Epitope Randomization Redefines the Functional Role of Glutamic Acid 110 in Interleukin-5 Receptor Activation*

Sequence randomization through functional phage display of single chain human interleukin (IL)-5 was used to investigate the limits of replaceability of the Glu110 residues that form a part of the receptor-binding epitope. Mutational analysis revealed unexpected affinity for IL-5 receptor α chain with variants containing E110W or E110Y. Escherichia coli-expressed Glu110variants containing E110W in the otherwise sequence-intact N-terminal half, including a variant with an E110A replacement in the sequence-disabled C-terminal half, were shown by their CD spectra to be folded into secondary structures similar to that of single chain human IL-5 (scIL-5). Biosensor kinetics analysis revealed that (E110W/A5)scIL-5 and (E110W/A6)scIL-5 had receptor α chain binding affinities similar to that of (wt/A5)scIL-5. However, (E110W/A6)scIL-5 had a significantly reduced bioactivity in TF-1 cell proliferation compared with both (wt/A5)scIL-5 and (E110W/A5)scIL-5, and this activity reduction was disproportionately greater than the much smaller effect of Glu110 mutation on receptor binding affinity. The marked and disproportionate decrease in TF-1proliferation observed with (E110W/A6)scIL-5 suggests a role for Glu110 in the biological activity mediated by the signal transducing receptor βc subunit of the IL-5 receptor. This is also consistent with the lack of stimulation of JAK2phosphorylation by the (E110W/A6)scIL-5 mutant in recombinant 293T cells, as compared with the concentration-dependent stimulation seen for scIL-5. The results reveal the dispensability of charge in the Glu110 locus of IL-5 for receptor α chain binding and, in contrast, its heretofore underappreciated importance for receptor activation.

[1]  R. Cook,et al.  Mutants of Single Chain Interleukin 5 Show Asymmetric Recruitment of Receptor α and βc Subunits* , 1996, The Journal of Biological Chemistry.

[2]  G Canziani,et al.  Randomization of the Receptor α Chain Recruitment Epitope Reveals a Functional Interleukin-5 with Charge Depletion in the CD Loop* , 1999, The Journal of Biological Chemistry.

[3]  J. G. Zhang,et al.  The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Doyle,et al.  Analysis of the interaction between human interleukin‐5 and the soluble domain of its receptor using a surface plasmon resonance biosensor , 1994, Journal of molecular recognition : JMR.

[5]  M. Doyle,et al.  Monomeric isomers of human interleukin 5 show that 1:1 receptor recruitment is sufficient for function. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Tavernier,et al.  Recombinant soluble human interleukin-5 (hIL-5) receptor molecules. Cross-linking and stoichiometry of binding to IL-5. , 1993, The Journal of biological chemistry.

[7]  M. Lambert,et al.  A novel dimer configuration revealed by the crystal structure at 2.4 Å resolution of human interleukin-5 , 1993, Nature.

[8]  J. Pandit,et al.  Three-dimensional structure of dimeric human recombinant macrophage colony-stimulating factor. , 1992, Science.

[9]  B. Violand,et al.  Three-dimensional structure of a genetically engineered variant of porcine growth hormone. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[10]  I. Chaiken,et al.  Single Chain Human Interleukin 5 and Its Asymmetric Mutagenesis for Mapping Receptor Binding Sites (*) , 1996, The Journal of Biological Chemistry.

[11]  R. Cook,et al.  Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor alpha chain recognition. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. S. Garrett,et al.  Three-dimensional solution structure of human interleukin-4 by multidimensional heteronuclear magnetic resonance spectroscopy. , 1993, Science.

[13]  S. Bass,et al.  Selecting high-affinity binding proteins by monovalent phage display. , 1991, Biochemistry.

[14]  I. Chaiken,et al.  Construction and Binding Kinetics of a Soluble Granulocyte-Macrophage Colony-stimulating Factor Receptor α-Chain-Fc Fusion Protein* , 1998, The Journal of Biological Chemistry.

[15]  J. Tavernier,et al.  A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific α chain and a β chain shared with the receptor for GM-CSF , 1991, Cell.

[16]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[17]  M. Migita,et al.  Molecular cloning and expression of the human interleukin 5 receptor , 1992, The Journal of experimental medicine.

[18]  R. Ames,et al.  Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library. , 1995, Journal of immunology.

[19]  J. Tavernier,et al.  Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Bazan,et al.  Haemopoietic receptors and helical cytokines. , 1990, Immunology today.

[21]  C. Dobson,et al.  Secondary structure and topology of human interleukin 4 in solution. , 1991, Biochemistry.

[22]  S. Carr,et al.  Binding interactions of human interleukin 5 with its receptor alpha subunit. Large scale production, structural, and functional studies of Drosophila-expressed recombinant proteins. , 1995, The Journal of biological chemistry.

[23]  D Eisenberg,et al.  3D domain swapping: A mechanism for oligomer assembly , 1995, Protein science : a publication of the Protein Society.

[24]  J. Wells,et al.  Production of an atrial natriuretic peptide variant that is specific for type A receptor. , 1994, The EMBO journal.

[25]  R. Kastelein,et al.  The amino‐terminal helix of GM‐CSF and IL‐5 governs high affinity binding to their receptors. , 1991, The EMBO journal.

[26]  M. Sliwkowski,et al.  Binding Interaction of the Heregulinβ egf Domain with ErbB3 and ErbB4 Receptors Assessed by Alanine Scanning Mutagenesis* , 1998, The Journal of Biological Chemistry.

[27]  M. Vadas,et al.  GM-CSF, IL-3 and IL-5: cross-competition on human haemopoietic cells. , 1992, Immunology today.

[28]  M. Baggiolini.  Guidebook to cytokines and their receptors , 1995 .

[29]  V. Mordvinov,et al.  Biological and molecular characteristics of interleukin-5 and its receptor. , 1998, International reviews of immunology.

[30]  T. Honjo,et al.  Characterization of high-affinity receptors for interleukin 5 on interleukin 5-dependent cell lines. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Bazan.  Unraveling the structure of IL-2. , 1992, Science.

[32]  T. Clackson,et al.  In vitro selection from protein and peptide libraries. , 1994, Trends in biotechnology.

[33]  M. Peitsch,et al.  Identification of Key Charged Residues of Human Interleukin-5 in Receptor Binding and Cellular Activation (*) , 1995, The Journal of Biological Chemistry.