophila cell cultures at levels comparable to shIL5R lacking the Fc region. The cell line was expanded to 4 liters for production of the Fc receptor chimera. In the present study, we evaluated the binding properties of the uncleaved and unreduced chimera (molecular mass 140 kDa), which retains both sIL5R fragments/molecule. Expression of Full-length hIL5R in Drosophila Cells The transmembrane and cytoplasmic region of hIL5R was cloned by reverse transcriptase polymerase chain reaction from a butyrate-induced eosinophilic subline of human promyelocytic HL-60 cells and the intended DNA sequence confirmed. This fragment was cloned into pMTAL-shIL5R to create a new vector, pMTAL-hIL5R, containing the full-length receptor (20 amino acid signal sequence followed by 400 amino acid mature protein). Western blot analysis of induced cells carrying this construct revealed a band with an apparent molecular mass of 50 kDa. Protein Purification Expression levels of hIL5, shIL5R, and shIL5R-Fc in Drosophila media used for large scale purification were estimated to be 22, 17, and 10 mg/liter, respectively, from Western blot analysis and Coomassie Blue staining of SDS-PAGE. Western blot analyses of all three products in culture media suggested heterogeneity in the carbohydrate content. Protein concentration of the starting material and at various stages of purification was estimated by the Micro BCA Protein assay. Extinction coefficients at 280 nm calculated from tryptophan and tyrosine contents matched well with those measured by amino acid analysis: E(cm - mg/ml)= 0.63 for hIL5, 1.9 for shIL5R, and 1.65 for shIL5R-Fc. Throughout this study, concentrations of purified proteins were based on absorbance at 280 nm.

Purification of hIL5

28 liters of sterile-filtered Drosophila media were diluted 3-fold with water, adjusted to pH 8.0, and loaded onto a Q-Sepharose FF column (11.4 14.1 cm, Pharmacia) equilibrated with 20 m M Tris-HCl, pH 8.0. hIL5 in the flow-through fractions was pooled, adjusted to pH 7.4 with 6 M HCl, and loaded onto a hydroxylapatite column (HA) equilibrated with Buffer T (20 m M Tris-HCl, pH 7.4). The column was washed with Buffer T, and bound hIL5 was eluted with 0.25 M potassium phosphate, pH 7.5. Pooled fractions from the HA column were mixed with an equal volume of 3 M ammonium sulfate and applied to phenyl-Sepharose ff (5.7 7 cm) equilibrated with 1.5 M NHSOin Buffer P (20 m M sodium phosphate, pH 7.0). hIL5 was eluted with a linear gradient of 1.5-0 M NHSOin Buffer P. The pooled fraction from phenyl-Sepharose (600 ml) was concentrated to 80 ml, loaded onto Superose 12, and eluted with Buffer T + 0.15 M NaCl. The final yield of hIL5, approximately 500 mg, was >98% electrophoretically homogeneous based on silver staining of SDS-PAGE gels; nucleic acid and endotoxin were undetectable.

Purification of shIL5R

4.3 liters of sterile-filtered conditioned Drosophila media (MRD3) were diluted 2-fold with Buffer T, adjusted to pH 7.4, and loaded onto a Q-Sepharose column (5 8.8 cm, Pharmacia) equilibrated with Buffer T. Receptor in flow-through fractions (9 L) was concentrated to 1900 ml using a Miniset 10K cutoff membrane (Filtron, Northborough MA). The concentrated Q-Sepharose pool was applied to an ABX-plus column (5 7 cm, J. T. Baker Chemical Co.) equilibrated with Buffer A (20 m M sodium acetate, pH 5.5), and washed with Buffer A. Bound receptor was eluted with a linear gradient of 0-1 M NaCl in Buffer A. Fractions containing receptor were immediately neutralized with pH 7.4 1 M Tris-HCl buffer. This ABX pool (240 ml) was adjusted to 5 m M CaCl, sterile filtered, and loaded onto two lentil lectin columns (2.5 5.8 cm) in tandem, equilibrated with Buffer C (20 m M Tris-HCl, pH 7.2, 0.15 M NaCl, and 5 m M CaCl). Bound receptor was eluted with 0.25 M methyl D-mannosylpyranoside in Buffer C. The lentil column pool (88 ml) was concentrated to 12 ml on a YM-10 membrane (Amicon) and loaded onto a Superdex 75 preparation grade column (2.6 96.4 cm) equilibrated in 0.1 M HEPES, pH 7.5. Pooled fractions (29 ml) were stored at -70 °C. The Superdex 75 column eluate contained 20 mg of receptor, with >98% electrophoretic homogeneity based on silver staining of SDS-PAGE gels.

Purification of shIL5R-Fc Chimera

4 liters of conditioned Drosophila media were diafiltered into 0.1 M Tris-HCl, pH 8.1, and concentrated to 600 ml. The diafiltered and concentrated media were loaded onto a Protein A-Sepharose ff column (2.5 5.8 cm, Pharmacia) equilibrated with 0.1 M Tris-HCl, pH 8.0, and washed with the same buffer. The Fc chimera was eluted with 0.1 M glycine HCl, pH 3.0, and fractions were collected in 1 M Trizma base to raise the final pH to 7.7. The chimera was fractionated on a Superose 6 column into 0.1 M HEPES, pH 7.5. 2.8 mg of Fc chimera (>95% electrophoretically homogeneous in SDS-PAGE) was obtained from 4 liters of media. Mass Spectroscopy Matrix-assisted laser desorption mass spectrometry (MALD-MS) data were obtained on a Vestec Research model mass spectrometer (Persceptive Biosystems, Boston, MA). Samples were prepared for analysis by mixing 1 µl of a 1-6 pmol/ml solution of protein in phosphate-buffered saline, pH 7.4, with 1 µl of the matrix, sinapinic acid, on the stainless steel target. The 337-nm line from a nitrogen laser (10 ns pulse width, 10 Hz repetition rate) was used for desorption/ionization of the sample. The spectra were the sum of 20-70 laser shots. Spectra were calibrated externally using the BSA (bovine serum albumin) and BSA dimer peaks.

