FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1965798918> ?p ?o ?g. }

• W1965798918 endingPage "15769" @default.
• W1965798918 startingPage "15762" @default.
• W1965798918 abstract "Interleukin-5 (IL-5) is a cytokine that plays a major role in the differentiation and activation of eosinophils. In order to identify which charged residues of human IL-5 are important in binding to its receptor and subsequent cellular activation, we have systematically replaced all of the clusters of charged amino acids with alanine residues. The mutants have been expressed in Escherichia coli, renatured, and purified. They were assayed for ability to cause proliferation of the erythroleukaemic cell line TF-1 and the up-regulation of eosinophil adhesion to ICAM-1. In addition, we studied receptor binding using either immobilized recombinant IL-5 receptor α-chain or the α/β-receptor complex expressed on TF-1 cells. The key charged residue involved in binding to the β-chain of the receptor is Glu-12. This residue is in an identical position to those previously identified in IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in binding to the receptor β-chain. The α-chain binding site is shown to involve the side chains Arg-90 and Glu-109, located in the second β sheet and after the end of the fourth helix, respectively. It is unique to IL-5 and does not occur in IL-3 or GM-CSF. Understanding the topology of the interaction of IL-5 with its receptor chains will help in the search for rationally designed antagonists of IL-5 function. Interleukin-5 (IL-5) is a cytokine that plays a major role in the differentiation and activation of eosinophils. In order to identify which charged residues of human IL-5 are important in binding to its receptor and subsequent cellular activation, we have systematically replaced all of the clusters of charged amino acids with alanine residues. The mutants have been expressed in Escherichia coli, renatured, and purified. They were assayed for ability to cause proliferation of the erythroleukaemic cell line TF-1 and the up-regulation of eosinophil adhesion to ICAM-1. In addition, we studied receptor binding using either immobilized recombinant IL-5 receptor α-chain or the α/β-receptor complex expressed on TF-1 cells. The key charged residue involved in binding to the β-chain of the receptor is Glu-12. This residue is in an identical position to those previously identified in IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in binding to the receptor β-chain. The α-chain binding site is shown to involve the side chains Arg-90 and Glu-109, located in the second β sheet and after the end of the fourth helix, respectively. It is unique to IL-5 and does not occur in IL-3 or GM-CSF. Understanding the topology of the interaction of IL-5 with its receptor chains will help in the search for rationally designed antagonists of IL-5 function. INTRODUCTIONInterleukin-5 (IL-5)1( 1The abbreviations used are: ILinterleukinCSFcolony-stimulating factorM-CSFmacrophage colony-stimulating factorsGM-CSFgranulocyte-macrophage colony-stimulating factor. )is a T-cell-derived cytokine that causes the differentiation of eosinophils from their bone marrow precursors and also plays a role in their activation processes. Normally, these cells are involved in the killing of parasites. However, in inflammatory diseases such as asthma and atopic dermatitis (Sanderson, 1992Sanderson C.J. Adv. Pharmacol. 1992; 23: 163-177Crossref PubMed Scopus (23) Google Scholar, Takatsu, 1992Takatsu K. Curr. Opin. Immunol. 1992; 4: 299-306Crossref PubMed Scopus (75) Google Scholar), eosinophil numbers are raised, and the presence of toxic eosinophil products has been detected. Furthermore, when monoclonal antibodies against IL-5 are given in guinea pig models of allergic asthma, there is a diminution of the eosinophil influx, and at high concentrations, a decrease in the bronchial hyperreactivity (a measure of the underlying inflammation (Mauser et al., 1993Mauser P.J. Pitman A. Witt A. Fernandez X. Zurcher J. Kung T. Jones H. Watnick A.S. Egan R.W. Kreutner W. Adams G.K. Am. Rev. Resp. Dis. 1993; 148: 1623-1627Crossref PubMed Google Scholar)). Based on these data, an IL-5 antagonist would be expected to have useful pharmacological properties.We have previously expressed and purified recombinant human IL-5 (Proudfoot et al., 1990Proudfoot A.E.I. Fattah D. Kawashima E.H. Bernard A. Wingfield P.T. Biochem. J. 1990; 270: 357-361Crossref PubMed Scopus (38) Google Scholar). The protein is a disulfide-linked homodimer, and, although it is glycosylated in its natural form, the deglycosylated IL-5 is equipotent in all in vitro assays. IL-5 has been crystallized, and the three-dimensional structure solved at a resolution of 2.4 Å (Hassell et al., 1993Hassell A.M. Wells T.N.C. Graber P. Proudfoot A.E.I. Anderegg R. Burkhart W. Jordan S.R. Milburn M.V. J. Mol. Biol. 1993; 229: 1150-1152Crossref PubMed Scopus (7) Google Scholar, Milburn et al., 1993Milburn M.V. Hassell A.M. Jordan S.R. Proudfoot A.E.I. Graber P. Wells T.N.C. Nature. 1993; 363: 172-176Crossref PubMed Scopus (229) Google Scholar). IL-5 has a novel two-domain structure, with each domain showing a striking similarity to the four-helix bundle seen for IL-2 (Bazan and McKay, 1992Bazan J.F. McKay D.B. Science. 1992; 257: 410-413Crossref PubMed Scopus (193) Google Scholar); IL-4 (Smith et al., 1992Smith L.J. Redfield C. Boyd J. Lawrence G.M.P. Edwards R.G. Smith R.A.G. Dobson C.M. J. Mol. Biol. 1992; 224: 899-904Crossref PubMed Scopus (113) Google Scholar); M-CSF (Pandit et al., 1992Pandit J. Bohm A. Jancaril J. Halenbeck R. Koths K. Kim S.-H. Science. 1992; 258: 1358-1362Crossref PubMed Scopus (162) Google Scholar) and GM-CSF (Diederichs et al., 1991Diederichs K. Boone T. Karplus P.A. Science. 1991; 254: 1779-1782Crossref PubMed Scopus (166) Google Scholar). Amongst these structures, IL-5 is unique in that the formation of each four-helix bundle domain requires the participation of two chains of IL-5.IL-5 binds to a heterodimeric receptor consisting of a specific α-chain, which binds its ligand at picomolar concentrations (Tavernier et al., 1991Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (493) Google Scholar; Murata et al., 1992Murata T. Takaki S. Migata M. Kikuchi Y. Tominaga A. Takatsu K. J. Exp. Med. 1992; 175: 341-351Crossref PubMed Scopus (162) Google Scholar). Signaling requires the presence of a β-chain that is also shared with IL-3 and GM-CSF (Tavernier et al., 1991Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (493) Google Scholar). Studies to define the binding site for either of the receptor chains on IL-5 have been limited. The most detailed ones published to date, a series of human/mouse chimerae, have shown that the carboxyl-terminal third of the molecule is important in controlling the human to mouse species selectivity (McKenzie et al., 1991McKenzie A.N.J. Barry S.C. Strath M. Sanderson C.J. EMBO J. 1991; 10: 1193-1199Crossref PubMed Scopus (55) Google Scholar). In this study, chimeric proteins in which the last 36 residues of the protein had been replaced with those from the species of interest had retained biological activity. In addition, the importance of the carboxyl terminus in the biological activity of human IL-5 had been suggested from experiments where CNBr cleavage of the carboxyl-terminal 8 amino acids results in the loss of biological activity (Kodama et al., 1991Kodama S. Tsuruoka N. Tsujimoto M. Biochem. Biophys. Res. Commun. 1991; 178: 514-519Crossref PubMed Scopus (20) Google Scholar), although this may simply be a problem of protein denaturation. Early experiments using hybrid GM-CSF and IL-5 constructs suggested that the amino-terminal helix-A of IL-5 was required for high affinity binding to the receptor (Shanafelt et al., 1991Shanafelt A.B. Miyajima A. Kitamura T. Kastelein R.A. EMBO J. 1991; 10: 4105-4112Crossref PubMed Scopus (76) Google Scholar). The binding site for the common β-chain of the receptor has been located on the first helix of IL-3 and GM-CSF (Lopez et al., 1992Lopez A.F. Shannon M.F. Barry S. Phillips J.A. Cambareri B. Dottore M. Simmons P. Vadas M.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11842-11846Crossref PubMed Scopus (35) Google Scholar, Barry et al., 1994Barry Jr., S.C. Bagley C.J. Phillips J. Dottore M. Cambareri B. Moretti P. D'Andrea R. Goodall G.J. Shannon M.F. Vadas M.A. Lopez A.F. J. Biol. Chem. 1994; 269: 8488-8492Abstract Full Text PDF PubMed Google Scholar; Hercus et al., 1994Hercus T.R. Cambareri B. Dottore M. Woodcock J. Bagley C.J. Vadas M.A. Shannon M.F. Lopez A.F. Blood. 