By
From Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121
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 BIAcoreTM 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.
Programmed cell death (apoptosis) mediated by the Fas-
FasL system is a mechanism used to control immune
responses. The Fas (CD95) antigen, a 45-kD protein of the
TNF receptor (TNFR) family, is widely expressed and binds
a TNF-like ligand (FasL) (1). Perturbations of the Fas-FasL
interaction have drastic functional consequences in lpr and
gld mice, leading to lymphadenopathy and severe immune
disregulation (2, 3). A human lymphoproliferative disorder,
the Canale-Smith syndrome, appears to be due to mutation of the signal transduction domain of Fas (4). Although FasL is expressed as a cell surface molecule, it is also released after cleavage by metalloproteinases (5), enabling FasL to act as a
soluble mediator of cell death. Fas-mediated cell death is
thought to be involved in the pathology of a number of
disease states, including fulminant hepatitis and chronic
liver disease (6, 7), multiple sclerosis (8), and it may also
have a role in neutrophil-mediated tissue destruction (9). In
addition, some tumors are able to escape immune surveillance by releasing FasL, which kills activated T cells infiltrating the tumor (10, 11).
Molecular details of the Fas-FasL interaction have yet to
be determined. Fas is a type I membrane protein, consisting of
three TNFR-like extracellular domains (D1, D2, and D3), a
hydrophobic transmembrane region, and a cytoplasmic tail
containing a death domain. The death domain binds Fas death
domain-binding protein (FADD, MORT1), which links Fas
to a cascade of IL-1 Monoclonal Antibodies and Fusion Proteins.
Soluble FasL was produced in a manner similar to that described for a closely related
TNF family member, gp39, the ligand for CD40 (18), by fusing
the extracellular domain of FasL to the extracellular domain of
murine CD8. cDNA encoding for the extracellular domain of human FasL (amino acids 105-281) was amplified by PCR from
monocyte cDNA using the primers CGC CGC GGA TCC CTT CCA CCT ACA GAA GGA GCT G (forward primer containing a BamHI site) and GGC TGC TCT AGA CCC AAA GTG
CTT CTC TTA GAG CTT ATA TAA GCC (reverse primer
containing a XbaI restriction enzyme site). Amplified cDNA was
digested with BamHI and XbaI, gel purified, and ligated into the
pCDM7-like proteolytic enzymes known as
caspases (12). Recently, the three dimensional structure of
the Fas death domain was solved using NMR spectroscopy, and was shown to consist of six antiparallel, amphipathic
helices arranged in a novel fold (13). Fas binds to FasL across
the murine and human species barrier, suggesting the conservation of amino acid residues important for binding. We
wished to investigate the structural basis for the Fas-FasL interaction. Using the TNFR three-dimensional structure as a
template, we were able to generate a model of the Fas extracellular domains (14). On this model, we were able to
map residues conserved between human and murine Fas,
and positions implicated in the interaction of TNFR with TNF (15), and/or positions implicated by mutagenesis analysis in the interaction of another family member, CD40, with
the CD40L (16,17). A surface was identified on the extracellular D2 of Fas and a part of D3, which consists of residues conserved in murine and human Fas, but not conserved between Fas and TNFR or CD40. Residues in this
region were considered potential candidates for FasL binding and were subjected to serine-scanning mutagenesis. We
found that binding of FasL is centered on D2 of Fas and involves a region that corresponds to the ligand binding sites
in TNFR and CD40.
vector containing cDNA encoding for murine CD8.
CD8-FasL was produced in COS cells following transfection by
the DEAE-Dextran chloroquine method. Supernatants containing CD8-FasL fusion proteins were harvested and passed through
a 0.22-µm filter. CD8-FasL was affinity purified on an anti-CD8
(53-6) column as previously described for CD8-CD40L (19).
1), consisting of the extracellular
domain of Fas fused to the hinge, CH2, and CH3 regions of human IgG1 containing a thrombin cleavage site, was constructed.
In brief, cDNA was amplified by PCR using oligonucleotide
primers 5
-CGC CCC AAG CTT CGG AGG ATT GCT CAA
CAA CC (containing a HindIII site) and 3
-CGC CGC GGA
TCC CCC AAG TTA GAC CTG GAC CCT TCC TC (naturally occurring BamHI and BglII sites were removed and a BamHI
site was added). Purified PCR products were digested with HindIII
and BamHI restriction enzymes, gel purified, and ligated into
CDM7
containing a thr-human IgG1 (thrR
1) cassette (20).
Fasthr R
1 supernatants were produced as described above for
CD8-FasL, affinity purified on protein A-Sepharose, eluted with
ImmunoPure elution buffer (Pierce, Rockford, IL), dialyzed in
PBS, and concentrated. The concentration of each protein was
determined using an anti-human IgG binding ELISA. Preparation
of murine CD6 SRCR-D1thrR
1 (mCD6D1thrR
1) used as a
negative control protein has been previously described (21).
Site-directed Mutagenesis.
Eight amino acid residues in the extracellular domain of Fas were selected for site-directed mutagenesis to serine based on the molecular model of the Fas extracellular region (14). FasthrR1 mutants were produced by encoding
the desired mutation in overlapping oligonucleotide primers, and
using FasthrR
1 cDNA as a template, the mutants were generated by PCR. The 5
and 3
oligonucleotide primers described
for the production of FasthrR
1 were also used for the generation of mutants. PCR products were digested with HindIII and
BamHI and ligated into the CDM7
-thrR
1 cassette as described above. Mutant proteins were produced as described above
for FasthrR
1. Each cDNA construct was sequenced to confirm
insertion of the appropriate mutation and to ensure that the PCR
had not produced unwanted mutation.
Stimulation of Jurkat Cell Death.
Jurkat cells that were susceptible to FasL-mediated apoptosis were added to the wells of 96well microtiter plates at a final concentration of 1 × 105 cells/
well in a total volume of 200 µl with 25 µl of a CD8-FasL containing supernatant and various concentrations of FasthrR1 or
mutant FasthrR
1. Microtiter plates were incubated overnight at
37°C, 5% CO2 before the addition of 25 µl/well of Alamar Blue (BioSource International, Camarillo, TX). Plates were incubated for a further 8-12 h at 37°C before measuring the OD (A570nm
A595nm).
Enzyme Immunoassays.
The gross structural integrity of the
fusion proteins was confirmed using the panel of mAbs described
above in an ELISA assay. Fusion proteins (300 ng/ml in carbonate/bicarbonate buffer, pH 9.6) were immobilized on Immunolon II (Dynatech Laboratories, Inc., Alexandria, VA) plates overnight at 4°C. Plates were blocked with 3% BSA-PBS, followed
by addition of mAb at various concentrations. Plates were washed,
followed by 1:5,000 dilution of HRP-conjugated goat anti-mouse
IgG (Jackson ImmunoResearch Labs., Inc., West Grove, PA). HRP
substrate (GIBCO BRL, Gaithersberg, MD) was added, the color
reaction stopped with H2SO4, and OD measured (A450nm A725nm).
Surface Plasmon Resonance Analysis. All experiments were run on a BIAcoreTM 1000 instrument (Pharmacia Biosensor, Uppsala, Sweden) at 25°C using PBS, pH 7.4, containing 0.005% surfactant P20 (Pharmacia Biosensor) as the running buffer. CD8-FasL was immobilized on research-grade CM5 sensor chips (Pharmacia Biosensor) using standard N-ethyl-N-dimethylaminopropyl carbodiimid/N-hydroxysuccinimide coupling. Purified CD8-FasL protein (15 µg/ml) was diluted in 10 mM sodium formate buffer, pH 4.0, and incubated with activated sensor chips for 3 min. After coupling, excess N-hydroxysuccinimide groups were inactivated with ethanolamine. Immobilization of ~6,000 RU of FasL was achieved.
