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Volume 272, Number 33, Issue of August 15, 1997 pp. 20611-20618
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Spatial Orientation of the  and _c Receptor Chain Binding Sites on Monomeric Human Interleukin-5 Constructs

(Received for publication, March 25, 1997, and in revised form, May 20, 1997)

Michael D. Edgerton , Pierre Graber , Derril Willard ^§ , Tom Consler ^§ , Murray McKinnon ^¶ , Iain Uings ^¶ , Christian Y. Arod , Frederic Borlat , Richard Fish , Manuel C. Peitsch , Timothy N. C. Wells and Amanda E. I. Proudfoot

From the  Geneva Biomedical Research Institute, 1228 Plan-les-Ouates, Geneva, Switzerland, the ^§ Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina 27709, and ^¶ Glaxo Wellcome Research and Development, Stevenage SG1 2NY, United Kingdom

Interleukin-5 (IL-5), a disulfide-linked homodimer, can be induced to fold as a biological active monomer by extending the loop between its third and fourth helices (Dickason, R. R., and Huston, D. P. (1996) Nature 379, 652-655). We have designed eight monomeric IL-5 proteins to optimize biological activity and stability of the monomer. This was achieved by (i) inserting the joining loop at three different positions, (ii) by introducing an additional intramolecular disulfide bridge onto these backbones, and (iii) by creating circular permutations to fix the position of the carboxyl-terminal helix relative to the three other helices. The proteins dimerize with K_d values ranging from 20 to 200 µM and are therefore monomeric at the picomolar concentrations where they are biologically active. Introduction of a second disulfide confers increased stability, but this increased rigidity results in lower activity of the protein. Contrary to wild type IL-5, mutation of the _c contact residue on the first helix, Glu¹², to Lys, into the circularly permutated constructs, did not abolish TF-1 proliferative and eosinophil activation activities. These results indicate that activation of the IL-5 receptor complex is not mediated solely by Glu¹² on the first helix, and alternative mechanisms are discussed.

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