1994; 83: 3500-3508Crossref PubMed Google Scholar). The key residue in GM-CSF is Glu-21, which is on the exposed face of helix-A. In IL-3, similar results have been obtained for Glu-22, which is predicted to be in the same position in helix-A.Previous studies of other four-α-helix bundle cytokines such as IL-4 or human growth hormone have shown that electrostatic interactions play a key role in the formation of the receptor-ligand complex (Cunningham and Wells, 1993Cunningham B.C. Wells J.A. J. Mol. Biol. 1993; 234: 554-563Crossref PubMed Scopus (485) Google Scholar; Demchuk et al., 1994Demchuk E. Mueller T. Oschkinat H. Sebald W. Wade R.C. Protein Sci. 1994; 3: 920-935Crossref PubMed Scopus (49) Google Scholar). The association rate constants for the receptor-ligand complexes in these cases predicted that electrostatic steering was important in the initial association event. We have therefore decided to initially study the charged residues of IL-5. In order to define which areas of the protein are important in binding to either the α- or the β-chain of the receptor, we have systematically analyzed all of the charged residues in IL-5. Using the alanine scanning mutagenesis technique (Cunningham and Wells, 1989Cunningham B.C. Wells J.A. Science. 1989; 244: 1081-1085Crossref PubMed Scopus (1079) Google Scholar; Hébert et al., 1991Hébert C.A. Vitangcol R.V. Baker J.B. J. Biol. Chem. 1991; 266: 18989-18994Abstract Full Text PDF PubMed Google Scholar), we have produced a set of mutant proteins where the charged side chains have been replaced by alanine. Alanine was chosen because it has a short nonpolar side chain, does not cause major conformational disturbances to the protein structure, and is also routinely found in both buried and solvent-exposed positions in proteins. By analyzing the activity of these mutant proteins in binding assays and cellular responses, we have been able to identify the two key charged interactions in the α-chain binding site and confirm the location of the β-chain binding site as Glu-12 on helix-A.EXPERIMENTAL PROCEDURESSite-directed Mutagenesis and Expression of IL-5 MutantsThe synthetic gene for IL-5 was subcloned from the plasmid pLTEX4 (Tavernier et al., 1989Tavernier J. Devos R. Van der Heyden J. Haquier G. Bauden R. Rache I. Kawashima E. Vanderkirkove J. Contreras R. Fiers W. DNA. 1989; 8: 491-501Crossref PubMed Scopus (39) Google Scholar, Proudfoot et al., 1990Proudfoot A.E.I. Fattah D. Kawashima E.H. Bernard A. Wingfield P.T. Biochem. J. 1990; 270: 357-361Crossref PubMed Scopus (38) Google Scholar) into the mutated M13mp8 supplied with the SculptorTM in vitro mutagenesis system (Amersham Corp.). Initially, we designed 15 mutants (designated A-O, see Fig. 1 and). These scanned the entire length of the IL-5 sequence, replacing the charged amino acids with alanine side chains. Multiple mutations were made where there was more than one charged side chain within a 5-amino acid stretch. All of the mutations therefore involved changes of between 1 and 3 amino acids, with the exception of mutant K, where there was a continuous run of 5 charged amino acids. Typical oligonucleotide lengths were 20-25 nucleotides. The mutated constructs were then subcloned back into the pLTEX4 vector for expression in the Escherichia coli strain W3110. Mutant proteins were expressed in E. coli, where all proteins formed inclusion bodies. Small scale cultures (500 ml) were grown in shaker flasks in all cases, and yields were typically 5 g of cell paste. Unfortunately, we were unable to obtain good expression of mutant K, and therefore we were obliged to make the five point mutations scanning the region Glu-87 to Arg-91, and express them individually (see Table II). Since the data from the initial set of mutations suggested that mutants B (K11A,E12A) and C (H20A,R21A) had impaired binding to the β-chain of the receptor, we made the four further single mutations, K11A, E12A, H20A, and R21A in order to more precisely define the binding site of the receptor. Also, since studies on IL-4 (Kruse et al., 1993Kruse N. Shen B.-J. Arnold S. Tony H.