Electrospray mass spectra were recorded on a Sciex API-III triple quadrupole mass spectrometer fitted with a standard pneumatically assisted nebulization probe and an atmospheric pressure ionization source (Sciex, Ontario, Canada). Buffers and salts were removed by high performance liquid chromatography prior to mass spectrometry. Sample was loaded in aqueous buffer onto a 2.1 mm inner diameter C8 guard column, flushed with 0.1% trifluoroacetic acid for 5 min and then step eluted with 60% CHCN, 40% HO, 0.1% trifluoroacetic acid. An aliquot of the sample was concentrated to near dryness and brought to a final concentration of 20 pmol/µl with methanol/water (50:50 v/v) containing 0.2% formic acid. Sample was introduced into the mass spectrometer by infusion with a syringe pump (Harvard Instruments) at 2 µl/min. Approximately 2 min of data were recorded and 40 pmol of the sample consumed. Enzymatic Deglycosylation and Cross-linking to I-hIL5 hIL5 was iodinated with Bolton-Hunter reagent to a specific activity of >350 Ci/mmol using the manufacturer's protocol (DuPont NEN). 100 µg of shIL5R or 10 µg of I-hIL5 were enzymatically deglycosylated by treating with 5,000 units of PNGaseF (New England Biolabs) in 50 m M sodium phosphate, pH 7.5, for 12-16 h at 37 °C in the presence of a mixture of protease inhibitors (1 m M phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 20 µ M leupeptin). Analysis of the shIL5R product by Western blot showed a reduction in molecular mass of 3-4 kDa relative to untreated receptor, consistent with loss of carbohydrate but absent proteolytic degradation (data not shown). The mobility was identical to shIL5R enzymatically deglycosylated in denaturing conditions (0.5% SDS, 1% -mercaptoethanol), suggesting that deglycosylation was complete. Similarly, deglycosylation of hIL5 led to 1 kDa reduction in mobility on Western blots (data not shown).

Cross-linking was performed by incubating 1 n M each of I-hIL5 and shIL5R together with 1 n M of the bifunctional cross-linker bis-suberimidate (Pierce) in 25 m M HEPES, pH 7.2, 0.1% BSA for 20-30 min at 4 °C. Products were analyzed by 12.5% SDS-PAGE in reducing conditions, followed by gel drying and autoradiography. B13 Cell Proliferation Assay The murine IL5/IL3-dependent cell line LyH7.B13 was subcultured twice weekly in RPMI 1640 medium (Life Technologies, Inc.), supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, penicillin-streptomycin (all Life Technologies, Inc.), plus 2-mercaptoethanol (5 10 M, Sigma), 10% fetal bovine serum (Globepharm), and 1-10 units of recombinant murine IL5 (Mitchell et al., 1993). For assays, cells were washed and maintained in medium without IL5 for a minimum of 2 h. They were cultured for 48 h in triplicate (5,000 cells/well) in 96-well round bottom plates in the presence of appropriately diluted test samples and pulsed with 0.5 µCi of [H]thymidine (Amersham) for the final 4 h. They were processed for scintillation counting in a 1205 Betaplate (LKB Wallac). Results are presented as Stimulation Index ± standard deviation, relative to unstimulated signal. Crystallization Crystals of deglycosylated hIL5 (from PNGaseF treatment in 0.1 M HEPES, pH 7.4, 25 °C, 48 h) and of the complex between glycosylated hIL5 and shIL5R were obtained using the hanging drop method of vapor diffusion (McPherson, 1976). Deglycosylated hIL5 was concentrated to approximately 10 mg/ml in 100 m M HEPES buffer, pH 7.2. The complex was prepared by mixing equimolar amounts of hIL5 (dimer) and shIL5R in 100 m M HEPES, pH 7.2. Typically, 2 µl of either protein solution were mixed with 2 µl of various precipitant concentrations and vapor equilibrated at several temperatures against the solution added to the protein. At each temperature, a three-dimensional matrix was established for each precipitant, in which protein concentration, precipitant concentration, and pH were varied. Ammonium sulfate, sodium chloride, and various polyethylene glycols were used as precipitants. Determination of Molecular Ratio of hIL5 and shIL5R in Cocrystals Cocrystals were harvested under a microscope, washed twice in crystallization solution P (27% PEG 600 (v/v) in 100 m M sodium acetate, pH 5.0), solubilized in SDS-sample buffer, and analyzed by 12% SDS-PAGE. The gels were either stained using Coomassie Blue or silver-stained. Intensities of the bands of IL5 and shIL5R in the cocrystals were compared with those of controls (hIL5, shIL5R, or combinations of both proteins) by scanning with IS-1000 Digital Imaging System (Alpha Innotech Co, San Leandro CA), and the peak areas were integrated. Crystal Structure Determination of Deglycosylated hIL5 Crystals of deglycosylated hIL5 belong to the space group C2 with a = 118.3 Å, b = 24.3 Å, c = 43.8 Å, and = 110°. Assuming one deglycosylated monomer/asymmetric unit, the Vvalue is 2.1 Å/dalton, which is within the range found for most protein crystals (Mathews, 1968). These crystals show diffraction beyond 2.6-Å resolution and are stable in the x-ray beam for several days.

X-ray diffraction data were measured from a single crystal using a Siemens two-dimensional position-sensitive detector. Approximately 5776 reflections were measured in 2 days to give 3054 unique reflections to 2.6-Å resolution, representing 84% of the data. The merging R factor for symmetry-related reflections was 0.042.

The structure of the deglycosylated hIL5 monomer was determined by molecular replacement using XPLOR (Brunger, 1992). The starting model consisted of all atoms of the dimer from the previously reported crystal structure of hIL5 (Milburn et al., 1993). Rotation functions were calculated using x-ray diffraction data from 10 to 5 Å and a radius of integration of 27 Å. Results from the cross-rotation function revealed two peaks ( , , are 166.9, 131.5, 159.3° and 345.7, 50.8, 200.7°) related by a 2-fold axis. Translation was done manually on the graphics by moving rotated dimer along the a and c axes, so that the b (2-fold) axis passed through the center of the dimer. Rigid body refinement of translated monomer gave an R factor of 0.49 using 2.0( F) data from 8- to 3- Å resolution.

The monomer was refined using XPLOR. The resulting phases were used to calculate Fourier maps with coefficients F- F and 2 F- F, into which the atomic model was analyzed. Four water molecules were found and included in the model. The R factor was reduced to 0.231 when data greater than 2( F), in the resolution range 8-2.6Å, were used to refine all atomic positions of the monomer and their temperature factors. The overall root-mean-square deviation from ideal geometry for bond lengths was 0.009 Å, while that for bond angles was 1.69°. Analytical Ultracentrifugation Equilibrium sedimentation experiments were performed with a Beckman XL-A analytical ultracentrifuge. Double sector cells with charcoal-filled epon centerpieces and sapphire windows were used. Sedimentation experiments were performed at 25.0 °C in 0.1 M HEPES, pH 7.4. The partial specific volumes of the monomers and the potential complexes were calculated using the partial specific volumes of the component amino acids (Cohn and Edsall, 1943), which yielded a value of 0.741 ml gfor IL5, 0.722 ml gfor shIL5R, 0.730 ml gfor the 1:1 (molar basis) complex, and 0.727 ml gfor 2:1 complex (molar basis). The solvent density, , was estimated to be 1.006 g ml. To test the hypothesis that IL5 could potentially interact with 2 mol of shIL5R, shIL5R and IL5 were mixed in two ratios. For one, shIL5R was in small excess of IL5, on a molar basis. For the other, shIL5R was in larger excess of IL5, i.e. approximately 2 mol of shIL5R/mol of IL5. For both experiments the total protein concentration was 10 M.

Sedimentation equilibrium data were analyzed using nonlinear least-squares methods (Johnson and Frasier, 1985; Brooks, et al., 1994a, 1994b) under the control of a modified version of IGOR-PRO (Wavemetrics, Lake Oswego, OR) running on an Apple MacIntosh computer. Data sets were collected after reaching equilibrium, usually about 18 h, at indicated rotor speeds. Equilibrium was established by determining that scans taken 4 h apart were superimposible.

At equilibrium, the concentration distribution of a single, homogeneous species is given by

   

On-line formulae not verified for accuracy

Kinetic data were evaluated using relationships described pre-viously (Karlsson et al., 1991; Morton et al., 1994). For the bimolecular interaction,

On-line formulae not verified for accuracy

   

On-line formulae not verified for accuracy


RESULTS

Structural Analysis and Sedimentation Behavior of Recombinant Proteins

Sequence Analysis

The amino acid compositions and NH-terminal sequences of all three proteins agreed well with those predicted from the cDNA sequences (Tanabe et al., 1987; Tavernier et al., 1991). For IL5 the NH-terminal sequence was NH-GARSEIPTSALVKET. The GARS sequence was from the TPA leader sequence used in expression (see ``Experimental Procedures''). The COOH-terminal peptide, produced by CNBr cleavage, was NTEWIIES-COH. The NH-terminal sequences of shIL5R and shIL5R-Fc chimera were NH-DLLPDEKISLLPV- x, where x is a potential site of N-linked glycosylation.

Molecular Mass

Molecular masses of the Drosophila-expressed recombinant proteins were estimated by SDS-PAGE as well as by gel filtration. Human IL5 migrates by SDS-PAGE as a single band at 32 kDa under non-reducing conditions and 16 kDa under reducing conditions (dithiothreitol, see Fig. 1 A), confirming that the Drosophila hIL5 is a disulfide-linked dimer. Human IL5 was eluted from Superose 12 as a dimer (data not shown). The molecular mass of hIL5 was measured by electrospray-mass spectrometry as 28,492 ± 2 Da (data not shown). Of this, 26,418 Da was calculated to have originated from the amino acid residues (Murata et al., 1992) and the remaining from carbohydrate. This mass difference fits precisely the combined residue masses of two N-linked ManGlcNAcFuc moieties and is constant with carbohydrate structures expressed in insect cell lines (see Carbohydrate Analysis, below) (Hsieh and Robbins, 1984; Kuroda, et al., 1990). A global analysis of equilibrium sedimentation data from three rotor speeds yielded a single molecular mass of 28.1 ± 0.4 kDa and demonstrated that hIL5 was a dimer in solution and did not aggregate further (Fig. 2 A).

The molecular mass estimated for shIL5R (315 residues, 35,870 Da) was 46 kDa by non-reducing SDS-PAGE (Fig. 1 A) and 44 kDa by size exclusion chromatography (data not shown), indicating that shIL5R was monomeric, in spite of the presence of 1 unpaired cysteine residue. A molecular mass of 42 kDa was determined by MALD-MS (data not shown). From these values, it is evident that 14% of the mass of shIL5R was carbohydrate. A global analysis of equilibrium sedimentation yielded a single molecular mass of 44 ± 0.8 kDa (Fig. 2 B). There appeared to be some tendency for shIL5R to self-associate to a tetrameric form at higher protein concentrations. The monomer was 50% associated at 1.0 10 M (data not shown). With this affinity, the receptor was <1% associated at 10 M.


Figure 1: SDS-PAGE analysis of Drosophila expressed recombinant proteins. A, purified soluble proteins, 20 µg, 12% gel stained with Coomassie blue: hIL5 ( lanes 4 and 7), shIL5R ( lanes 3 and 6), and shIL5R-Fc ( lanes 2 and 5). Protein standards are in lanes 1 and 8. Lanes 1-4 were run under nonreducing conditions and lanes 5-8 under reducing conditions. B, full-length hIL5R on Drosophila cells, gel stained by Western blotting. Protein was detected using a rabbit antiserum raised against an E. coli expressed fusion protein, containing residues 196-293 of mature sIL5R following 81 amino acids of influenza virus structural protein NS1 (Shatzman et al., 1990). Samples are: lane 1, pMT-shIL5R culture medium from induced cells; lane 2, pMT-hIL5R cells uninduced; and lane 3, pMT-hIL5R cells induced.



shIL5R-Fc (553 residues, 62,542 Da) migrated by SDS-PAGE at 67.7 kDa under reducing conditions and 145 kDa under non-reducing conditions (Fig. 1 A), indicating the molecule was a disulfide-linked immunoglobulin-like dimer. It eluted from Superose 6 with an apparent mass of 178 kDa (data not shown). MALD-MS showed a mass of 140,280 Da, indicating that 11% of the mass was from carbohydrate (data not shown). Analytical ultracentrifuge analysis showed that shIL5R-Fc had a tendency to associate at higher concentrations. These data were best fit to a monomer-tetramer equilibrium, with 50% tetramer formation at 6 10 M, under these solvent conditions. With this affinity, the shIL5R-Fc was <4% associated at 10 M. Thus, self-association of receptor was concluded not to be a complicating factor in the subsequent biophysical experiments.

