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 Fas ligand (FasL). When human soluble FasL (sFasL, 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.
Schneider
P
P
Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center, CH-1066 Epalinges, Switzerland. pascal.schneider@ib.unil.ch
Bodmer
J L
JL
Holler
N
N
Mattmann
C
C
Scuderi
P
P
Terskikh
A
A
Peitsch
M C
MC
Tschopp
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D053222:Fas Ligand Protein; D006031:Glycosylation; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011392:Proline / Q000378:metabolism; D016415:Sequence Alignment; D012995:Solubility; D013045:Species Specificity; D014443:Tyrosine / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008024:Ligands; D008562:Membrane Glycoproteins; D019014:fas Receptor; D014443:Tyrosine; D011392:Proline