Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex - PubMed (original) (raw)

. 2003 Feb;23(4):1428-40.

doi: 10.1128/MCB.23.4.1428-1440.2003.

Aubry Tardivel, Magdalena Kovacsovics-Bankowski, Sylvie Hertig, Olivier Gaide, Fabio Martinon, Antoine Tinel, David Deperthes, Silvio Calderara, Therese Schulthess, Jürgen Engel, Pascal Schneider, Jürg Tschopp

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Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex

Nils Holler et al. Mol Cell Biol. 2003 Feb.

Abstract

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.

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Figures

FIG. 1.

FIG. 1.

Determination of the molecular masses of various FasL fusion proteins. (A) Schematic representation of the different FasL constructs used in this study. The number of amino acid residues in each domain is indicated. “Cryptic” means that the cytotoxic activity remains cryptic until the ligand is cross-linked. (B) SDS-PAGE analysis of purified recombinant FasL. Proteins (5 μg per lane) were analyzed under reducing conditions. Fc:FasL was also analyzed under nonreducing conditions. Gels were stained with Coomassie blue. Molecular mass markers are in kilodaltons. (C) Gel permeation chromatography of purified recombinant FasL. Ligands (200 μg) were applied to a Superdex-200 column and eluted in PBS. Cytotoxic activity was measured on Jurkat cells in the presence of anti-Flag antibodies and is expressed as the percentage of maximal response. The elution positions of molecular mass markers are indicated at the top of the figure (molecular masses in kilodaltons).

FIG. 2.

FIG. 2.

Rotary shadowing electron microscopy of recombinant FasL. Six representative images of FasL, ACRP:FasL, ACRPΔ:FasL, and Fc:FasL preparations are shown, with a schematized interpretation of the picture. Filled circles, trimeric FasL; black rods, collagen domain of ACRP or ACRPΔ; open circles, dimeric Fc portion of IgG1; thin lines, possible connectivity between FasL and Fc domains. For Fc:FasL, alternative interpretations are possible regarding the identification of FasL and Fc domains and regarding the connectivity between domains. Pictures were taken at a magnification of ×150,000, and a scale of 10 nm is indicated (bars). Schematic representations of the hexameric forms of ACRP:FasL and Fc:FasL are shown at the bottom of the figure.

FIG. 3.

FIG. 3.

Cytotoxicity of various recombinant FasLs. OD, optical density. (A) The T-lymphoblastoma Jurkat cell line was exposed to the indicated amount of purified Fc:FasL (total preparation) or to the high- and low-molecular-weight (high MW and low MW) fractions of Fc:FasL (fractions 7 and 15 of Fig. 1C, respectively). The reducing capacity of mitochondria was monitored with PMS/MTS reagents as a measure of cell viability. (B) Raji Burkitt lymphoma, HeLa epitheloid cervix carcinoma, A431 epidemoid carcinoma, and Jurkat cell lines and primary human T cells from peripheral blood activated for 2 days were exposed to recombinant FasL (squares) or ACRP:FasL (circles) at the indicated concentration, in the presence (filled symbols) or absence (open symbols) of cross-linking anti-Flag M2 monoclonal antibody. For cell lines, viability was measured with PMS/MTS reagents. Primary T cells were stained with annexin V, and viable cells were quantified by fluorescence-activated cell sorter analysis. (C) Left three panels: the cytotoxic activity of the indicated FasL constructs was monitored on Jurkat cells in the presence (filled symbols) or absence (open symbols) of anti-Flag M2 antibody. Cell viability was quantified with PMS/MTS reagents. Right panel: anti-Flag Western blot of various FasL preparations separated by SDS-PAGE under reducing (+DTT) or nonreducing (−DTT) conditions.

FIG. 4.

FIG. 4.

Biological activity of ACRP:FasL, ACRP:CD40L, and ACRP:Tweak. (A) FasL-sensitive 293T-6 cells transfected with a Flag-JNK expression plasmid were treated for the indicated period of time with FasL or ACRP:FasL in the presence or absence of cross-linking antibody. Cell lysates were analyzed by Western blotting using an anti-phospho-JNK antibody. Equal expression levels of JNK were confirmed by reprobing the membrane with anti-Flag antibody. (B) Jurkat cells were treated with FasL and ACRP:FasL as described in the Fig. 3B legend, except that the caspase inhibitor Z-VAD-fmk was added to reveal caspase-independent cell death. (C) Purified murine splenic B cells were incubated for 48 h with the indicated concentration of murine CD40L (squares) or ACRP:CD40L (circles) in the presence (filled symbols) or absence (open symbols) of cross-linking anti-Flag M2 antibody. Proliferation was measured by [3H]thymidine incorporation. Controls include LPS (triangles) and heat-inactivated ACRP:CD40L (diamonds). Figure shows the mean plus or minus standard deviation of triplicates. (D) The Kym-1 rhabdomyosarcoma cell line was treated with Tweak and ACRP:Tweak, as indicated. Cell viability was monitored using PMS/MTS reagents.

FIG. 5.

FIG. 5.

FasL and ACRP:FasL bind Fas equally well. (A) The interaction between Flag-tagged FasL, ACRP:FasL, and ACRPΔ:FasL on the one hand and coated Fas:Fc (filled symbols) or TRAILR2:Fc (open symbols, negative control) on the other hand was monitored by ELISA. (B) Burkitt lymphoma Raji cells were incubated for 15 min with FasL, ACRP:FasL, or PBS alone. Washed cells were lysed and FasL bound to the cells was detected by anti-Flag Western blotting. Cross-linking antibody was added during the incubation (+) or after cell lysis (−). (C) The binding of [125I]-labeled ZB4 anti-Fas antibody to BJAB cells was competed with the indicated concentrations of recombinant FasL (squares) or ACRP:FasL (circles). (D) The binding of Flag-tagged FasL or ACRP:FasL (at 200 ng/ml) to recombinant Fas:Fc was competed with the indicated amounts of untagged Fc:FasL. Bound ligands were detected via the Flag tag. (E) HeLa cells were incubated for 16 h with a constant, lethal concentration of ACRP:FasL in the presence of various amounts of FasL. Cell viability was monitored with the PMS/MTS reagent.

FIG. 6.

FIG. 6.

DISC analysis in Raji cells. Raji cells were treated for 15 min with 0.5 μg of FasL or ACRP:FasL or with PBS alone in the presence (+) or absence (−) of cross-linking anti-Flag M2 antibody and were then washed in PBS. After cell lysis and sampling of soluble cell extracts, lysates of untreated cells were supplemented with 0.5 μg of FasL or ACRP:FasL (postlysis). Anti-Flag antibody was added to all samples to allow immunoprecipitation (IP) of Flag-tagged FasL. Immunoprecipitates and cell extracts were analyzed by Western blotting (WB) for Fas, FADD, caspase 8, and Flag-tagged FasL. IgG, heavy chain of the immunoprecipitating antibody. Molecular masses are indicated in kilodaltons.

FIG. 7.

FIG. 7.

DISC analysis in wild-type (wt) and mutant Jurkat cells. Jurkat cells (wild type, FADD deficient [def], and caspase 8 deficient) were treated for 15 min with FasL, ACRP:FasL, or PBS alone, in the presence (+) or absence (−) of cross-linking anti-Flag M2 antibody, and were then washed in PBS. After lysis, samples treated without cross-linker were supplemented with anti-Flag antibody and ligands were immunoprecipitated and analyzed by Western blotting for Fas, FADD, and caspase 8. For each condition (except PBS alone), 1% of the postnuclear extract was loaded for comparison. The migration positions of procaspase 8 (Casp-8), FADD, and Fas and the high-molecular-mass modification of Fas (Fas hmw) are indicated by black arrowheads. The open arrowhead points to the heavy chain of the immunoprecipitating antibody (IgG).

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