-P. Müller T. Sebald W. EMBO J. 1993; 12: 5121-5129Crossref PubMed Scopus (115) Google Scholar) had shown that replacement of the carboxyl-terminal aromatic group Tyr-124 with an acid produced an antagonist, we made the equivalent mutation (W110D) in IL-5.Table II: Open table in a new tab The proteins were purified and renatured as described previously (Proudfoot et al., 1990Proudfoot A.E.I. Fattah D. Kawashima E.H. Bernard A. Wingfield P.T. Biochem. J. 1990; 270: 357-361Crossref PubMed Scopus (38) Google Scholar), adapting the column sizes for 5 g of cell paste. The refolding was carried out at slightly lower protein concentrations (2-5 μg/ml), and including an oxidation step to facilitate dimerization typically gave a 40% yield (Graber et al., 1993Graber P. Bernard A.R. Hassell A.M. Milburn M.V. Jordan S.R. Proudfoot A.E.I. Fattah D. Wells T.N.C. Eur. J. Biochem. 1993; 212: 751-755Crossref PubMed Scopus (17) Google Scholar). The quantity of each mutant protein was calculated from its UV absorbance using a specific absorbance value based on the published amino acid sequence. The amino acid composition of mutant proteins was verified in each case by overnight hydrolysis at 105°C in 6 M HCl and quantified using a Beckman 6300 amino acid analyzer with a SICA integrator. The correct amino terminus was verified for several of the mutants using Edman degradation using an ABI 477A pulsed liquid phase sequencer. To confirm that the mutant proteins were correctly folded, 100-μg samples were placed in Centricon 10 filters and washed 5 times with 2 ml of Tris-d11-DCl buffer, pH 8.5, to remove proton-containing buffers and other exchangeable protons. NMR was carried out on a Brucker AMX-600 spectrometer using a 3-mm triple resonance microprobe. Jump-Return spectra (τ = 120 microsiemens, excitation maximum at 11 ppm), accumulated 2000 transients with a sweep width of 13 157.89 Hz.2( 2M. Banks, P. Graber, A. Proudfoot, C. Arod, B. Allet, A. Bernard, E. Sebille, M. McKinnon, T. Wells and R. Solari, submitted for publication. )Bioassays of IL-5 Mutant Proteins Radiolabeling of Interleukin-5Recombinant human IL-5 was iodinated by a modification of the chloramine-T method as described by Hébert et al., 1990Hébert C. Luskinskas F.W. Kiely J.-M. Luis E.A. Darbonne W.C. Bennett G.L. Liu C.C. Obin M.S. Gimbrone M.A. Baker J.B. J. Immunol. 1990; 145: 3033-3040PubMed Google Scholar. Three micrograms of IL-5 were incubated with 500 μCi of [125I]NaI (Amersham Corp., IMS-30) in 50 μl of 1.5 M potassium phosphate buffer, pH 7.4, in a siliconized glass tube. Following the addition of 20 μl of chloramine T solution (0.1 mg/ml) the sample was incubated at room temperature for 2 min. The reaction was terminated by the addition of 100 μl of 1 M KI solution, and the 125I-labeled IL-5 was separated from the free [125I]NaI by gel filtration on a PD10 size exclusion column (Pharmacia Biotech Inc.), using 10 mM potassium phosphate buffer, pH 7.5, containing 1% (w/v) bovine serum albumin. The specific activity of these preparations was routinely between 1500 and 2000 Ci●mmol⁻1. In later experiments, commercially labeled IL-5 (Amersham Corp.) was used and gave identical results.Binding of IL-5 to TF-1 cellsTF-1 cells were washed once with medium and resuspended in the binding medium (RPMI 1640 containing 0.5% (w/v) bovine serum albumin, 0.05% NaN3, and 20 mM Hepes buffer, pH 7.4). Binding experiments were performed at room temperature in 300 μl of binding medium containing 1 × 106 cells and 150-200 pM125I-labeled IL-5, in the presence of increasing concentrations of unlabeled wild-type IL-5 or mutant IL-5. After a 2-h incubation period, the binding mixture was layered over 200 μl of oil (84% silicone oil, 16% paraffin oil) in 500-μl tubes and centrifuged at 12,000 rpm in a bench-top microcentrifuge for 3 min. After rapid freezing, the tips of the tubes were cut just above the cell pellet and placed in vials, which were directly counted in a γ counter. Nonspecific binding was defined as the residual radioactivity when the assay was carried out in a 300-fold molar excess of unlabeled IL-5. Total specific binding was fitted to the equation for competition of ligand binding, using a single site model B = Bmaxˈ/(IC50+ [L]) where Bmaxˈ is the binding seen in the absence of competing ligand. Under circumstances where the formation of receptor ligand complex does not significantly lower the concentration of free ligand, the IC50 value is equal to the sum of the