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• W2075045013 abstract "Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) proteins with substitutions at residues in the first α-helix were examined for biological activity and receptor binding properties. Substitution at the buried residue His affected both bioactivity and receptor binding. Of the four surface-exposed positions examined (Arg, Lys14, Lys, and Glu) only substitutions at Glu impaired bioactivity. Proteins with charge reversal substitutions at this position were partial agonists and weak antagonists of native mGM-CSF action. All substitutions at Glu abrogated high affinity binding. Lys14 and Lys substitution proteins showed various receptor binding defects. Qualitative and quantitative measurement of these binding defects identified Lys14 as a residue that interacts specifically with the β subunit of the mGM-CSF receptor, whereas Lys appeared to exist at the GM-R α-subunit/GM-R β-subunit interface as substitutions at this position produce both high and low affinity binding losses. These determinations permitted the design of a more potent mGM-CSF antagonist. Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) proteins with substitutions at residues in the first α-helix were examined for biological activity and receptor binding properties. Substitution at the buried residue His affected both bioactivity and receptor binding. Of the four surface-exposed positions examined (Arg, Lys14, Lys, and Glu) only substitutions at Glu impaired bioactivity. Proteins with charge reversal substitutions at this position were partial agonists and weak antagonists of native mGM-CSF action. All substitutions at Glu abrogated high affinity binding. Lys14 and Lys substitution proteins showed various receptor binding defects. Qualitative and quantitative measurement of these binding defects identified Lys14 as a residue that interacts specifically with the β subunit of the mGM-CSF receptor, whereas Lys appeared to exist at the GM-R α-subunit/GM-R β-subunit interface as substitutions at this position produce both high and low affinity binding losses. These determinations permitted the design of a more potent mGM-CSF antagonist. INTRODUCTIONGranulocyte-macrophage colony-stimulating factor (GM-CSF) 1The abbreviations used are: GM-CSFgranulocyte-macrophage colony-stimulating factormGM-CSFmouse GM-CSFhGM-CSFhuman GM-CSFGM-RGM-CSF receptorGM-RαGM-R α-subunitGM-RβGM-R β-subunitIL-2interleukin-2IL-3interleukin-3IL-4interleukin-4GHgrowth hormoneGH-RGH receptorMTT3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. is a pleiotropic cytokine made by various cell types, such as T cells and monocytes, and is likely to play an important role in the regulation of hemeatopoiesis(1Metcalf D. Science. 1985; 229: 16-22Crossref PubMed Scopus (734) Google Scholar, 2Metcalf D. Blood. 1986; 67: 257-267Crossref PubMed Google Scholar). GM-CSF interacts with specific high affinity cell surface receptors (GM-Rαβ, K 5 × 10M) that are complexes containing at least two subunits, both of which are members of the cytokine receptor superfamily(3Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1869) Google Scholar). The receptor α chain (GM-Rα) binds GM-CSF specifically with low affinity (K 3 × 10M), whereas the receptor β chain (GM-Rβ), common to the receptors for GM-CSF, IL-3, and IL-5, does not bind this protein detectably by itself, but confers high affinity binding when co-expressed with GM-Rα(4Hayashida K. Kitamura T. Gorman D.M. Arai K. Yokota T. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9655-9659Crossref PubMed Scopus (518) Google Scholar, 5Kitamura T. Sato N. Arai K. Miyajima A. Cell. 1991; 66: 1165-1174Abstract Full Text PDF PubMed Scopus (505) Google Scholar, 6Park L.S. Martin U. Sorensen R. Luhr S. Morrissey P.J. Cosman D. Larsen A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4295-4299Crossref PubMed Scopus (93) Google Scholar). Formation of this complex with GM-CSF is required for receptor activation and cellular signaling(7Kitamura T. Hayashida K. Sakamaki K. Yokota T. Arai K. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5082-5086Crossref PubMed Scopus (170) Google Scholar).Structurally, GM-CSF has been characterized as a four anti-parallel α-helical bundle protein(8Diederichs K.S. Boone T. Karplus P.A. Science. 1991; 254: 1779-1782Crossref PubMed Scopus (166) Google Scholar). This topological fold is shared by many of the known cytokine structures, including interleukins-2(9McKay D.B. Science. 1992; 257: 412-413Crossref PubMed Google Scholar), −4 (10Smith 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), and −5(11Milburn M.V. Hassell A.M. Lambert M.H. Jordon S.R. Proudfoot A.E.I. Graber P. Wells T.N.C. Nature. 1993; 363: 172-176Crossref PubMed Scopus (229) Google Scholar), macrophage colony-stimulating factor(12Pandit J. Bohm A. Jancarik J. Halenbeck R. Koths K. Kim S. Science. 1992; 258: 1358-1362Crossref PubMed Scopus (162) Google Scholar), and growth hormone(13Abdel-Meguid S.S. Shieh H. Smith W.W. Dayringer H.E. Violand B.N. Bentle L.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6434-6437Crossref PubMed Scopus (430) Google Scholar).Molecular genetic studies and studies with neutralizing anti-GM-CSF antibodies have identified regions and residues in both mouse and human GM-CSF that interact with specific subunits of GM-Rαβ(14Shanafelt A.B. Johnson K.E. Kastelein R.A. J. Biol. Chem. 1991; 266: 13804-13810Abstract Full Text PDF PubMed Google Scholar, 15Shanafelt A.B. Miyajima A. Kitamura T. Kastelein R.A. EMBO J. 1991; 10: 4105-4112Crossref PubMed Scopus (76) Google Scholar, 16Shanafelt A.B. Kastelein R.A. J. Biol. Chem. 1992; 267: 25466-25472Abstract Full Text PDF PubMed Google Scholar, 17Meropol N.J. Altmann S.W. Shanafelt A.B. Kastelein R.A. Johnson G.D. Prystowsky M.B. J. Biol. Chem. 1992; 267: 14266-14269Abstract Full Text PDF PubMed Google Scholar, 18Lopez A.F. Shannon M.F. Hercus T. Nicola N.A. Cambareri B. Dottore M. Layton M.J. Eglinton L. Vadas M.A. EMBO J. 1992; 11: 909-916Crossref PubMed Scopus (89) Google Scholar, 19Brown C.B. Hart C.E. Curtis D.M. Bailey M.C. Kaushansky K. J. Immunol. 1990; 144: 2184-2189PubMed Google Scholar, 20Kanakura Y. Cannistra S.A. Brown C.B. Nakamura M. Seelig G.F. Prosise W.W. Hawkins J.C. Kaushansky K. Griffin J.D. Blood. 1991; 77: 1033-1043Crossref PubMed Google Scholar, 21Seelig G.F. Prosise W.W. Scheffler J.E. J. Biol. Chem. 1994; 269: 5548-5553Abstract Full Text PDF PubMed Google Scholar). These studies suggest the functional importance of the first and fourth helix of GM-CSF; in particular, the N-terminal helix of mGM-CSF interacts directly with mGM-Rβ in the context of mGM-Rαβ to form the high affinity ligand-receptor complex(15Shanafelt A.B. Miyajima A. Kitamura T. Kastelein R.A. EMBO J. 1991; 10: 4105-4112Crossref PubMed Scopus (76) Google Scholar). Alanine scanning mutagenesis of this region identified Glu as essential for this interaction in both mouse and human GM-CSF(16Shanafelt A.B. Kastelein R.A. J. Biol. Chem. 1992; 267: 25466-25472Abstract Full Text PDF PubMed Google Scholar, 17Meropol N.J. Altmann S.W. Shanafelt A.B. Kastelein R.A. Johnson G.D. Prystowsky M.B. J. Biol. Chem. 1992; 267: 14266-14269Abstract Full Text PDF PubMed Google Scholar, 18Lopez A.F. Shannon M.F. Hercus T. Nicola N.A. Cambareri B. Dottore M. Layton M.J. Eglinton L. Vadas M.A. EMBO J. 1992; 11: 909-916Crossref PubMed Scopus (89) Google Scholar). Although substitution of Glu with alanine in mGM-CSF results in a loss of high affinity binding, biological activity remains essentially intact. The biological activity of hGM-CSF Glu substitution proteins is reduced, and a basic side chain at this position causes the most severe defect(18Lopez A.F. Shannon M.F. Hercus T. Nicola N.A. Cambareri B. Dottore M. Layton M.J. Eglinton L. Vadas M.A. EMBO J. 1992; 11: 909-916Crossref PubMed Scopus (89) Google Scholar).In this report we examine further the biological and receptor binding properties of mGM-CSF mutant proteins with substitutions at residues in the first helix of mGM-CSF. We show that all Glu substitution proteins display only low affinity binding, but that the biological activity depends on the nature of the substituted side chain. Substitutions at several other examined residues lead to a loss in either high or low affinity binding. We also show that this knowledge can be used to design mGM-CSF derivatives with antagonistic properties.MATERIALS AND METHODSBacterial Host Strains, Cell Lines, and VectorsThe Escherichia coli K12 strains XL-1 (Stratagene) and JM101 (22Messing J. Methods Enzymol. 