Title : Characterization of Fas ( Apo-1 , CD95 )- Fas ligand interaction
Abstract :
The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand ( FasL )
When human soluble FasL (s FasL , containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion
Based on the structure of the closely related lymphotoxin alpha- tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods
Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites
The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas
Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins
Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas
Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells