A CD40-CD95L fusion protein interferes with CD40L-induced prosurvival signaling and allows membrane CD40L-restricted activation of CD95.
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 microg/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.
Assohou-Luty
Constance
C
Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Gerspach
Jeanette
J
Siegmund
Daniela
D
Müller
Nicole
N
Huard
Bertrand
B
Tiegs
Gisa
G
Pfizenmaier
Klaus
K
Wajant
Harald
H
D000818:Animals; D019013:CD40 Antigens / Q000276:immunology* / Q000378:metabolism; D023201:CD40 Ligand / Q000276:immunology* / Q000378:metabolism; D016923:Cell Death; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D053222:Fas Ligand Protein / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053245:RANK Ligand / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D019014:fas Receptor / Q000276:immunology* / Q000378:metabolism
D019013:CD40 Antigens; D053222:Fas Ligand Protein; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D019014:fas Receptor; D023201:CD40 Ligand