Single Chain Human Interleukin 5 and Its Asymmetric Mutagenesis for Mapping Receptor Binding Sites

Abstract

Wild type human (h) interleukin 5 (wt IL5) Human interleukin 5 (hIL5)1 is a T cell-derived cytokine which plays an important role in the differentiation, proliferation, and activation of eosinophils (Sanderson et al., 1992;Bentley et al., 1992). Natural hIL5 is a disulfide-linked, homodimeric glycoprotein with 115 residues per chain. The high resolution crystal structures of both Escherichia coli-expressed (Milburn et al., 1993) and Drosophila-expressed hIL5 (Johanson et al., 1995) have revealed a core of two four-helix bundles. Each four-helix bundle resembles the four-helix bundle seen in IL2 (Bazan and McKay, 1992), IL4 (Smith et al., 1992), and GM-CSF (Diederichs et al., 1991). However, the bundle organization in IL5 is unique in that helix D of one monomer combines with helices A, B, and C of the second monomer, and vice versa. In the two-bundle structure, the A and D helices form one face, the B and C helices a second. The organization of structural features on each face is palindromic, as observed for restriction sites in DNA.Human IL5 receptor is composed of two different chains, denoted ␣ and ␤ (Tavernier et al., 1991). The ␣ chain is specific for IL5 (Murata et al., 1992) and has a K d in the 0.1-1 nM range depending on the assays and receptor forms examined (Li et al., 1996). In contrast, the ␤ chain of hIL5R is not cytokine-specific but is shared with the receptors for IL3 and GM-CSF (Tavernier et al., 1991) and appears needed for signal transduction. A soluble form of hIL5 receptor ␣ chain (shIL5R␣), which has only the extracellular domain of IL5R␣, also has been described. This binds to hIL5 with nanomolar affinity (Tavernier et al., 1991;Johanson et al., 1995). Despite the dimeric nature of IL5, a 1:1 binding stoichiometry between shIL5R␣ and hIL5 has been reported (Devos et al., 1993;Johanson et al., 1995).New insights into the binding mechanism of IL5 to its receptor are emerging with the availability of high resolution structure and mutagenesis techniques. Data from hybrid constructs of mouse/human IL5 suggest that the carboxyl-terminal 36 residues of IL5 interact directly with the IL5R␣ and confer species specificity (McKenzie et al., 1991). By Ala-scanning mutagenesis of the carboxyl-terminal region of hIL5, we found previously that Glu-110 and Trp-111 contribute significantly to receptor binding . In addition, mutation of residues (Glu-89 and Arg-91) in the loop between helices C and D have been found to affect binding to IL5R␣ (Tavernier et al., 1995;Graber et al., 1995). All of these residues cluster around the interface between the two 4-helix bundles of hIL5 and appear to constitute a central patch for binding to a single molecule of hIL5R␣ . In contrast, residue Glu-13, which is at the distal ends of the IL5 dimer away from the helix bundle interface, was suggested to interact with the ␤ chain of IL5R, since mutation at this position resulted in loss of biological activity but did not affect the binding affinity to the ␣ chain (Tavernier et al., 1995;Graber et al., 1995).While emerging...

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

“…(wt/A5)scIL-5 was expressed in E. coli as an active molecule and was also expressed displayed on the surface of phage in a form able to bind the IL-5 receptor ␣ chain. The retention of substantial function by (wt/A5)scIL-5 is consistent with previous observations of activity in asymmetrically mutated forms of scIL-5 (12,24) as well as in several monomeric forms of the protein (15,31,32).…”

“…The ratio of k off /k on yielded a K d value of 10.0 nM. These rates and consequent K d are comparable with the proteins of COS-expressed wtIL-5 and scIL-5 (12,15,24), indicating that the recombinant scIL-5 expressed in E. coli retained full binding activity to the hIL-5 receptor ␣ chain.…”

“…Phage preparations from pMK-IL-4-G3 plasmid-transformed cells specifically bind to the soluble IL-4R␣ and anti-IL-4 antibodies by enzyme-linked immunosorbent assay (ELISA). 2 The single chain IL-5 DNA was derived by BglII digestion of pCDN-IL-5(sc) (12). The 350-bp scIL-5 BglII fragment was ligated into the BglII site of pMK-IL-5, yielding pMK-scIL-5.…”

“…pMK-scIL-5 encodes a scIL-5 protein containing two tandem IL-5 sequences linked by a GlyGly sequence. This scIL-5 sequence is the same as the COS-expressed scIL-5 (12) except that there are two extra amino acids (Met-Ala) at the N terminus of E. coli -expressed scIL-5.…”

“…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 binding affinities for shIL-5R␣.…”