Carbohydrate Analysis

Monosaccharide composition was determined as described previously (Anumula and Taylor, 1989). Oligosaccharide map analysis indicated that the major oligosaccharide of both proteins had the structure of ManGlcNAcFuc (data not shown). The high mannose content, lack of sialic acid, and presence of two glucosamines and a fucose at each glycosylation site are typical for proteins expressed in Drosophila (Culp et al., 1991). Bioactivity of Recombinant hIL5 and Receptor Proteins

An IL5- and IL3-dependent B cell proliferation assay was used to verify the functional activity of the recombinant proteins. Drosophila-expressed hIL5 caused the proliferation of murine Bcells (specific activity = 1.7 10units/mg), consistent with the cross-recognition of hIL5 by the murine receptor. The level of activity obtained with Drosophila-expressed protein was similar to that seen with native hIL5 produced by peripheral blood T lymphocytes stimulated with mitogen. Soluble hIL5R was able to antagonize the activity of hIL5 in the same Bassay, with an ICof 12 n M. In contrast, the response elicited by murine IL3 was unaffected by shIL5R at concentrations up to 40 n M. Anti-IL3 antibody, on the other hand, completely inhibited the response to IL3.

The adequacy of Drosophila-specific glycosylation for binding activity was assessed by enzymatic deglycosylation of shIL5R and assay by cross-linking to I-hIL5. As expected from previous data (Proudfoot et al., 1990), PNGaseF-treated I-hIL5 produced a 1:1 cross-linked product with untreated shIL5R. However, using similar enzymatic deglycosylation with PNGaseF, untreated I-hIL5 could not be cross-linked to deglycosylated shIL5R (data not shown). The results suggest that receptor glycosylation, although not hIL5 glycosylation, is required to stabilize receptor-ligand interaction. Crystal Structure of Deglycosylated hIL5

Crystals of deglycosylated hIL5 were grown from 27% PEG-600 or PEG-1000 in 100 m M Tris buffer, pH 8.0, at 25 °C. The structure determined using these crystals is essentially identical to that previously reported by Milburn et al. (1993) for E. coli-expressed hIL5, except in two regions (Fig. 3). These are the NH-terminal helix and the loop between helix 2 and helix 3. Superposition of the 216 -carbon atoms from the two structures gives an root-meam-square deviation of 1.6 Å, while superposition of 1,742 backbone and side chain atoms gives an root-mean-square deviation of 2.1 Å. Overall, the crystal structure of deglycosylated, Drosophila-expressed hIL5 confirms its basic structure as a covalent dimer of two 4-helix bundles, similar to that determined before for E. coli-expressed hIL5 (Milburn et al., 1993). Determination of Stoichiometry of IL-5shIL5R Complex by Analytical Ultracentrifugation

A species analysis of sedimentation equilibrium data (see ``Experimental Procedures'') was used to examine the stoichiometry of the hIL5receptor interaction. The results of analyses for two different mixtures of shIL5R and IL5 are given in Fig. 4, A and B. In both cases, the data fit best to a mixture of the two components, hIL5 receptor (44.0 kDa) and the 1:1 complex of shIL5R and IL5 homodimer (72.1 kDa). There is no evidence from either data set for the presence of a 2:1 complex (116.1 kDa) or free IL5 (28.1 kDa). These data show that shIL5R and IL5 (dimer) associate in solution as a 1:1 molar complex at concentrations up to 40 µ M. The data show no evidence of weak complexes, which likely would be observed under the conditions of the ultracentrifuge experiments if they had Kvalues of 0.1 m M or less. Cocrystallization of hIL5 and shIL5R and Cocrystal Analysis

Needle crystals were grown from a mixture of Drosophila-expressed hIL5 and shIL5R in the presence of 24% PEG 1000 (v/v), 100 m M sodium acetate/acetic acid, pH 5.0, incubated at 7 °C. Slightly larger crystals were grown from 27% PEG 600 (v/v) in the same buffer.

We analyzed the contents of the cocrystals for the abundance of both putative components of the hIL5shIL5R complex by thoroughly washing and dissolving them as described under ``Experimental Procedures'' and then examining their contents by SDS-PAGE. The gel scan of the Coomassie-stained gel is shown in Fig. 5. Gel scanning along with standards containing various ratios of the two components showed that the ratio of peaks of hIL5 to shIL5R in the cocrystals was 1.39, compared to a ratio of 1.26 for an equimolar mixture of the two proteins. In silver-stained SDS-PAGE, the ratio for the equimolar mixture was 1.58 while that of cocrystal was 1.59. These results indicate that the stoichiometric ratio of hIL5 dimer versus shIL5R in the cocrystal is 1:1, consistent with the results obtained by analytical ultracentrifugation. Equilibrium Binding Properties by Titration Microcalorimetry

We examined further aspects of the equilibrium of the hIL5receptor interaction using titration calorimetry, particularly to obtain a measure of the equilibrium affinity of the solution interaction. Fig. 6 shows representative titration microcalorimetric data for the equilibrium binding reaction of hIL5 with its soluble receptor. Data were fit according to a single-site binding model. Titration experiments were carried out as a function of temperature in order to determine the heat capacity change for the reaction of hIL5 with shIL5R. High quality data were obtained from 17 to 35 °C. Data could not be measured at lower temperatures due to the decrease in signal-to-noise with temperature. This is a consequence of the heat capacity change of the reaction. High temperature data could not be measured either, presumably due to thermal inactivation of shIL5R. At 45 °C no heat of binding could be detected, suggesting the shIL5R was unfolded.

