title: Epitope-labeled soluble human interleukin-5 (IL-5) receptors. Affinity cross-link labeling, IL-5 binding, and biological activity.
abstract: The human receptor for the potent eosinophilopoietic cytokine interleukin-5 (IL-5) consists of two components: a 60-kDa ligand-binding alpha chain (IL-5 alpha R) and a 130-kDa beta chain (IL-5 beta R). Three ectodomain constructs of the alpha chain (alpha RED) bearing C-terminal epitope tags were engineered and expressed in baculovirus-infected Sf9 cells. Each recombinant alpha chain was secreted into the medium, maximum expression occurring 72 h post-infection. The various soluble alpha chains were shown by affinity cross-link labeling and competition with unlabeled IL-5 to bind recombinant human (rh) 125I-IL-5 specifically with an ED50 of 2-5 nM. The epitope tag provided a simple purification of the receptor from conditioned medium using immunoaffinity chromatography. The purified material had an apparent molecular mass of 43 kDa and was heterogeneously glycosylated. Sedimentation analysis revealed a 1:1 association of the purified epitope-tagged soluble receptor with its ligand, resulting in the formation of a 70-74-kDa complex. Circular dichroism analysis revealed that the soluble alpha chain existed with a significantly ordered structure consisting of 42% beta-sheet and 6% alpha-helix. Such analyses combined with fluorescence spectrometry suggested that ligand-receptor complex formation in solution resulted in minimal conformational changes, consistent with the suggestion that the membrane-associated form of the alpha chain itself has minimal signal transduction capability. Surface plasmon resonance studies of the interaction of the purified alpha RED with immobilized rhIL-5 revealed a specific, competable interaction with a dissociation constant of 9 nM. Preincubation of an IL-5-dependent cell line with the epitope-tagged alpha RED also dose-dependently neutralized rhIL-5-induced proliferation. These data demonstrate that biologically active epitope-tagged recombinant soluble IL-5 receptors are facile to produce in large quantities and may have therapeutic utility in the modulation of IL-5-dependent eosinophilia in man.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002942:Circular Dichroism; D000939:Epitopes; D006801:Humans; D015848:Interleukin-5 / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D018123:Receptors, Interleukin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D000939:Epitopes; D015848:Interleukin-5; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011994:Recombinant Proteins