CD95L concatemers highlight different stoichiometries of CD95-mediated apoptotic and nonapoptotic pathways

To better understand the stoichiometry of CD95L required to trigger apoptotic and nonapoptotic signals, we generated several CD95L concatemers from dimer to hexamer conjugated via a flexible link (GGGGS)2. These ligands reveal that although the hexameric structure is the best stoichiometry to trigger cell death, a dimer is sufficient to induce the apoptotic response in CD95-sensitive Jurkat cells. Interestingly, only trimeric and hexameric forms can implement a potent Ca2+ response, suggesting that while CD95 aggregation controls the implementation of the apoptotic signal, both aggregation and conformation are required to implement the Ca2+ pathway. Using the extracellular region of CD95L (FasL), concatemers were designed, synthesized, and expressed. The biological effects of these molecules on the apoptotic and nonapoptotic (intracellular Ca2+ response) signals were analyzed. The findings highlight the major role of conformation (nonapoptotic) and aggregation (apoptotic) in the CD95-mediated signals. image