The thermodynamics for hIL5 binding to shIL5R and shIL5R-Fc are summarized in Table I. The tabulated Kvalues at 25 °C are based on measurements of Kvalues at higher temperature where the affinity was a little weaker, and therefore somewhat easier to measure. The Kvalue for IL5 binding to sIL5R was measured as 5.9 n M at 33 °C, and the Kvalue for IL5 binding to sIL5R-Fc was measured as 3.2 n M at 35 °C. Both measurements were done in triplicate and gave results within a factor of two of the reported values. These experimental Kvalues were corrected to 25 °C according to the known temperature dependence (Robert et al., 1989) using enthalpy and heat capacity changes determined from analysis of the data in Fig. 7. The error associated with any single measurement of the Kwas about a factor of 2-3, due to the relatively high affinity of the binding interaction and the small enthalpy change for the reaction. Nevertheless, repeat measurements of the Kconsistently gave values well within the factor of 2-3 error limit.

The observed molar ratio of hIL5 bound to shIL5R and shIL5R-Fc was 0.98 ± 0.03 and 1.61 ± 0.05, respectively. These values are consistent with stoichiometries of 1:1 and 2:1 for hIL5 binding to shIL5R and shIL5R-Fc, respectively. The higher stoichiometry for the Fc chimera (molecular mass 140 kDa) is expected since there are two receptor chain domains/chimera. The molar ratio of 0.98 found for shIL5R is a demonstration of its high functional purity given the 1:1 stoichiometry determined by analytical ultracentrifugation. On the other hand, the ratio of 1.61 for the shIL5R-Fc case suggests that 20% of the potential binding sites were inactive.

Fig. 7 shows the temperature dependence of the enthalpy change for the reaction of hIL5 with shIL5R and shIL5R-Fc. The slope of this temperature dependence is equal to the heat capacity change for the equilibrium reaction and is due in large part to changes in buried surface area of polar and apolar amino acid side chains (see ``Discussion''). Regression analysis of the data in Fig. 7 gives the best-fit H and Cvalues for the reaction of hIL5 with shIL5R and shIL5R-Fc listed in . Correction of the measured enthalpies for buffer ionization heats was not necessary, by virtue of the low ionization heat of phosphate (Christensen et al., 1976) and the pH independence of the hIL5-shL5R binding affinity in the neutral pH range (Morton et al., 1994).


Figure 7: Enthalpy change for IL-5 binding to shIL5R and shIL5R-Fc versus temperature. Circles are for shIL5R and squares for shIL5R-Fc. Slope of the best-fit line yields the heat capacity change, which is identical for the two receptor constructs, -0.65 kcal/mol hIL5/degree. The heat capacity change for hIL5 binding is related to the amount of surface area buried upon complexation with receptor (see text). Each enthalpy change value was measured by titration calorimetry as indicated in Fig. 6.



It can be seen from Fig. 7that the absolute values of the IL5-binding enthalpies and the temperature dependences for shIL5R and shIL5R-Fc are, within error, identical. These findings, in combination with affinity measurements (), are strong evidence that the molecular details of the reactions of hIL5 with both types of soluble receptor constructs are the same. Furthermore, the linearity of temperature dependence of the binding enthalpy supports the simple binding model. Comparison of the tabulated binding thermodynamics () for the interaction of soluble receptor with both the glycosylated or deglycosylated form of IL5 show the absence of any appreciable changes upon conversion to the deglycosylated form. The data for the deglycosylated form were obtained from a single measurement at 28 °C. A small temperature correction to 25 °C was made by assuming that the Cof the reaction is the same as it is for the glycosylated hIL5. Kinetics and Equilibrium Binding Properties of hIL5 with Soluble Receptor from Optical Biosensor Analysis

The kinetics of binding of hIL5 to shIL5R-Fc were examined with the receptor components immobilized on sensor chips of a BIAcore biosensor. Sensorgrams and replots of the data according to Equations 5 and 6 are shown in Fig. 8. The linearizing replots for both the association and dissociation processes showed nonlinearity (Fig. 8, B and C). This type of curvilinearity also is evident in the previously reported biosensor analysis of hIL5 interaction with shIL5R (Morton et al., 1994). The measured dissociation rate (slope of the plot in Fig. 8C) is increased by including shIL5R as a competitor in the dissociation buffer, but the curvilinearity persists. The 400 n M concentration of competitor used in the dissociation phase in Fig. 8 C is great enough (close to two orders of magnitude greater) compared to Kthat little or no rebinding is likely to occur under these conditions. This result argues that the curvilinearity in the dissociation phase is not due to rebinding.


Figure 8: Kinetic analysis of on and off rates of interaction of hIL5 with immobilized shIL5R-Fc. A, sensorgrams showing binding of hIL5 (in response units), obtained at hIL5 concentrations of 30, 20, 15, 10, 5, and 2 n M, to shIL5R-Fc immobilized to the sensor chip via Protein A. For each sensorgram, buffer was pumped over the sensor chip at 5 µl/min. At 100 s the buffer was replaced by the hIL5 solution. The increase in response shows the binding of hIL5 from solution to the immobilized receptor (the association phase). At 460 s the injection ended and was replaced by buffer. The decay in response represents the dissociation of bound hIL5 (the dissociation phase). B, association. The association phases of the sensorgrams in A were replotted, according to Equation 5, as the slope of the curve at a given time ( dR/dt) versus relative response at that time ( R). The replot is shown for the experiment with 27 n M hIL5. The curvilinearity of this replot, typical of the data obtained at all concentrations in particular at the five highest concentrations, is not expected from Equation 4 for a single exponential process. The dashed line is the linear fit to the data from 235 to 295 s used to calculate k. Inset, plot of k values (see ``Experimental Procedures,'' Equation 5) obtained at all concentrations. The kvalue was determined from the slope of this plot. C, dissociation. The dissociation phase of the sensorgram obtained at highest hIL5 concentration in A (30 n M) was replotted as the log (response at start of dissociation/response at time n after dissociation) versus time (Equation 6). The data shown are for dissociation after the injection of 30 n M hIL5 on immobilized shIL5R-Fc. A comparison also is shown of dissociation phases for hIL5-shIL5R-Fc interaction with and without 400 n M shIL5R in dissociation buffer. Curvilinearity of the plots in C is evident, suggesting that dissociation does not behave according to Equation 4.