Apparent association and dissociation rates for wild-type FasthrRFas belongs to the TNFR
superfamily and its extracellular region includes three domains (D1-D3) with distinct sequence homology to TNFR
(1). On this basis, a detailed three-dimensional model was
generated (14) using the TNFR x-ray structure (15) as a
template. Eight residues that were spatially adjacent in the model (K78, K84, R86, R87, L90, E93, H95 in D2, and
H126 in D3) were selected as candidates for mutagenesis.
These eight residues are conserved in murine and human
Fas, but they are not conserved between family members
(14). Six of these positions (all except R86 and H95) correspond to residues that are implicated in the TNFR and/or
CD40-ligand interactions (14, 16, 17). The molecular model
and mutated residues are shown in Fig. 1. Color-coding of this model is based upon the results of the mutagenesis
studies described below and discussed at the end of this report. Mutant proteins were generated, expressed as Ig fusion proteins (FasthrR1), and purified for use in FasL
binding and functional assays.
Binding of Mutant Proteins to mAb.
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 FasthrR1 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 FasthrR
1 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.
FasL Binding Capabilities of FasthrR
The functional capabilities and binding characteristics of FasthR1 mutants were compared with wild-type FasthR
1 in vitro.
Supernatants of CD8-FasL were able to kill Jurkat cells
in a dose-dependent manner (data not shown), and the killing could be inhibited by wild-type FasthrR1. FasthrR
1
wild-type and mutants were titered at a constant concentration of CD8-FasL, and the viability of the Jurkat cells
was determined by measuring the change in color of Alamar
Blue added to the microtiter wells. In this assay, FasthrR
1
inhibited apoptosis in a concentration-dependent manner,
whereas mutant proteins showed a range of inhibitory activities (Fig. 3). Mutation of residues K78 and H95 to
serine had little effect on the ability of the fusion proteins
to inhibit killing, whereas mutation of residues, K84, L90,
E93, and H126 to serine markedly reduced the ability of
FasthrR
1 to inhibit the apoptotic activity of CD8-FasL.
Mutation to serine at positions R86 and R87 completely
abolished the inhibitory effect of the fusion proteins, suggesting they are critical for the Fas-FasL interaction. A control fusion protein, mCD6D1thrR
1, had no protective effect
in these assays.
To measure directly the binding of FasthrR1 mutants
to FasL, an ELISA assay was developed. FasthrR
1 and
mutants were captured on anti-human Ig-coated plates, followed by addition of CD8-FasL-containing supernatant.
Bound FasL was detected using an anti-FasL mAb, NOK-2, and subsequently anti-mouse IgG. The results parallel those
obtained in the biological assay (Fig. 4). K78S and H95S,
which showed wild-type inhibitory activity of FasL-mediated killing bound at similar levels to wild type. Although
saturation of binding by K78S was higher than wild type,
the linear portions of the binding curves were similar. Mutants R86S and R87S, which were unable to protect Jurkat
cells from FasL-mediated lysis, were likewise unable to bind
FasL in the ELISA. Their binding characteristics were identical to the mCD6D1thrR
1 control fusion protein, indicating that positions 86 and 87 are critical for FasL binding.
Mutants with intermediate inhibitory activity in the viability
assay also showed intermediate binding to FasL, thus indicating a strong correlation of binding and functional characteristics. Of these intermediate mutants, H126S showed the
least binding, whereas the binding levels of K84S, L90S, and
E93S were roughly equivalent but ~10-fold reduced compared with wild-type FasthrR
1. To confirm that the hierarchy of mutant to binding FasL was not a consequence of
differential capture of the mutants, duplicate plates were analyzed for amount of proteins captured by detecting the
Fc portion of the fusion proteins with an anti-human IgG
reagent. Equivalent binding profiles were obtained for each
fusion protein (data not shown), confirming that the differential FasL binding observed was due to the mutation in
the Fas region and not due to differences in protein concentration or ability to bind anti-human Ig.
