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A CD40–CD95L fusion protein interferes with CD40L-induced prosurvival signaling and allows membrane CD40L-restricted activation of CD95

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Abstract

We analyzed a novel bifunctional fusion protein, CD40ed–CD95Led, consisting amino-terminally of the extracellular domain of CD40 and carboxy-terminally of the extracellular domain of CD95L. On cells lacking CD40L, this fusion protein is poorly active with respect to CD95 activation [median effective dose (ED50)>1 μg/ml], but it stimulates CD95 signaling with high efficiency upon binding to membrane-expressed CD40L (ED50<1 ng/ml). Thus, cell surface immobilization mediated by the CD40 part of the molecule unmasks the high-latent, CD95-stimulating capacity of the otherwise poorly active CD95L fusion protein. Moreover, interaction of the CD40 part of CD40ed–CD95Led with CD40L prevents the activation of cellular CD40. The CD40ed–CD95Led fusion protein therefore simultaneously blocks antiapoptotic CD40 activation and induces CD95-mediated apoptosis. Indeed, T47D cells displaying an antiapoptotic autocrine CD40–CD40L signaling loop were significantly more sensitive toward CD40ed–CD95Led than toward soluble CD95L artificially activated by crosslinking. Fusion proteins of RANK and CD95L (RANKed–CD95Led) and CD40 and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (CD40ed–TRAILed), with domain architectures similar to CD40ed–Cd95Led, displayed RANKL-dependent CD95 and CD40L-dependent TRAILR2 activation, respectively, indicating the principle feasibility of this fusion protein design.

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Abbreviations

ed:

extracellular domain

TRAIL:

tumor necrosis factor-related apoptosis inducing ligand

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Acknowledgements

This work was supported by Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 487, project B7), Deutsche Krebshilfe (grant 10-1751-Wa 3), Wilhelm–Sander–Stiftung (grant 2003.120.1), and Interdisziplinären Zentrum für Klinische Forschung Wuerzburg (project B-32). We were further supported by APOXIS S.A. (Epalinges, Switzerland).

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Authors and Affiliations

  1. Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    Constance Assohou-Luty, Jeanette Gerspach & Klaus Pfizenmaier

  2. Department of Molecular Internal Medicine, Medical Clinic and Polyclinic II, University of Wuerzburg, Roentgenring 11, 97070, Wuerzburg, Germany

    Daniela Siegmund, Nicole Müller & Harald Wajant

  3. Dermatology Department, Geneva University Medical Center, 1 rue Michel-Servet, CH-1211, Geneva 4, Switzerland

    Bertrand Huard

  4. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuremberg, Fahrstrasse 17, 91054, Erlangen, Germany

    Gisa Tiegs

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  1. Constance Assohou-Luty
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Correspondence to Harald Wajant.

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Klaus Pfizenmaier and Harald Wajant contributed equally to this work.

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Assohou-Luty, C., Gerspach, J., Siegmund, D. et al. A CD40–CD95L fusion protein interferes with CD40L-induced prosurvival signaling and allows membrane CD40L-restricted activation of CD95. J Mol Med 84, 785–797 (2006). https://doi.org/10.1007/s00109-006-0073-1

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