CHARACTERIZATION OF FAS (APO-1, CD95)-FAS LIGAND INTERACTION

The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Pas Ligand (FasL). When human soluble FasL (s FasL, containing the extracellular portion) was expressed in human emb ryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the struc ture of the closely related lymphotoxin alpha-tumor necrosis factor re ceptor I complex, a molecular model of the FasL homotrimer bound to th ree Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect t he receptor-ligand interaction were introduced at three sites. The F27 5L mutant, mimicking the loss of function murine gld mutation, exhibit ed a high propensity for aggregation and was unable to bind to Fas. Mu tants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fa s-positive cells with a concomitant decrease in the binding affinity f or the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was in active, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interest ingly, mutant Y218F could induce apoptosis in murine, but not human ce lls.