Article: article from journal or magazin.
Characterization of Fas (Apo-1, CD95)-Fas ligand interaction.
Journal of Biological Chemistry
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.
Amino Acid Sequence, Animals, Antigens, CD95/metabolism, Binding Sites, Chromatography, Gel, Enzyme-Linked Immunosorbent Assay, Fas Ligand Protein, Glycosylation, Humans, Jurkat Cells, Ligands, Membrane Glycoproteins/genetics, Membrane Glycoproteins/metabolism, Mice, Models, Molecular, Molecular Sequence Data, Mutagenesis, Proline/metabolism, Sequence Alignment, Solubility, Species Specificity, Tyrosine/metabolism
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