1983; 101: 20-78Crossref PubMed Scopus (3400) Google Scholar) were used as host strains for the propagation and maintenance of plasmid and M13 DNA. Strain CJ236 (23Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4543) Google Scholar) was used to prepare uracil-DNA for use in site-directed mutagenesis procedures. M13mp19 containing the ompA leader sequence and the entire mGM-CSF gene was used as the template for site-directed mutagenesis(24Shanafelt A.B. Kastelein R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4872-4876Crossref PubMed Scopus (36) Google Scholar). Strain AB1899 (25Howard-Flanders P. Simson E. Theriot L. Genetics. 1964; 49: 237-246Crossref PubMed Google Scholar) was used as the host for expression of mutant GM-CSF proteins. The plasmid pINIIIompA2 (26Lundell D. Greenberg R. Alroy Y. Condon R. Fossetta J.D. Gewain K. Kastelein R. Lunn C.A. Reim R. Shah C. Van Kimmenade A. Narula S.K. J. Indust. Microbiol. 1990; 5: 215-228Crossref PubMed Scopus (9) Google Scholar) was used as the expression vector for mutant GM-CSF proteins. The use of this secretory E. coli system to express biologically active, mature GM-CSF has been described elsewhere (27Greenberg R. Lundell D. Alroy Y. Bonitz S. Condon R. Fossetta J.D. Frommer B. Gewain K. Katz M. Leibowitz P.J. Narula S.K. Kastelein R. Van Kimmenade A. Curr. Microb. 1988; 17: 321-332Crossref Scopus (10) Google Scholar). A mouse GM-CSF-dependent myeloid leukemia cell line, NFS60, was maintained in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum and 50 μM 2-mercaptoethanol in the presence of mGM-CSF (1 ng/ml) (Schering-Plough).Construction of mGM-Rα Expression Plasmid and Transfection to Mouse L-cellsThe mouse GM-Rα clone 71 cDNA (6Park L.S. Martin U. Sorensen R. Luhr S. Morrissey P.J. Cosman D. Larsen A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4295-4299Crossref PubMed Scopus (93) Google Scholar) (kindly provided by L. Park, Immunex) was inserted downstream of the SRα promoter in the expression vector pME18-Neo, a neomycin-resistant derivative of the vector pCEV4 (28Itoh N. Yonehara S. Schreurs J. Gorman D.M. Maruyama K. Ishii A. Yahara I. Arai K. Miyajima A. Science. 1990; 247: 324-327Crossref PubMed Scopus (289) Google Scholar) (kindly provided by A. Miyajima, DNAX). Mouse L-cells washed with 20 mM HEPES-buffered saline (pH 7.1) were resuspended at 107 cells/ml, and 50 μg of linearized plasmid DNA was added to 0.8 ml of cell suspension in a 0.4-cm electroporation cuvette (Bio-Rad). Electroporation was performed using a Gene Pulser (Bio-Rad) at 960 microfarads and 400 V. Transfectants were selected with G418 (1.5 mg/ml) (Schering-Plough) and cells expressing mGM-Rα were confirmed by their ability to bind I-mGM-CSF.Mutagenesis, Recombinant DNA, and Sequencing ProtocolsSite-directed mutagenesis was performed using the method described by Kunkel et al.(23Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4543) Google Scholar). Oligonucleotides, 21 nucleotides in length, corresponding to mGM-CSF sequences incorporating the desired amino acid substitution were used as primers in the mutagenesis reactions. Individual clones were sequenced using the method and modifications described in the Sequenase (United States Biochemical Corp., Cleveland, OH) protocol(29Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52338) Google Scholar). M13 (replicative form) DNA containing confirmed mutations was digested with XbaI and BamHI and subcloned into pINIIIompA2.Preparation and Quantitation of Protein SamplesExpression and quantitation of mutant proteins has been described previously(24Shanafelt A.B. Kastelein R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4872-4876Crossref PubMed Scopus (36) Google Scholar). Briefly, mutant proteins were expressed in E. coli AB1899 and periplasmic extracts were generated by osmotic shock. The concentration of mutant protein in the periplasmic extracts was determined by quantitative immunoblot analysis. Extracts were titrated along with a standard curve of purified E. coli derived recombinant GM-CSF. Either hybridoma mg 1.8.2 (30Miyajima A. Otsu K. Schreurs J. Bond M.W. Abrams J.S. Arai K. EMBO J. 1986; 5: 1193-1197Crossref PubMed Scopus (75) Google Scholar) or 22E9 (31Abrams J.S. Roncarolo M-G. Yssel H. Andersson U. Gleich G.J. Silver J.E. Immunol. Rev. 1992; 127: 5-44Crossref PubMed Scopus (331) Google Scholar) were used as primary antibody, and the secondary antibody was I-labeled sheep anti-rat IgG (Amersham Corp.). The amount of immunoreactive protein was quantified by integrating the area × density from an exposed screen using a PhosphorImager (Molecular Dynamics). The error in the calculated concentrations of GM-CSF mutant proteins by this method did not exceed 2-fold based on repetitive analysis of individual samples.Biological Assays for GM-CSF ActivityProtein extracts were assayed for their ability to stimulate the proliferation of the mouse GM-CSF- dependent cell line NFS60. Samples were titrated in quadruplicate using serial 3-fold dilutions. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) assay (32Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (45437) Google Scholar) was used to measure the extent of proliferation, and the absorbance value difference of 570-650 nm was measured with a Vmax kinetic microplate reader (Molecular Devices). The concentration of each mutant and wild-type mGM-CSF protein that gave 50% maximal response was determined using the Softmax program (Molecular Devices). The correlation coefficient of the 4-parameter curve fit to the measured values exceeded 0.990 for all curves presented. Protein extracts were assayed for their ability to antagonize the GM-CSF proliferative response of NFS60 cells. Samples were titrated in quadruplicate using serial 3-fold dilutions in the presence of 3.5 × 10M mGM-CSF using the MTT assay format.Competitive Receptor Binding Assays of Mutant GM-CSF ProteinsMouse GM-CSF was labeled with I using the Bolton and Hunter reagent (Amersham; specific activity 400 mCi/mmol) to a specific activity of 20-40 μCi/μg as described previously(16Shanafelt A.B. Kastelein R.A. J. Biol. Chem. 1992; 267: 25466-25472Abstract Full Text PDF PubMed Google Scholar). Competition binding assays were performed as follows: NFS60 cells maintained in media containing mGM-CSF were harvested, washed, and incubated with 1 ml of ice-cold 10 mM NaPO4, 150 mM NaCl (pH 3.0) for 2 min then diluted to 50 ml with 10 mM NaPO4, 150 mM NaCl (pH 7.0). Following centrifugation the cells were resuspended in 1 × Hanks' balanced salts solution (Life Technologies, Inc.) containing 0.1% bovine serum albumin, 0.02% NaN3, and 10 mM HEPES (pH 7.5) (HBAH buffer). 2.5 × 106 cells in 200 μl of HBAH buffer were incubated with decreasing concentrations of unlabeled competitor in the presence of 3.0 × 10MI-mGM-CSF at 4°C with continuous agitation for 4 h. Samples were titered in triplicate using serial 3-fold dilutions. Nonspecific binding was determined from the data collected for the competition of unlabeled wild-type mGM-CSF. Cell-bound radioactivity was separated from free ligand by centrifugation at 4°C (2 min, 12,000 ×g) through dioctylphthalate/dibutylphthalate (2:3). Bound and total radioactivity was measured with a Cobra 5010 -counter (Packard). Low affinity receptor competition binding assays were performed in a similar manner except 7.5 × 105 mouse L-cells expressing mGM-Rα were incubated with unlabeled competitors in the presence of 2.0 × 10MI-mGM-CSF for 2 h. The equilibrium binding data were analyzed using the Ligand program (33Munson P.J. Methods Enzymol. 