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 Glu(110) residues that form a part of the receptor-binding epitope. Mutational analysis revealed unexpected affinity for IL-5 receptor alpha chain with variants containing E110W or E110Y. Escherichia coli-expressed Glu(110) variants 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 alpha 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 Glu(110) mutation on receptor binding affinity. The marked and disproportionate decrease in TF-1 proliferation observed with (E110W/A6)scIL-5 suggests a role for Glu(110) in the biological activity mediated by the signal transducing receptor betac subunit of the IL-5 receptor. This is also consistent with the lack of stimulation of JAK2 phosphorylation 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 Glu(110) locus of IL-5 for receptor alpha chain binding and, in contrast, its heretofore underappreciated importance for receptor activation.
Wu
S J
SJ
Department of Medicine, University of Pennsylvania School of Medicine, 909 Stellar Chance Laboratories, Philadelphia, Pennsylvania 19104, USA.
Tambyraja
R
R
Zhang
W
W
Zahn
S
S
Godillot
A P
AP
Chaiken
I
I
D000939:Epitopes; D018698:Glutamic Acid; D006801:Humans; D015848:Interleukin-5 / Q000378:metabolism; D053614:Janus Kinase 2; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D018123:Receptors, Interleukin / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D053649:Receptors, Interleukin-5; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D000939:Epitopes; D015848:Interleukin-5; D011518:Proto-Oncogene Proteins; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D018698:Glutamic Acid; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2