Surface Plasmon Resonance Analysis of FasthrR
Surface plasmon resonance analysis
of the Fas-FasL interaction was undertaken by BIAcoreTM
analysis to obtain an approximation of the binding constant
for this interaction. Purified CD8-FasL was immobilized
on a sensor chip and various concentrations of FasthrR1
in fluid phase was passed over the chip. Association and dissociation rates were calculated, and a Kd of value of ~7 × 10
8 M was obtained from the ratio of dissociation/association. This is a relatively weak interaction compared with Kd
values obtained for the TNFR(p55)Ig-TNF interaction
(6.5 × 10
11 M for TNF-
and 6.4 × 10
10 M for TNF
) obtained by Scatchard analysis (22). The Kd of the
FasthrR
1-CD8-FasL interaction is also considerably weaker than that reported for CD40-CD40L (Kd, ~5 × 10
10 M
[23]) but similar to another TNFR-TNF family member,
4-1BB/4-1BBL (Kd of low affinity sites using recombinant
ligand ~7 × 10
8 M and Kd of high affinity sites ~3 × 10
10 M [24]), each Kd value obtained by binding Ig fusion
proteins to ligand expressed on cells.
Equilibrium binding curves (Fig. 5) showed results equivalent to the ELISA. K78S saturation binding was higher
than wild type due to a slower off rate from FasL. H95S
binding was similar to wild type; however, the curves were
steeper on each side of the equilibrium plateau, suggesting
faster on and off rates. R86S and R87S showed no binding
to the chip, whereas mutants K84S, L90S, E93S, and H126S
showed reduced binding to the chip-bound FasL. Again,
the hierarchy of the intermediate mutants was consistent between different assays, with K84S, L90S, E93S having
roughly equivalent RU values, whereas binding H126S
was markedly lower than the other three mutants.
Outline of the Fas Ligand-binding Site.
Mutants with deleterious effects on ligand binding were mapped with the aid of the three-dimensional Fas model (see Fig. 1). Two adjacent arginine residues (R86 and R87; colored magenta in Fig. 1) are critical for binding. Four other residues support binding (K84, L90, E93, H126; colored in gold in Fig. 1) but are not critical as mutation of these residues does not obliterate binding to ligand. In the model, these six residues form a surface that is likely to constitute a center of Fas- FasL interactions. Therefore, binding of ligand is centered on extracellular D2, but residues in D3 may support ligand binding. Mutation of residues K78 and H95 have no deleterious effects on ligand binding and have been colored in green in the model (see Fig. 1). Positions equivalent to H95 have not been implicated in the ligand binding of TNFR or CD40. Residues critical for ligand binding in Fas (R86 and R87) are adjacent to a disulfide bond, which is conserved in TNFR and CD40 (14); however, positions equivalent to only one of these residues (R87) are implicated in ligand binding for the other family members (15). Although the surface of ligand binding for TNFR, CD40, and Fas are located in a similar location, the amino acid composition of the binding surfaces differ. Different residues at equivalent positions alter the ligand binding surface and therefore determine the ligand binding specificity. At present, it is unclear whether all of the identified residues are directly involved in FasL recognition, or whether some mutations introduce local conformational changes sufficient to compromise binding to ligand but not to mAb. Additionally, other residues may be expected to contribute to the interaction.
In summary, individual residues in Fas have been identified as critical for ligand binding and an important role of charged residues in mediating Fas-FasL interactions has been demonstrated. Residues important for binding are conserved in murine and human Fas, which provides a rationale for the observed cross-species Fas-ligand interactions. However, these residues are not conserved in TNFR or CD40, thus explaining the specificity of the Fas receptor-ligand interaction. On the basis of our study, Fas D2 includes two residues that are critical for ligand binding and three residues that support ligand binding. Four of these residues (except R86) correspond to residues that contribute to CD40 (17) and TNFR ligand binding (15). This suggests that although specific residue contributions differ, equivalent regions are utilized by these receptors for mediating different ligand binding specificities.
Address correspondence to Gary C. Starling, Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, WA 98121.