1983; 92: 543-576Crossref PubMed Scopus (275) Google Scholar). Curve fitting for both one- and two-site models were performed on each sample. Curves were either fit using a fixed high and low affinity equilibrium binding constants (K; 5.0×10M, K; 3.0×10M) or by fitting the control wild-type mGM-CSF and the mutant protein competition curve simultaneously. Binding curves are presented as the concentration of competitor versus the specific counts/minute bound as a percentage of total bound. Analysis of the binding data for Lyssubstitutions exposed a limitation of the Ligand software. Substitutions for Lysshowed a range of defects in high affinity binding. For substitutions resulting in small changes a two site model with reliable high and low affinity Kvalues was statistically significant. For substitutions with reduced high affinity binding approaching that of the low affinity, the Ligand program was unable to discriminate a two-site model. This is reflected in the unreliable binding constants with small F and large p (>0.05) values as well as high affinity Kvalues with values higher than would be expected from examination of the binding curves (Table 1). We presume that this is the result of combining both high and low affinity values into a single binding constant. Only by measuring the affinity of Lyssubstitution proteins for the low affinity receptor alone (mGM-Rα) were we able to asign binding defects of Lysmutant proteins to the mGM-Rβ(Table 4).Table 1 Open table in a new tab Table 4View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Mouse GM-CSF Molecular ModelingThe coordinates for hGM-CSF were kindly provided by Dr. A. Karplus (Cornell University, Ithaca, NY). The mouse GM-CSF Cα backbone conformation was modeled using SegMod (34Levitt M. J. Mol. Biol. 1992; 226: 507-533Crossref PubMed Scopus (514) Google Scholar) followed by refinement of side chain conformations using the program CARA (35Lee C. Subbiah S. J. Mol. Biol. 1991; 217: 373-388Crossref PubMed Scopus (248) Google Scholar) within the LOOK suite of programs (Molecular Applications Group, Palo Alto, CA). The structure was displayed using Insight II software (Biosym Technologies, San Diego, CA).RESULTSBiological Response of mGM-CSF Mutant ProteinsA panel of amino acid substitutions was generated for selected residues in the first α-helix of mGM-CSF. Based on previous studies (16Shanafelt A.B. Kastelein R.A. J. Biol. Chem. 1992; 267: 25466-25472Abstract Full Text PDF PubMed Google Scholar) the following residues were targeted: Arg (R11), Lys14 (K14), His (H15), Lys (K20), and Glu (E21). Position Glu, although surface exposed and located between Glu14 and Glu, was not included in this analysis, since all available evidence points to at best a minor involvement of this residue(15Shanafelt A.B. Miyajima A. Kitamura T. Kastelein R.A. EMBO J. 1991; 10: 4105-4112Crossref PubMed Scopus (76) Google Scholar, 16Shanafelt A.B. Kastelein R.A. J. Biol. Chem. 1992; 267: 25466-25472Abstract Full Text PDF PubMed Google Scholar). Mutant protein was produced in E. coli and quantitated as described previously(24Shanafelt A.B. Kastelein R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4872-4876Crossref PubMed Scopus (36) Google Scholar). Biological activity was assessed by measuring the proliferative response of the mGM-CSF-dependent myeloid cell line NFS60. Of the four surface-exposed residues (Arg, Lys14, Lys, and Glu), only substitution of Glu significantly altered the biological activity of mGM-CSF (Fig. 1, panel 5A, Table 3). Charge reversal substitutions at this position (E21R and E21K) reduced the biological activity to <1% and reduced the magnitude of the plateau. Several other Glu substitution proteins (Ala, Gly, Ser, and Leu) had lower biological activity (10-30%), although the magnitude of the response was not affected. All other substitutions at these four positions had only a marginal effect on the biological activity (>30%) with the exception of proline substitutions of Lys and Glu. The introduction of proline residues at these positions in the first α-helix reduced the activity of the resulting proteins to less than 1% (Fig. 1, panels 4A and 5A, Table 2, Table 3). The magnitude of the plateau for E21P but not K20P was reduced. Proline substitutions of Arg and Lys14, located just outside and just within the first α-helix, respectively, did not significantly alter the biological activity (Fig. 1, panels 1A and 2A, Table 1).Figure 1:Biological activity and competition binding of wild-type and mutant proteins. A, proliferation of NFS60 cells was measured as a function of protein concentration using the MTT assay(32Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (45437) Google Scholar). Assays were performed in quadruplicate with symbol error bars indicating standard deviation. Curves were generated using a 4-parameter logistical fit and all correlation coefficients exceeded 0.990. B, competition of I-mGM-CSF on NFS60 cells were performed with a constant concentration of 3.0 × 10MI-mGM-CSF in triplicate with symbol error bars indicating standard deviation. Under these conditions both high and low affinity sites were detected using the Ligand program (33Munson P.J. Methods Enzymol. 1983; 92: 543-576Crossref PubMed Scopus (275) Google Scholar). Each pair of A and B panels represents multiple amino acid substitutions for a specific residue as indicated Arg (1), Lys14 (2), His (3), Lys (4); and Glu (5). Substitutions are represented by each symbol as follows: •, wild-type; , Gly; , Met; Pro; ○, Ala; □, Leu; , Val; ◬, His; , Phe; ▿., Trp; ., Tyr; ▵, Gln; ▿, Ser; ⬢., Arg; , Glu; ⬢, Lys.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 3View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Based on homology to hGM-CSF, His of mGM-CSF appears to occupy a buried position in the first α-helix. This buried position is less tolerant to substitution than the surface exposed residues (with the exception of Glu). Substitution at His with charged or polar residues resulted in a reduced biological activity, whereas hydrophobic or aromatic substitutions are well tolerated (Fig. 1, panel 3A). All His mutants reached full plateau.Competitive Receptor Binding of mGM-CSF Mutant ProteinsThe effects of amino acid substitution on the ability of mGM-CSF to bind to the high affinity mGM-CSF receptor (mGM-Rαβ) on NFS60 cells was measured using conditions designed to detect both high and low affinity binding (see “Materials and Methods”). Some proteins had no loss in high affinity binding, and such mutant proteins were not investigated further. Some proteins had decreased high affinity binding, and the mutant protein was then tested for its ability to bind to the low affinity mGM-CSF receptor (mGM-Rα) on stably transfected L-cells (see “Materials and Methods”). This step was necessary so that the observed reduction in affinity could be assigned to either a loss of binding to the mGM-Rβ component in the mGM-Rαβ complex, to a loss of binding to the mGM-Rα component, or to a loss of binding to both.For most substitution mutants at Arg, Lys14, His, and Lys, a two-site fit of the high affinity receptor binding data was statistically significant (data shown for Lys14 and Lys; Table 1, Table 2). Substitution at Arg with either a negatively charged (R11E) or positively charged (R11K) residue, neutral (R11G), hydrophobic (R11L), or aromatic (R11W) residue had no effect on either high or low affinity binding constants (data not shown). This residue was not studied further.Table 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Mutant proteins with charged and polar substitutions at position His showed a reduced ability to compete for I-mGM-CSF (Fig. 1, panel 3B), in agreement with the observed loss in biological activity (Fig. 1, panel 3A). Since this is a buried position in the protein, losses in activity and binding are probably due to structural perturbations of the protein core. Proteins mutated at this position were also not studied further.Mutations at position Lys14 did not alter noticeably the biological activity of the resulting mutant protein. However, significant binding defects were observed (Fig. 1, panels 2A and 2B, Table 1). For some Lys14 mutants, only one affinity site is detected (Glu, Gly, Ser, Val, Trp, and Tyr). For others, two sites were still detected but both were reduced in affinity (e.g. Phe and Gln). In general, reduction of the high affinity binding constant was less than 10-fold, while values for the low affinity Kd showed wider variation with a concomitant increase in the %CV, indicating that the calculated value for the low affinity binding constant was less reliable under these conditions. Several Lys14 mutants displaying a range of high affinity defects were selected and analyzed for their low affinity binding to