dissociation constant and the concentration of hot ligand added. Data were analyzed by Grafit 3.01 (Leatherbarrow, 1992Leatherbarrow R.J. Grafit version 3.01. Erithacus Software, Staines, United Kingdom1992Google Scholar) using simple weighting. Since some of the mutants showed significant deviations from this model, all data were also fitted to a four-parameter logistic equation B = Bmax′/IC50+ [L])n+ background.Binding to Immobilized IL-5 Receptor α SubunitThe extracellular domains of the IL-5 receptor α-subunit were expressed as a fusion protein containing the IgG binding ZZ-domain at its carboxyl terminus (Moks et al., 1987Moks T. Abrahmsén B. sterlöf B. Josephson S. stling M. Enfors S.-O. Persson I. Nilsson B. Uhlén M. BioTechnology. 1987; 5: 37-42Google Scholar). This was expressed into the culture medium of baculovirus-infected Sf9 cells and purified as will be described elsewhere.3( 3B. Allet, A. Bernard, P. Graber, and T. N. C. Wells, unpublished results. )Binding of mutant versions of IL-5 was assessed by their ability to compete with radiolabeled wild-type IL-5 binding to receptor α-chains immobilized onto scintillation beads (Amersham Corp.). The linked scintillation proximity assay was carried out in white clear bottom microtitre plates in a final volume of 200 μl.2 Briefly, 5 ng●ml⁻1 of the fusion protein, 6.5 μg●ml⁻1 rabbit IgG- and 625 μg●ml⁻1 anti-rabbit IgG-coated fluoromicrospheres (Amersham Corp.), in 50 mM Hepes buffer, pH 8.5, containing 100 mM NaCl and 0.5% (v/v) bovine serum albumin were incubated for 2 h at room temperature. 125I-labeled IL-5 (Amersham Corp.) was added to a final concentration of 100 pM and incubated at room temperature for between 4 and 24 h before being counted. Nonspecific binding was measured using a 500-fold molar excess of IL-5. Data were fitted to a single-site model for competition of binding using Grafit 3.01 (Leatherbarrow, 1992Leatherbarrow R.J. Grafit version 3.01. Erithacus Software, Staines, United Kingdom1992Google Scholar).TF-1 Proliferation AssayThe human erythroleukaemia cell line TF-1 proliferates in response to IL-5 (Kitamura et al., 1989Kitamura T. Tonge T. Terasawa T. Chiba S. Kuwaki T. Miyagawa K. Piao Y.-F. Miyazono K. Urabe A. Takaku F. J. Cell. Physiol. 1989; 140: 323-334Crossref PubMed Scopus (713) Google Scholar). Proliferation assays were performed by incubating 5 × 103 cells in 100 μl of medium (RPMI 1640 containing 25 mm Hepes, 1% L-glutamine and 10% heat-inactivated fetal calf serum), and IL-5 wild-type or mutant was added in doubling dilutions with initial concentrations of 1-10 μg/ml. Plates were incubated in 5% CO2 at 37°C for 60-72 h. The level of proliferation induced was measured using Cell Titer 96TM nonradioactive cell proliferation assay (Promega), which monitors the conversion of tetrazolium blue into its formazan product. Data were fitted to a four-parameter logistic curve using Grafit 3.01 with simple weighting.Adhesion of Purified Human EosinophilsThe adhesion of purified human eosinophils to an immobilized ICAM-ZZ fusion protein coated onto 96-well polystyrene plates was measured as described previously.4( 4D. Fattah, K. R. Page, S. Bezbaruah, R. C. Priest, C. M. Horgan, and R. Solari, submitted for publication. )Briefly, 5000 purified human eosinophils were mixed with IL-5 wild-type or mutant in a total volume of 100 μl and added to a 96-well microtitre plate that had been coated with the fusion protein ICAM-1-ZZ. After incubation at 37°C for 30 min, the induction of adhesion was assessed by measuring the endogenous peroxidase activity of the adherent eosinophils.RESULTSDefining the Regions of IL-5 Important in Biological ResponsesWe have systematically replaced the charged amino acids of IL-5 (aspartic acid, glutamic acid, arginine, lysine, and histidine) to alanine in groups of 1, 2 or 3 residues (Fig. 1). These proteins were expressed in E. coli, and the protein was purified and refolded as described previously (Proudfoot et al., 1990Proudfoot A.E.I. Fattah D. Kawashima E.H. Bernard A. Wingfield P.T. Biochem. J. 1990; 270: 357-361Crossref PubMed Scopus (38) Google Scholar). All of the mutants expressed at between 1 and 20% of total cell protein, except for the mutant K, which was designed to remove all five charges from the second β-sheet. This was therefore subsequently made as a set of five single mutants: E87A, E88A, R89A, R90A, and R91A.Initially, all of the mutant proteins were tested in an assay for biological response in their ability to stimulate proliferation of the human erythroleukaemic cell line TF-1 (Kitamura et al., 1989Kitamura T. Tonge T. Terasawa T. Chiba S. Kuwaki T. Miyagawa K. Piao Y.