Randomization of the Receptor α Chain Recruitment Epitope Reveals a Functional Interleukin-5 with Charge Depletion in the CD Loop

Abstract

1 is a T cell-derived cytokine that specifically stimulates differentiation, proliferation, and activation of eosinophils (1) and appears to play an important role in the pathogenesis of asthma. IL-5 is a homodimer that folds into a cylindrical molecule containing two four-helix bundles (2), each of which resembles the four-helix bundle seen in the monomeric cytokines IL-3, IL-4, granulocyte-macrophage colony-stimulating factor, and growth hormone.The cell receptor for IL-5 is composed of two subunits, ␣ and ␤ c . The ␣ chain is cytokine-specific and by itself can bind IL-5 with high affinity (3). Extensive site-directed mutagenesis studies have shown that residues clustered near the helix bundle interface, in the CD loop, including Glu 89 and Arg 91 , and at the carboxyl-terminal end of helix D, notably Glu 110 , engage in receptor ␣ chain binding (4 -6). Glu 13 , which is at each of the distal ends of the IL-5 cylinder away from the interface between the two four-helix bundles and is not involved directly in ␣ chain interaction, is a key residue mediating productive interaction of the ␤ c chain of IL-5 receptor (4, 5). It is the ␤ c recruitment into ␣␤ c that leads to triggering of the intracellular signaling cascade.Although site-directed mutagenesis studies have identified important residues for receptor binding, the one-residue-at-atime substitution allowed by this technique limits a full mechanistic understanding of the specific structural and electrostatic features of the IL-5 surface required for receptor-ligand interaction. However, through the use of randomly generated side chain libraries, it should be possible to measure the effect of replacing individual side chains or sets of side chains in local regions on the IL-5 surface and hence determine which combinations allow productive binding. Such random epitope mutagenesis can be achieved with sequence libraries formed by phage display. This technique has been applied successfully to in vitro antibody maturation and protein engineering (7). Typically a foreign gene is fused in frame with phage coat protein pIII encoded by a phagemid vector, and the surface-displayed recombinant foreign protein selected by affinity selection procedures (biopanning). Human growth hormone has been displayed on phage and higher receptor affinity variants selected through random mutagenesis and phage panning (8). Phage display of multichain proteins also has been achieved, for example for antibody Fab fragments (9) and vascular epidermal growth factor (10). Nonetheless, in our hands, phage display of the dimeric wild type IL-5 has proven difficult, perhaps reflecting folding difficulties that previously have led to insoluble inclusion bodies in intracellular Escherichia coli expression (11).We have sought to overcome limitations in phage display of IL-5 by using a single chain form of human IL-5 (scIL-5), in which two IL-5 molecules are tandemly linked by a Gly-Gly dipeptide linker (12). Single chain IL-5 and wtIL-5 have virtually identical biological activities and bindin...

Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

“…7) The dominant importance of the opposing negative and positive charge distribution in IL5R␣ correlates with previously elucidated IL5 mutations showing that Arg 91 (and the overall positive charge balance of the CD turn region), and Glu 110 are two main IL5 charged sites for IL5R␣ recognition (15)(16)(17). Although the detailed structural description of the IL5⅐IL5R␣ interaction must await high resolution structure determination of the IL5⅐IL5R␣ complex by x-ray crystallography or NMR spectroscopy, it is tempting to speculate that the side chains of Arg 91 and Glu 110 of IL5 might come into contact with the negatively charged patch in D1 and the positively charged patch in D2D3, respectively (Fig.…”

“…7). It has been found that the known functionally important CD turn sequence 88 EERRR 92 in IL5 can be replaced by 88 SLRGG 92 and that the positive charge in this turn is thought to be the key factor promoting IL5R␣ recruitment (17). Among other things, we can speculate that the positive charge balance in the CD turn region interacts directly with the negatively charged patch of the D1 domain.…”

“…Site-directed mutagenesis analyses have shown the importance of charged residues in helix B (His 38 (14 -16). Moreover, phage-based CD turn randomization (17) has led to identification of a functionally active IL5 mimetic containing the CD turn sequence 88 SL-RGG 92 , instead of the native sequence 88 EERRR 92 . In the CD turn of this IL5 mutant, the only charged residue is the Arg 90 .…”

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

“…We have displayed scIL-5 on phage using a two-stage construction that now allows asymmetric randomization mutagenesis of the IL-5 molecule and therefrom a deeper understanding of the IL-5-receptor recognition process (29 , that the CD loop can be simplified in considering design of structure-based mimetics, and that phage display has utility to examine the IL-5 receptor recruitment mechanism.…”

“…We consider two mechanistic possibilities by which Glu 110 may function in receptor activation, including its direct involvement in ␤ c contact or alternatively its more indirect impact on ␤ c recruitment via the mode of ␣ chain binding that it helps to effect. 110 Mutants by Site-directed Mutagenesis-The phagemid vector, pMK-wt/A5-G3 (29), was used as a starting point to prepare different Glu 110 mutants. Two working vectors, pCR-IL-5A and pCR-IL-5B encoding one copy of IL-5 sequence, have been used for asymmetric mutation as reported previously (29).…”