mGM-Rα expressed alone on stably transfected L-cells (Fig. 2A). Of the five mutant proteins tested, four had a low affinity Kd similar to mGM-CSF (Kd = 7.2 × 10M; Table 4). From this analysis we conclude that the reduction seen in the high affinity binding constant for Lys14 substitution proteins results from losses in binding to the mGM-Rβ in the mGM-Rαβ complex. Protein K14P is the exception. A proline substitution at this position affected the low as well as the high affinity binding constant (Table 1).Figure 2:Competition binding of mutant and wild-type mGM-CSF proteins. A, competition of I-mGM-CSF on L cells transfected with mGM-Rα by •, mGM-CSF; ○, K14A; , K14E; , K14G; , K14P; and ▿, K14S. B, competition of I-mGM-CSF on L cells transfected with mGM-R-α by •, mGM-CSF; , K20G; , K20M; , K20P; ○, K20A; □, K20L; , K20V; □+, K20F; ▵+, K20H; ▿+, K20W; +, K20Y; ▵, K20Q; ▿, K20S; ⬢+, K20R; , K20E. Assays were performed in triplicate using a constant concentration of 2.0 × 10MI-mGM-CSF with symbol error bars indicating standard deviation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutant proteins with Lys substitutions also showed losses in both high and low affinity binding as measured on NSF60 cells (Fig. 1, panel 4B, Fig. 2B, Table 2, Table 3, Table 4). To distinguish between losses in binding to the mGM-Rβ and/or mGM-Rα, Lys substitution proteins were analyzed on mGM-Rα-expressing L-cells. Whereas most substitutions at Lys did not affect the low affinity binding constant as measured on L-cells, small amino acid substitutions (Ala, Gly, and Ser) at this position led to a 10-fold reduction in mGM-Rα binding (Fig. 2B, Table 4). The introduction of proline at this position profoundly disturbed the binding of the resulting protein to both high and low affinity receptor.In contrast to the other mutant proteins examined, all Glu substitution proteins competed I-mGM-CSF for only a single class of binding sites on NSF60 cells (Fig. 1, panel 5B, Table 3). The affinity of Glu substitution proteins (Kd = 1-4 × 10M) for these sites suggested that binding is to mGM-Rα. Scatchard analysis of one of the Glu substitution proteins, E21A, has shown that both affinity and number of binding sites were consistent with binding of this mutant protein to mGMRα only(16Shanafelt A.B. Kastelein R.A. J. Biol. Chem. 1992; 267: 25466-25472Abstract Full Text PDF PubMed Google Scholar). Again, the exception was E21P. This protein had a low affinity binding constant that was at least three orders of magnitude higher than the other Glu proteins (Fig. 5B, Table 3), probably caused by severe structural changes(36Altmann S.W. Johnson G.D. Prystowsky M.B. J. Biol. Chem. 1991; 266: 5333-5341Abstract Full Text PDF PubMed Google Scholar).Figure 5:Two views of the mGM-CSF Cα backbone indicating important structural features and identifying specific amino acid side chains. Helix A (red) and helix D (blue) form an anti-parallel helix pair and comprise the receptor binding site(38Hercus 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). Disulfide bonds are indicated in yellow. Amino acid residues significant to this study are colored according to the properties of their side chains, negatively charged (E), red; positively charged (K and R), blue; and aromatic (H), green.View Large" @default.
• W2075045013 created "2016-06-24" @default.
• W2075045013 creator A5018250332 @default.
• W2075045013 creator A5042411440 @default.
• W2075045013 date "1995-02-01" @default.
• W2075045013 modified "2023-10-16" @default.
• W2075045013 title "Rational Design of a Mouse Granulocyte Macrophage- Colony-stimulating Factor Receptor Antagonist" @default.
• W2075045013 cites W1502459142 @default.
• W2075045013 cites W1502629197 @default.
• W2075045013 cites W1513939725 @default.
• W2075045013 cites W1547742399 @default.
• W2075045013 cites W1557721783 @default.
• W2075045013 cites W1560065907 @default.
• W2075045013 cites W1601687245 @default.
• W2075045013 cites W1675276494 @default.
• W2075045013 cites W167658880 @default.
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• W2075045013 cites W63256448 @default.
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