-F. Miyazono K. Urabe A. Takaku F. J. Cell. Physiol. 1989; 140: 323-334Crossref PubMed Scopus (713) Google Scholar). The data were fitted to a four-parameter logistic equation, which fits to a background value, a maximum response, an EC50 (the concentration required for a 50% maximal response), and a slope of the curve. In this assay, the wild-type IL-5 gave a geometric mean EC50 of 4.1 pM and a slope of n = 0.78 ± 0.06. The assay to assay variation of EC50 values showed a relatively large variation, and so these proliferation assays were repeated at least 3 times (normally 5). The results for these assays are shown in Table I:, Table II:. The EC50 of the mutants is expressed as the ratio of the wild-type EC50 in that particular assay. In all cases except for mutant B, the mutants were capable of a complete agonist effect. That is to say, that the maximum proliferative response seen was not significantly different from the wild-type. Mutant B showed only 93 ± 2.8% maximal response. However, there were clear differences in the EC50 as can be seen in Table I and Fig. 2a, where a selection of the mutants showing a decrease in potency are shown (not all data are shown in the figure in the interests of clarity).Table I:0p4in cWe were unable to express this multiple mutant, and so the residues were mutated individually. Open table in a new tab Figure 2:Proliferation of the human erythroleukaemic cell line TF-1 by wild-type and selected mutant of IL-5. a, the abilities of wild-type IL-5 (○) and mutants M (+); R90A (×); E87A (●); and W110D (♦) cause proliferation of TF-1 cells with a clear loss of potency while retaining a full agonist ability. EC50 values were calculated as described in the text and are shown in Table I:, Table II:. The data shown are taken from a single experiment, but they are typical of those seen in three or more experiments. b, effect of double mutant B (K11A,E12A) (■) and the single mutation E12A (□) compared with wild-type IL-5 (○) showing that the single mutation produces a protein with partial agonist activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Four of the mutant proteins (B, C, M, and R90A) showed a greater than 10-fold reduction in potency in the TF-1 proliferation bioassays. In order to further define which of the residues in these multiple mutants were important in the interaction, single mutants were made for B (K11A,E12A) and C (H20A,R21A). Since previous studies have shown that deletion of the carboxyl-terminal region as far as residue 112 has no effect on receptor binding or bioactivity, the important residue in mutant M can be assumed to be Glu-109. The mutant K11A was not well expressed in E. coli, and in addition we were unable to refold the H20A mutant. These mutants were all tested in TF-1 proliferation and receptor binding assays, as described previously, and the data are shown in Table II and Fig. 2a. These mutants also showed large shifts in potency. The E12A mutant exhibited partial agonist activity, since even at very high concentrations of ligand, the maximal proliferative response seen was only 64.7 ± 4.0% of that seen for the wild-type and the other mutants (Fig. 2b).In addition, we mutated Trp-110 to aspartate. This is the only tryptophan in the protein, and it aligns well with Tyr-124 in human IL-4. The mutant Y124D produces an antagonist in IL-4, and we were interested to know whether the same effect could be seen in IL-5. The protein shows a 10-fold reduction in activity in the TF-1 proliferation assay as compared to wild-type.Eosinophil Activation AssayAll mutants that showed a non-wild-type response in the TF-1 proliferation assay were also tested for their ability to activate LFA-1 on human eosinophils and therefore enhance the adherence to ICAM-1-ZZ-coated plastic.4 The wild-type protein showed an EC50 of 5 pm, using a four-parameter logistic equation (Fig. 3). Although the shape of the dose-response curve is steeper in