Single chain human interleukin 5 and its asymmetric mutagenesis for mapping receptor binding sites.
Wild type human (h) interleukin 5 (wt IL5) is composed of two identical peptide chains linked by disulfide bonds. A gene encoding a single chain form of hIL5 dimer was constructed by linking the two hIL5 chain coding regions with Gly-Gly linker. Expression of this gene in COS cells yielded a single chain IL5 protein (sc IL5) having biological activity similar to that of wt IL5, as judged by stimulation of human cell proliferation. Single chain and wt IL5 also had similar binding affinity for soluble IL5 receptor alpha chain, the specificity subunit of the IL5 receptor, as measured kinetically with an optical biosensor. The design of functionally active sc IL5 molecule. Such mutagenesis was exemplified by changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor binding and bioactivity data obtained are consistent with a model in which residues from both IL5 monomers interact with the receptor alpha chain, while the interaction likely is asymmetric due to the intrinsic asymmetry of folded receptor. The results demonstrate a general route to the further mapping of receptor and other binding sites on the surface of human IL5.
Li
J
J
Molecular Immunology Department, SmithKline Beecham, King of Prussia, Pennsylvania 19406, USA.
Cook
R
R
Dede
K
K
Chaiken
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D015848:Interleukin-5 / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D018123:Receptors, Interleukin / Q000378:metabolism; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D015848:Interleukin-5; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins