Randomization of the receptor alpha chain recruitment epitope reveals a functional interleukin-5 with charge depletion in the CD loop.
We report the functional phage display of single chain human interleukin-5 (scIL-5) and its use for receptor-binding epitope randomization. Enzyme-linked immunosorbent assays and optical biosensor analyses verified expression of scIL-5 on the phage surface and binding of scIL-5 phage to interleukin-5 receptor alpha chain. Furthermore, an asymmetrically disabled but functional scIL-5 mutant, (wt/A5)scIL-5, was displayed on phage. (wt/A5)scIL-5 was constructed from an N-terminal half containing the original five charged residues (88EERRR92) in the CD loop, including the Glu89 and Arg91 believed key in the alpha chain recognition site, combined with a C-terminal half containing a disabled CD loop sequence (88AAAAA92) missing the key recognition residues. This asymmetric variant was used as a starting point to generate an scIL-5 library in which the intact 88-92 N-terminal CD loop was randomized. From this epitope library, a receptor-binding variant of IL-5 was detected, (SLRGG/A5)scIL-5, in which the only charged residue in the CD loop is an Arg at position 90. Characterization of this variant expressed as a soluble protein in E. coli shows that the IL-5 pharmacophore for receptor alpha chain binding can function with a single positive charge in the CD loop. Charge-depleted CD loop mimetics of IL-5 suggest the importance of charge distribution in functional IL-5 receptor recruitment.
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
S J
SJ
Wu
J
J
Li
P
P
Tsui
R
R
Cook
W
W
Zhang
Y
Y
Hu
G
G
Canziani
I
I
Chaiken
D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002455:Cell Division; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015848:Interleukin-5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000737:chemistry / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D000939:Epitopes; D015848:Interleukin-5; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5