this assay (typically giving a slope of 2-3), the relative EC50 values calculated in the two biological response experiments are similar. Data are shown for mutants B and M, which show the largest decrease in potency, and the parameters are listed in Table I.Figure 3:Dose-response curve for effect of wild-type and mutant IL-5 on the adhesion of eosinophils to ICAM-ZZ coated plates. The cytokines were added at the beginning of the assay, and incubated with 5000 eosinophils/well for 30 min at 37°C. After washing to remove nonadherent cells and medium, eosinophil peroxidase activity of the adherent eosinophils was measured. Data were fitted to a four-parameter logistic equation and are shown for wild-type IL-5 (○); mutant B (■); and mutant M (+). Data from mutant proteins giving values similar to wild-type are not shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Binding to the Receptor α-ChainIn order to measure the affinity of the mutant proteins for their receptor, all mutants were tested for their ability to compete with wild-type 125I-labeled IL-5 for binding to the IL-5 receptor α-chain immobilized at the surface of scintillation proximity assay beads.2 In this assay, the wild-type protein competes with an IC50 value of 1 nm (Fig. 4). The data were fitted to a single-site model for competition of binding and showed no significant improvement if additional sites were added to the model or if the slope was allowed to vary. The data for the wild-type protein and for seven of the mutants that showed a significant difference from the wild-type activity are shown in Fig. 4 and Table I:, Table II:. The largest changes are for the mutants in the carboxyl-terminal region W110D, R90A, and mutant M (E109A,E113A) where 210-370-fold changes can be seen. The magnitudes of these changes were much larger than those seen in either of the biological response assays used. The only mutants where there was a much larger effect on the potency in the biological response assays than in the receptor α-chain binding assays were the mutant B (K11A,E12A) and the single mutant E12A.Figure 4:Competition of 125I-labeled IL-5 binding to immobilized IL-5 receptor α-chain by wild-type and selected mutants of IL-5. The curves are calculated using the equation for ligand competition from a single site. Data are shown for wild-type IL-5 (○) and for mutants showing a large change in binding affinity: (●), mutant C; (□), mutant D; (■), mutant B; (△), mutant E; (+), mutant M; (×), R90A; and (♦) W110D. Assays were performed at room temperature, and samples were incubated for 4 h.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Binding to the α/β Receptor ComplexMutant proteins that showed a significant deviation from the wild-type response in the TF-I proliferation assay were then tested for their ability to compete with 125I-labeled IL-5 for its binding sites on TF-1 cells, where both the α- and the β-chains of the receptor are present (Fig. 5a,). All data were initially fitted to an equation describing competition from a single binding site. However, in some cases, a better fit to the data was found using a four-parameter logistic equation, and this improvement in fit was found to be statistically significant (p < 0.05) for mutants D (E28A,R31A), E (H37A,K38A,H40A), W110D, and R90A (Fig. 5b). In these cases, the value for the exponential function used" @default.
• W1965798918 created "2016-06-24" @default.
• W1965798918 creator A5036488639 @default.
• W1965798918 creator A5041076535 @default.
• W1965798918 creator A5049795360 @default.
• W1965798918 creator A5050934674 @default.
• W1965798918 creator A5060493134 @default.
• W1965798918 creator A5073156947 @default.
• W1965798918 creator A5075957870 @default.
• W1965798918 creator A5081330050 @default.
• W1965798918 creator A5087065312 @default.
• W1965798918 creator A5089030305 @default.
• W1965798918 date "1995-06-01" @default.
• W1965798918 modified "2023-09-29" @default.
• W1965798918 title "Identification of Key Charged Residues of Human Interleukin-5 in Receptor Binding and Cellular Activation" @default.
• W1965798918 cites W1437694810 @default.
• W1965798918 cites W1459230013 @default.
• W1965798918 cites W1490744061 @default.
• W1965798918 cites W1513939725 @default.
• W1965798918 cites W1525221108 @default.
