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.

• Authors: P M Brown, P Tagari, K R Rowan, V L Yu, G P O'Neill, C R Middaugh, G Sanyal, A W Ford-Hutchinson, D W Nicholson
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 270 Issue 49 Pg. 29236-43 (Dec 08 1995) ISSN: 0021-9258 [Print] United States
• PMID: 7493953 (Publication Type: Journal Article)

Chemical References

• Epitopes
• Interleukin-5
• Receptors, Interleukin
• Receptors, Interleukin-5
• Recombinant Proteins

Topics

• Amino Acid Sequence
• Animals
• Base Sequence
• Cell Division (drug effects)
• Circular Dichroism
• Epitopes
• Humans
• Interleukin-5 (metabolism, pharmacology)
• Mice
• Molecular Sequence Data
• Receptors, Interleukin (chemistry, immunology, metabolism)
• Receptors, Interleukin-5
• Recombinant Proteins (biosynthesis)