The curvilinearity of both the association and dissociation data as shown in Fig. 8 suggests that binding does not fit to a simple (A + B) to AB binding model (Equation 4). The results could fit to a conformational isomerization model as suggested by thermodynamics analysis from titration calorimetry; this possibility is addressed under ``Discussion.''

In a previous study, analysis of data from the early or faster phases of the association (excluding the very early phase which likely is dominated by mass transport) and dissociation processes yielded kand kvalues, and consequent Kvalues from kand k, which fit with the Kobtained by steady state analysis (Morton et al., 1994). Hence, in the current work, we estimated kand kvalues from, respectively, the pseudolinear portion of the association phase (from 175 to 255 s, corresponding to 187.2-299.7 response units, in the example shown in Fig. 8B) and the first 50 s of the dissociation phase. The average values obtained from two similar experiments (including that in Fig. 8) were k= 4.9 ± 0.3 10 Msand k= 3.7 ± 0.1 10s. These calculated values are taken as averages for the hIL5shIL5R-Fc interaction in the sensor assay given the multiphasic nature of the kinetics. The Kcalculated from the rate constant estimates is 7.6 ± 0.3 n M, which is in reasonable agreement with the titration calorimetry results. hIL5 Binding to Full-length Chain Receptor in Drosophila Cells

The affinity of hIL5 binding to membrane-bound receptor was examined using the full-length chain expressed in Drosophila. As shown in Fig. 9, iodinated hIL5 bound to Drosophila cell membranes containing full-length hIL5R, with the binding being saturable and competed by unlabeled hIL5. Nonlinear regression analysis of the binding data yielded a Kvalue of 6 n M, a value within a factor of 2-3 of the values determined by both titration calorimetry and biosensor kinetic analysis. This agreement underscores that the soluble component of the receptor chain is sufficient for hIL5 recognition and that the membrane-spanning and intracellular domains contribute relatively little energetically to direct hIL5 interaction.


DISCUSSION

Drosophila Expression Yields Functionally Active Recombinant hIL5 and hIL5 Receptor

In this study, we produced large quantities of recombinant hIL5 and two forms of its receptor, shIL5R and shIL5R-Fc, in the Drosophila expression system and used this material to determine interaction properties of the hIL5receptor complex. Drosophila-expressed proteins (van der Straten et al., 1989; Angelichio et al., 1991; Culp et al., 1991) are glycosylated, hence enabling correlation of their properties with those of naturally glycosylated proteins. Carbohydrate analysis of the hIL5 and receptor forms produced here revealed molecular masses somewhat lower than their fully human counterparts. Nonetheless, the Drosophila-derived hIL5 was highly active in the B cell proliferation assay, and the Drosophila-expressed shIL5R and shIL5R-Fc both inhibited the cellular activity of the hIL5. Hence, the properties determined for the Drosophila-expressed proteins can be considered close reflections of those of fully human proteins.

The Drosophila expression system also allowed the production of cells containing full-length receptor incorporated into the extracellular membrane surface. The binding affinity of soluble and full-length receptor could be compared, with the expectation that the carbohydrate structures would be similar for these two Drosophila-expressed forms of the receptor and hence that the only difference would be the presence of the membrane spanning region and cytoplasmic tail in the full-length form versus their absence in the soluble form.

The Stoichiometry of hIL5 and shIL5R is 1:1

In this study, we confirmed that hIL5 dimer and shIL5R form a 1:1 complex. This was judged initially (data not reported) by chemical cross-linking of I-hIL5, an experiment similar to that reported previously for COS- and baculovirus-expressed proteins (Devos et al., 1993). We then obtained direct evidence of a 1:1 complex between hIL5 and its receptor by molecular weight analysis of the complex using equilibrium sedimentation (Fig. 4). Furthermore, both titration calorimetry experiments and analysis of hIL5-shIL5R cocrystals showed a 1:1 molar ratio for the reaction of hIL5 with its receptor. These data demonstrate the absence of higher stoichiometries and aggregates for concentrations that approach millimolar.


Figure 4: Stoichiometry of hIL5 and shIL5R by analytical ultracentrifugation. A, equilbrium analytical ultracentrifuge data for a mixture of shIL5R and hIL5, total protein concentration 10 M, with a small molar excess of shIL5R over hIL5, as discussed under ``Experimental Procedures.'' Data obtained were fit to Equation 3 in terms of the species which can be identified (see ``Experimental Procedures''). In this experiment, only terms for shIL5R and the shIL5RhIL5 1:1 complex were needed to fit the data. When terms for the other species, e.g. hIL5 and shIL5RhIL5 2:1 complex, were included, the fit did not converge. B, a similar experiment to Fig. 5 A except that initially shIL5R was in larger excess to hIL5 (total protein concentration 6 10 M).



The 1:1 stoichiometry for the hIL5receptor interaction is unusual, given that the IL5 dimer has two 4-helix bundle domains and each resembles the 4-helix bundle domain of monomeric growth factor proteins (Abdel Meguid et al., 1987; Redfield et al., 1991; de Vos et al., 1992). One might therefore expect each IL5 4-helix bundle to bind at least one receptor molecule. While the data here for 1:1 stoichiometry are for soluble receptor, the observation of similar affinities of soluble and full-length receptor (see below) suggests that hIL5 also binds to the latter in the same 1:1 manner. By extrapolation from other cytokine receptor systems, signal transduction by IL5 may require aggregation of occupied receptors (Boulay and Paul, 1992). Since the stoichiometry of the hIL5receptor chain interaction is 1:1, receptor aggregation leading to signal transduction presumably must involve factors other than IL5 and chain alone. An --IL5 heterotrimerization could provide the aggregation needed to trigger signal transduction.

Affinity of Soluble and Membrane-associated Receptor Subunit Are Similar But Lower Than That for Heterocomplex

The affinity of IL5 for receptor was measured with both shIL5R and shIL5R-Fc by titration calorimetry and real-time biosensor kinetic analysis. In both instances, the Kvalues obtained were in the range of 2-8 n M at 25 °C. We also observed that the IL5 binding affinity of the soluble form of the receptor is very similar to that of the membrane-bound, full-length form. Of note, the 2-8 n M affinities reported here for soluble and membrane-associated Drosophila-expressed receptor chain are somewhat lower than the 0.6-1 n M values reported before for chain expressed in mammalian cells (Tavernier et al., 1991, 1992; Takaki et al., 1993). The reason for this difference is not fully understood at present.

