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Binding of the Drosophila cytokine Spätzle to Toll is direct and establishes signaling

Nature Immunology volume 4, pages 794–800 (2003)Cite this article

Abstract

The extracellular protein Spätzle is required for activation of the Toll signaling pathway in the embryonic development and innate immune defense of Drosophila. Spätzle is synthesized as a pro-protein and is processed to a functional form by a serine protease. We show here that the mature form of Spätzle triggers a Toll-dependent immune response after injection into the hemolymph of flies. Spätzle specifically bound to Drosophila cells and to Cos-7 cells expressing Toll. Furthermore, in vitro experiments showed that the mature form of Spätzle bound to the Toll ectodomain with high affinity and with a stoichiometry of one Spätzle dimer to two receptors. The Spätzle pro-protein was inactive in all these assays, indicating that the pro-domain sequence, which is natively unstructured, acts to prevent interaction of the cytokine and its receptor Toll. These results show that, in contrast to the human Toll-like receptors, Drosophila Toll requires only an endogenous protein ligand for activation and signaling.

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Figure 1: Expression of Toll and Spätzle in a baculovirus expression system.
Figure 2: Partial proteolysis of Spätzle pro-protein generates a stable 106-amino-acid C-terminal fragment.
Figure 3: Recombinant Spätzle protein is biologically active and capable of mediating immune responses in vivo.
Figure 4: The processed form of Spätzle, but not the pro-protein, binds to and activates cells expressing the Toll receptor.
Figure 5: Spätzle C-106 but not pro-protein binds with high affinity to the Toll ectodomain and with a stoichiometry of one Spätzle dimer to two Toll receptor molecules.

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Acknowledgements

We thank G. Shah, T. Chapman, P. Lowe, G. Praefcke and M. Moncrieffe for advice on baculovirus expression and biophysical techniques, as well as J. Royet for reagents and advice for hemolymph transfer experiments. We also thank J. Ladbury and T. Blundell for discussions and critical reading of this manuscript. We thank M. Sims for access to facilities in the GlaxoSmithKline Immunology Department in Stevenage. This work was supported by Centre National de la Recherche Scientifique and the National Institutes of Health (grant 5P01AI44220-02). S.T.-D. was supported by a short-term fellowship from La Ligue Contre le Cancer. A.W. was supported by GlaxoSmithKline, The Cambridge European Trust and St. John's College, Cambridge.

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Author notes

  1. Alexander N R Weber and Servane Tauszig-Delamasure: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom

    Alexander N R Weber & Nicholas J Gay

  2. UPR 9022–CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, Strasbourg, 67084, cedex, France

    Servane Tauszig-Delamasure, Jules A Hoffmann & Jean-Luc Imler

  3. Rheumatoid Arthritis Biology, RI CEDD, GlaxoSmithKline Medicines Research Centre, Stevenage, SG1 2NY, United Kingdom

    Keith P Ray

  4. Immunopharmacology, RI CEDD, GlaxoSmithKline Medicines Research Centre, Stevenage, SG1 2NY, United Kingdom

    Alexander N R Weber & Mary A Morse

  5. INSERM U.564, CHU Angers, Angers, 49033, Cedex 01, France

    Eric Lelièvre & Hugues Gascan

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Weber, A., Tauszig-Delamasure, S., Hoffmann, J. et al. Binding of the Drosophila cytokine Spätzle to Toll is direct and establishes signaling. Nat Immunol 4, 794–800 (2003). https://doi.org/10.1038/ni955

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