• W1965798918 cites W1547742399 @default.
• W1965798918 cites W1579558830 @default.
• W1965798918 cites W1971500432 @default.
• W1965798918 cites W1974607640 @default.
• W1965798918 cites W2024849805 @default.
• W1965798918 cites W2025864476 @default.
• W1965798918 cites W2027286533 @default.
• W1965798918 cites W2029559458 @default.
• W1965798918 cites W2032371271 @default.
• W1965798918 cites W2042655455 @default.
• W1965798918 cites W2048362048 @default.
• W1965798918 cites W2056527553 @default.
• W1965798918 cites W2067377498 @default.
• W1965798918 cites W2068187158 @default.
• W1965798918 cites W2071560930 @default.
• W1965798918 cites W2076224430 @default.
• W1965798918 cites W2087831434 @default.
• W1965798918 cites W2090030418 @default.
• W1965798918 cites W2090881803 @default.
• W1965798918 cites W2094869608 @default.
• W1965798918 cites W2096843631 @default.
• W1965798918 cites W2098648394 @default.
• W1965798918 cites W2124276056 @default.
• W1965798918 cites W2126812410 @default.
• W1965798918 cites W2149419600 @default.
• W1965798918 cites W2161045200 @default.
• W1965798918 cites W2161335440 @default.
• W1965798918 cites W2167988611 @default.
• W1965798918 cites W2211545673 @default.
• W1965798918 cites W2314777223 @default.
• W1965798918 cites W2399343855 @default.
• W1965798918 cites W302000954 @default.
• W1965798918 cites W311204152 @default.
• W1965798918 cites W4231829525 @default.
• W1965798918 cites W63256448 @default.
• W1965798918 cites W992611408 @default.
• W1965798918 doi "https://doi.org/10.1074/jbc.270.26.15762" @default.
• W1965798918 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7797578" @default.
• W1965798918 hasPublicationYear "1995" @default.
• W1965798918 type Work @default.
• W1965798918 sameAs 1965798918 @default.
• W1965798918 citedByCount "57" @default.
• W1965798918 countsByYear W19657989182012 @default.
• W1965798918 countsByYear W19657989182019 @default.
• W1965798918 countsByYear W19657989182020 @default.
• W1965798918 crossrefType "journal-article" @default.
• W1965798918 hasAuthorship W1965798918A5036488639 @default.
• W1965798918 hasAuthorship W1965798918A5041076535 @default.
• W1965798918 hasAuthorship W1965798918A5049795360 @default.
• W1965798918 hasAuthorship W1965798918A5050934674 @default.
• W1965798918 hasAuthorship W1965798918A5060493134 @default.
• W1965798918 hasAuthorship W1965798918A5073156947 @default.
• W1965798918 hasAuthorship W1965798918A5075957870 @default.
• W1965798918 hasAuthorship W1965798918A5081330050 @default.
• W1965798918 hasAuthorship W1965798918A5087065312 @default.
• W1965798918 hasAuthorship W1965798918A5089030305 @default.
• W1965798918 hasBestOaLocation W19657989181 @default.
• W1965798918 hasConcept C116834253 @default.
• W1965798918 hasConcept C170493617 @default.
• W1965798918 hasConcept C185592680 @default.
• W1965798918 hasConcept C18903297 @default.
• W1965798918 hasConcept C26517878 @default.
• W1965798918 hasConcept C55493867 @default.
• W1965798918 hasConcept C70721500 @default.
• W1965798918 hasConcept C86803240 @default.
• W1965798918 hasConcept C95444343 @default.
• W1965798918 hasConceptScore W1965798918C116834253 @default.
• W1965798918 hasConceptScore W1965798918C170493617 @default.
• W1965798918 hasConceptScore W1965798918C185592680 @default.
• W1965798918 hasConceptScore W1965798918C18903297 @default.
• W1965798918 hasConceptScore W1965798918C26517878 @default.
• W1965798918 hasConceptScore W1965798918C55493867 @default.
• W1965798918 hasConceptScore W1965798918C70721500 @default.
• W1965798918 hasConceptScore W1965798918C86803240 @default.
• W1965798918 hasConceptScore W1965798918C95444343 @default.
• W1965798918 hasIssue "26" @default.
• W1965798918 hasLocation W19657989181 @default.
• W1965798918 hasOpenAccess W1965798918 @default.

• next