Bacteria displaying interleukin-4 mutants stimulate mammalian cells and reflect the biological activities of variant soluble cytokines.
We describe a novel procedure that allows the rapid determination of cytokine activity on cells that express their cognate receptor. The four-helix bundle cytokine interleukin-4 (IL-4) was inducibly expressed as a fusion with the E. coli outer-membrane protein intimin, such that IL-4 was presented on the surfaces of the bacteria. Expression and accessibility of the cytokine on the cell exteriors were monitored by Western blotting and fluorescence microscopy, making use of two epitopes flanking the IL-4 component of the fusion protein. To demonstrate the biological activity of the immobilized cytokine, a Ba/F3-derived cell line stably transfected with both the bipartite human IL-4 receptor and an IL-4-specific luciferase reporter gene construct was employed. Bacterial cells displaying interleukin-4 elicited a specific, dose-dependent response in the reporter cells. Two variants of IL-4 with previously characterized (partial) antagonistic properties were also expressed as membrane-bound fusion proteins and were tested for their activity in the immobilized state. In comparison with bacteria displaying wild-type IL-4, E. coli clones presenting variants IL-4 Y124G and Y124D showed diminished or abolished activity, respectively, on murine reporter cells. The relative signaling potencies of the immobilized IL-4 variants thus closely mirror the agonistic properties of the corresponding soluble cytokines. This approach should be generally applicable for the mutational analysis of numerous signal mediators that trigger cellular responses through dimerization of transmembrane receptors.
Krause
Sebastian
S
University of Jena Medical School, Institute of Biochemistry I, 07743 Jena, Germany.
Würdemann
Dieco
D
Wentzel
Alexander
A
Christmann
Andreas
A
Fehr
Holger
H
Kolmar
Harald
H
Friedrich
Karlheinz
K
D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D015847:Interleukin-4 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D009092:Mucous Membrane / Q000378:metabolism; D009154:Mutation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D016207:Cytokines; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D015847:Interleukin-4