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Identification of amino acid residues important for ligand binding to Fas.

Starling GC, Bajorath J, Emswiler J, Ledbetter JA, Aruffo A, Kiener PA - J. Exp. Med. (1997)

Bottom Line: Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas.Two mutants (K78S and H95S) bound FasL comparably to wild type.Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.

ABSTRACT
The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas-FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

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A panel of anti-Fas mAbs  bind FasthrRγ1 and FasthrRγ1 mutants. Fusion proteins were immobilized, and binding was detected by  anti-Fas mAbs at various concentrations. Data is shown for mAb at 1  μg/ml, but similar relative binding  was observed at lower concentrations of mAb. Open bar, SW1/17;  shaded bar, SW1/1; closed bar, DX-2.  Negative control mAb OD values  for each fusion protein were ∼0.1.
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Figure 2: A panel of anti-Fas mAbs bind FasthrRγ1 and FasthrRγ1 mutants. Fusion proteins were immobilized, and binding was detected by anti-Fas mAbs at various concentrations. Data is shown for mAb at 1 μg/ml, but similar relative binding was observed at lower concentrations of mAb. Open bar, SW1/17; shaded bar, SW1/1; closed bar, DX-2. Negative control mAb OD values for each fusion protein were ∼0.1.

Mentions: The binding of each mutant protein to a panel of anti-Fas mAbs, which recognize three epitopes based on the following (a) their ability to block CD8–FasL-mediated apoptosis (DX-2); (b) their ability to induce apoptosis (SW1/1); or (c) their inability to block or induce apoptosis (SW1/17) in our system (data not shown), was examined. Each mAb was unable to immunoblot FasthrRγ1 when the protein was reduced before loading on the gel, indicating that the epitopes the mAb recognize are conformationally sensitive. Therefore, these mAbs are suitable to monitor the overall structural integrity of expressed mutant proteins. Each mAb bound to each mutant and to wild-type FasthrRγ1 equivalently as determined by ELISA (Fig. 2), indicating that the overall structural integrity of the proteins was not significantly compromised as a consequence of the mutations.


Identification of amino acid residues important for ligand binding to Fas.

Starling GC, Bajorath J, Emswiler J, Ledbetter JA, Aruffo A, Kiener PA - J. Exp. Med. (1997)

A panel of anti-Fas mAbs  bind FasthrRγ1 and FasthrRγ1 mutants. Fusion proteins were immobilized, and binding was detected by  anti-Fas mAbs at various concentrations. Data is shown for mAb at 1  μg/ml, but similar relative binding  was observed at lower concentrations of mAb. Open bar, SW1/17;  shaded bar, SW1/1; closed bar, DX-2.  Negative control mAb OD values  for each fusion protein were ∼0.1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196280&req=5

Figure 2: A panel of anti-Fas mAbs bind FasthrRγ1 and FasthrRγ1 mutants. Fusion proteins were immobilized, and binding was detected by anti-Fas mAbs at various concentrations. Data is shown for mAb at 1 μg/ml, but similar relative binding was observed at lower concentrations of mAb. Open bar, SW1/17; shaded bar, SW1/1; closed bar, DX-2. Negative control mAb OD values for each fusion protein were ∼0.1.
Mentions: The binding of each mutant protein to a panel of anti-Fas mAbs, which recognize three epitopes based on the following (a) their ability to block CD8–FasL-mediated apoptosis (DX-2); (b) their ability to induce apoptosis (SW1/1); or (c) their inability to block or induce apoptosis (SW1/17) in our system (data not shown), was examined. Each mAb was unable to immunoblot FasthrRγ1 when the protein was reduced before loading on the gel, indicating that the epitopes the mAb recognize are conformationally sensitive. Therefore, these mAbs are suitable to monitor the overall structural integrity of expressed mutant proteins. Each mAb bound to each mutant and to wild-type FasthrRγ1 equivalently as determined by ELISA (Fig. 2), indicating that the overall structural integrity of the proteins was not significantly compromised as a consequence of the mutations.

Bottom Line: Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas.Two mutants (K78S and H95S) bound FasL comparably to wild type.Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.

ABSTRACT
The interaction of Fas (CD95), a member of the tumor necrosis factor receptor (TNFR) family, and its ligand (FasL) triggers programmed cell death (apoptosis) and is involved in the regulation of immune responses. Although the Fas-FasL interaction is conserved across species barriers, little is currently known about the molecular details of this interaction. Our aim was to identify residues in Fas that are important for ligand binding. With the aid of a Fas molecular model, candidate amino acid residues were selected in the Fas extracellular domain 2 (D2) and D3 and subjected to serine-scanning mutagenesis to produce mutant Fas molecules in the form of Ig fusion proteins. The effects of these mutations on FasL binding was examined by measuring the ability of these proteins to inhibit FasL-mediated apoptosis of Jurkat cells and bind FasL in ELISA and BIAcore assays. Mutation of two amino acids, R86 and R87 (D2), to serine totally abolished the ability of Fas to interact with its ligand, whereas mutants K84S, L90S, E93S (D2), or H126S (D3) showed reduced binding compared with wild-type Fas. Two mutants (K78S and H95S) bound FasL comparably to wild type. Therefore, the binding of FasL involves residues in two domains that correspond to positions critical for ligand binding in other family members (TNFR and CD40) but are conserved between murine and human Fas.

Show MeSH