The similarity of affinities for hIL5 binding to both soluble and membrane-bound (full-length) chain forms strongly suggests the absence of structure-energy coupling between IL5 binding and membrane-induced structural constraints in the receptor. This argues against the possibility of transmembrane signal transduction occurring through the receptor alone, in agreement with the cell proliferation studies of Takaki et al. (1993).

The data reported here confirm that the affinity of hIL5 for its recognition receptor subunit, , is of quite high affinity. Nonetheless, the IL5 affinity of cells expressing both and chains of receptor has been reported to be greater than for the receptor alone by a factor of 2 to 4 (Tavernier et al., 1991, 1992; Takaki et al., 1993). Although this difference is rather modest, an increase in IL5 affinity for versus alone is in agreement with the binding energetics expected for a ligand-induced oligomerization equilibrium ( cf., Wyman and Gill, 1990). The ability of IL5 to induce - heterodimerization requires its affinity for the complex to be higher than that for either or alone. Of note, while the increase is modest in hIL5 affinity for cells expressing both and chains versus chain alone, the intrinsic affinity increase for the heterocomplex may be considerably higher but offset by free energy needed to drive the receptor heterodimerization equilibrium. IL5-induced receptor oligomerization is a part of the predicted model of IL5-receptor interaction put forth below.

Binding Thermodynamics Suggest Receptor Isomerization in the hIL5-shIL5R Interaction

The interaction analyses carried out in this work suggest that conformational change occurs upon IL5receptor chain interaction. This conclusion was drawn mainly from titration calorimetry data based on analysis of the binding thermodynamics, in particular the binding entropy change (see below). Biosensor-based kinetic analysis provided data consistent with this conformational isomerization model.

The total surface area buried in the binding reaction of hIL5 and its soluble receptor chain can be estimated from the thermodynamics of the interaction. Burial of apolar residues in particular has long been known to be proportional to the heat capacity change of a given protein-protein interaction ( cf., Tanford, 1980; Privalov and Gill, 1988). Recently, Murphy and Freire (1992) have extended this approach by accounting for the change in accessible surface area ( ASA) of both apolar and polar residues from the experimental binding Caccording to the empirical relationship,

  

On-line formulae not verified for accuracy

The very large value of 4160 Åfor the total amount of surface area buried during the binding of IL5 to the shIL5R implies either a very large interfacial surface between the two proteins or structure changes coupled to the binding reaction. Previous work suggests that much of the structural determinants for IL5-receptor binding lie in the A and D helices of IL5 (Kodoma et al., 1991; McKenzie et al., 1991; Shanafelt et al., 1991), and a model proposing the involvement of the A and D helices has recently been put forward (Goodall et al., 1993). The size of the accessible surface area of a single A and D helix pair, as judged by visual inspection of the crystal structure, is approximately 500 Å, while an entire face of the IL5 dimer exposes approximately 1900 Å. Hence, either an entire face of the IL5 dimer is involved in receptor binding, or conformational changes are coupled to binding which cause burial of surface area in other regions of IL5 and/or its receptor. From mutagenesis studies,() at least significant numbers of residues on the hIL5 AD surface can be changed to Ala without loss of shIL5R binding. This argues against binding through the entire face of the IL5 dimer and, rather, for conformational change. Since IL5 is comprised of two 4-helix bundles, with helix sharing between the bundles, and shows considerable thermal stability relative to shIL5R,() significant conformational changes seem more likely to occur in the receptor than in IL5 (although some degree on conformational isomerization in IL5 itself cannot at present be excluded). Cytokine receptor isomerization has been proposed recently for leukemia inhibitory factor, and multiple kinetic dissociation rates of leukemia inhibitory factor from its receptor as observed in a cell-binding filter assay were proposed to reflect this isomerization (Layton et al., 1994). For the IL5-receptor system, allosteric modification of IL5 upon chain binding has been hypothesized as a possible way to explain both the lack of binding to a second chain and increased affinity for the chain (Devos et al., 1993). Such IL5 allosterism, if it occurs, could involve subtle conformational change and might occur in addition to the conformational change of receptor proposed in the present study to occur upon ligand binding.

Further analysis of the possibility that conformational changes are coupled to the IL5-shIL5R binding reaction is provided from their binding entropy changes. The total entropy change (S) for binding has contributions from several terms (Spolar and Record, 1994):

 

On-line formulae not verified for accuracy

The multiphasic association and dissociation steps observed in the current study in biosensor kinetic analysis of hIL5shIL5R-Fc interaction (and also previously for the interaction of hIL5 with shIL5R (Morton et al., 1994)) are consistent with conformational isomerization such as that concluded from titration calorimetry. Interestingly, in a previous biosensor study (Fisher et al., 1994), the kinetics of binding of oncoproteins to DNA also demonstrated multiphasic kinetics, and a similar conclusion of conformational isomerization was proposed. It must be cautioned however that, while multiple on and off rates suggest a more complex binding model than as shown in Equation 4, the deviation from this simple model may be caused by factors other than conformational isomerization, for example those that derive from the biosensor method itself. These include immobilized or soluble analyte heterogeneity, rebinding after dissociation, and rate-limiting mass transport. In general, independent evidence is required to test the hypothesis of receptor isomerization when this is suggested by biosensor data. The results obtained in the current study by titration calorimetry provide a step in that direction. Interaction studies with mutant forms of hIL5 and receptor also would be of help.

Implications for the Mechanism of IL5 Receptor Interaction and Signal Transduction

The data in the current work, combined with the prevailing state of understanding on the interaction of IL5 and its receptors ( cf. Kioke and Takatsu, 1994; Goodall et al., 1993), lead to a prediction of IL5receptor interaction and signal transduction shown in Fig. 10. Fig. 10 A depicts schematically both the 1:1 stoichiometry of hIL5 chain interaction and the conformational isomerization which have been observed in this study. Fig. 10 B represents a prediction of how conformational change, in conjunction with binding of chain, could lead to signal transduction. This model accounts for the higher affinity of hIL5 for , heterocomplex versus subunit alone (Tavernier et al., 1991, 1992; Takaki et al., 1993). It also accounts for evidence showing that IL5 can be cross-linked with either or (Migita et al., 1991; Devos et al., 1991). That IL5 can bind directly to the receptor chain fits with the view that, while the IL5 does not bind to chain alone, it can do so upon binding to the chain (Shanefelt et al., 1991; Lopez et al., 1992). The model in Fig. 10B shows an absence of a tightly preformed heterocomplex, a feature which also can be argued from the previously observed cross-competition among IL5 and GM-CSF on cells coexpressing the common chain and the chains specific for the two cytokines (Tavernier et al., 1991). The presence of preformed but weakly associated heterocomplexes cannot be ruled out by the current data. Fig. 10 B indicates that IL5 may either bridge the and receptors or may induce a conformational change in the receptor which then allows for direct interaction between the two receptor molecules. Both are possible given the current data.


Figure 10: Models of 1:1 complex of IL5 with receptor chain and the conformational isomerization of IL5 receptor that may promote receptor heterodimerization with the chain and consequent signal transduction. A, model showing 1:1 stoichiometry and conformational isomerization in hIL5- chain interaction. This model is drawn from the experimental data of this study. B, an extension of model A, based on data in this study and prevailing published data, depicting how the and chains may interact with each other upon association with hIL5. Receptor subunit association is viewed as a possible trigger for signal transduction. This view is analogous to the association of human growth hormone receptor subunits upon interaction with human growth hormone (Wells and de Vos, 1993).



The model as shown in Fig. 10B for IL5 receptor activation resembles that of growth hormone, in which signal transduction is implicated to require hormone-induced receptor homodimerization (Wells and de Vos, 1993). On the other hand, the model contrasts with that of the closely related system GM-CSF, where pre-existence of a receptor heterodimer has been postulated (Ronco et al., 1994). However, in that work, the data were indirect, and at any rate the preformed complex postulated could well be weak, at least in the absence of GM-CSF. Finally, it should be noted further that, while the model in Fig. 10B suggests formation of a 1:1:1 complex of hIL5 with and chains, greater states of aggregation, as suggested by Boulay and Paul (1992), cannot be ruled out in particular as they may occur on the cell surface. This possibility is suggested by the observed tendency of both shIL5R and shIL5R-Fc to aggregate in solution (Fig. 2). The potential for chain to dimerize in the absence of ligand (or chain) has been hypothesized recently (D'Andreas et al., 1994).


Figure 2: Equilibrium sedimentation of hIL-5 and shIL5R. A, global analysis of equilibrium sedimentation data for IL-5. The upper three subpanels are the distributions of residuals (observed-fitted values) for the analyses of the data in terms of Equation 1 (see ``Experimental Procedures'') at 30,000, 20,000, and 25,000 revolutions/min, respectively. The lower panel is the primary data taken at the same rotor speeds. Data were taken 23, 73, and 91 h after the start of the run. The concentration range examined in this analysis was from 10to 10 M IL-5. The initial loading concentration was 2 10 M ( A= 0.4). B, global analysis of equilibrium sedimentation data for shIL5R. The upper three subpanels are the distributions of residuals for the analyses of the data in terms of Equation 1 (see ``Experimental Procedures'') at 15,000, 20,000, and 25,000 revolutions/min, respectively. The lower panel contains the primary data taken at the same rotor speeds. Data were taken 22, 44, and 66 h after the start of the run. The concentration range examined in this analysis was from 10to 10 M shIL5R. The initial loading concentration was 6 10 M ( A= 0.4). The solvent was 10 m M sodium phosphate buffer, 150 m M NaCl, pH 7.4.



Final Comments

The Drosophila expression system established in this study has enabled quantitative questions to be addressed concerning the interaction of hIL5 and its receptor, by providing both large scale production of soluble recombinant proteins as well as membrane-incorporated full-length recombinant receptor. The functional activity and, for hIL5, crystal structure confirm that the Drosophila-expressed proteins are adequate surrogates of the naturally occurring proteins for interaction analysis. The data show the formation of a 1:1 hIL5shIL5R complex of n M affinity, which poses the possibility that one receptor molecule can bind to both 4-helix bundle units of the hIL5 dimer simultaneously. (The same may be true for the chain.) The data also show that essentially all of the binding energy of interaction of full-length hIL5R derives from its soluble domain. Finally, analysis of the binding thermodynamics and kinetics suggests that conformational change is likely to accompany hIL5receptor interaction. Such conformational isomerization could be an important component in signal transduction. Beyond this study, the Drosophila expression system promises to enable further mechanistic studies at both the molecular and cell level, including high resolution structure determination of hIL5shIL5R complex, effects of mutagenesis on IL5-receptor recognition, and affinity measurements of the chain when expressed in membranes.

  
Table: Binding thermodynamics of human IL5 and its soluble -receptor at 25 °C, pH 7.4, 20 m M potassium phosphate, 150 m M NaCl



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: IL5, interleukin 5; EDC, N-ethyl- N`-(3-diethylaminopropyl)carbodiimide; GM-CSF, granulocyte-macrophage colony stimulating factor; hIL5, human interleukin 5; IgG, immunoglobulin G; IL3, interleukin 3; MALD-MS, matrix-assisted laser desorption mass spectrometry; NHS, N-hydroxysuccinimide; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; PNGase, N-glycanase; shIL5R, soluble human interleukin 5 receptor chain; shIL5R-Fc, soluble human interleukin 5 receptor -Fc chimera; tPA, tissue plasminogen activator.

T. Morton, J. Li, R. Cook, and I. Chaiken, unpublished results.

We have recently measured the half-unfolded temperatures for hIL5 and shIL5R as 67 and 44 °C, respectively, by monitoring changes in circular dichroism.


ACKNOWLEDGEMENTS

We are grateful to Dr. Steven R. Jordan (Glaxo, Research Triangle Park, NC) for atomic coordinates of E. coli-expressed human IL5 and to Dr. Kalyan Anumula (at SmithKline Beecham) for his assistance in carbohydrate analysis.


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