The Role of CD95 Glycosylation in CD95 Signaling

The CD95 (APO-1/Fas)-mediated apoptotic pathway is one of the well-studied death receptor pathways. CD95 is a highly glycosylated type I transmembrane protein and has a molecular mass of 37.5 kDa. The N-terminal domain of CD95 contains two putative N-linked glycosylation sites at N118 and N136 and posttranslational glycosylation leads to an increase of molecular mass of CD95 to 45–52 kDa. Upon stimulation of CD95, either with its natural ligand CD95L or with agonistic antibodies such as anti-APO-1, a Death-Inducing Signaling Complex (DISC) is formed. The DISC consists of oligomerized, most probably trimerized, CD95, the DD-containing adaptor molecule Fas associated death domain containing protein (FADD), procaspase-8, procaspase-10 and cellular FLICE inhibitory protein (c-FLIP). The interactions between the molecules in the DISC are based on homophilic contacts. The death domain (DD) of CD95 interacts with the DD of FADD while the death effector domain (DED) of FADD interacts with the N-terminal tandem DED of procaspase-8. Binding of procaspase-8 to the DISC results in its autocatalytic cleavage with the formation of the mature caspase-8 which is released into the cytosol to propagate the apoptotic signal. The data presented here show that N-glycosylation of CD95 influences death-inducing signaling complex (DISC) formation and procaspase-8 activation at the CD95 DISC. Treatment with the chemical inhibitors of N-glycosylation tunicamycin and Deoxymannojirimycin (DMM), neuraminidase from Vibrio cholerae and generation of CD95 glycosylation mutants at N136 and N118 show that N deglycosylation of CD95 results in the reduction of caspase-8 processing at the DISC. Modelling of the core CD95 DISC structure and the structure of the CD95 DISC network on the membrane indicated that the glycan attached to N118 could be important for the contacts between neighboring DISCs forming a network. A glycan moiety attached to N136 of CD95 could be important for the complex formation and/or stability as it is located in close proximity to one of the CD95L molecules. The present study shows that the CD95 glycostructure can contribute to the amount of generated caspase-8, which defines cell death initiation. This regulation might be important for cancer cells when a subtle difference in the amount of activated caspase-8 regulates life or death of the cell