Received for publication 29 January 1997.
We thank G. Whitney for mCD6D1thrR1, R. Peach for helpful discussions, and T. Nelson for assistance
in preparation of the manuscript.
1. | Nagata, S., and P. Golstein. 1995. The Fas death factor. Science (Wash. DC). 267: 1449-1456 [Medline]. |
2. | Watanabe-Fukunaga, R., C.I. Brannan, N.G. Copeland, N.A. Jenkins, and S. Nagata. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature (Lond.). 356: 314-317 [Medline]. |
3. | Takahashi, T., M. Tanaka, C.I. Brannan, N.A. Jenkins, N.G. Copeland, T. Suda, and S. Nagata. 1994. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell. 76: 969-976 [Medline]. |
4. |
Drappa, J.,
A.K. Vaishnaw,
K.E. Sullivan,
J.-L. Chu, and
K.B. Elkon.
1996.
Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated
with autoimmunity.
N. Engl. J. Med.
335:
1643-1649
|
5. | Kayagaki, N., A. Kawasaki, T. Ebata, H. Ohmoto, S. Ikeda, S. Inoue, K. Yoshino, K. Okumura, and H. Yagita. 1995. Metalloproteinase-mediated release of human Fas ligand. J. Exp. Med. 182: 1777-1783 [Abstract]. |
6. | Ogasawara, J., R. Watanabe-Fukunaga, M. Adachi, A. Matsuzawa, T. Kasugai, Y. Kitamura, N. Itoh, T. Suda, and S. Nagata. 1993. Lethal effect of the anti-Fas antibody in mice. Nature (Lond.). 364: 806-809 [Medline]. |
7. | Galle, P.R., W.J. Hofmann, H. Walczak, H. Schaller, G. Otto, W. Stremmel, P.H. Krammer, and L. Runkel. 1995. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J. Exp. Med. 182: 1223-1230 [Abstract]. |
8. | Dowling, P., G. Shang, S. Raval, J. Menonna, S. Cook, and W. Husar. 1996. Involvement of the CD95 (APO-1/Fas) receptor/ligand system in multiple sclerosis brain. J. Exp. Med. 184: 1513-1518 [Abstract]. |
9. | Liles, W.C., P.A. Kiener, J.A. Ledbetter, A. Aruffo, and S.J. Klebanoff. 1996. Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils. J. Exp. Med. 184: 429-440 [Abstract]. |
10. | O'Connell, J., G.C. O'Sullivan, J.K. Collins, and F. Shanahan. 1996. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J. Exp. Med. 184: 1075-1082 [Abstract]. |
11. |
Hahne, M.,
D. Rimoldi,
M. Schroter,
P. Romero,
M. Schreier,
L.E. French,
P. Schneider,
T. Bornand,
A. Fontana,
D. Lienard, et al
.
1996.
Melanoma cell expression of Fas
(Apo-1/CD95) ligand: implications for tumor immune escape.
Science (Wash. DC).
274:
1363-1366
|
12. | Nagata, S.. 1996. Apoptosis: telling cells their time is up. Curr. Biol. 6: 1241-1243 [Medline]. |
13. | Huang, B., M. Eberstadt, E.T. Olejniczak, R.P. Meadows, and S.W. Fesik. 1996. NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain. Nature (Lond.) 384: 638-641 [Medline]. |
14. | Bajorath, J., and A. Aruffo. 1997. Prediction of the threedimensional structure of the human Fas receptor by comparative molecular modeling. J. Computer-Aided Mol. Design 11: 3-8 [Medline]. |
15. |
Banner, D.W.,
A. D'Arcy,
W. Janes,
R. Gentz,
H.-J. Schoenfeld,
C. Broger,
H. Loetscher, and
W. Lesslauer.
1993.
Crystal structure of the soluble human 55 kd TNF receptor-human
TNF![]() |
16. | Bajorath, J., N.J. Chalupny, J.S. Marken, A.W. Siadak, J. Skonier, M. Gordon, D. Hollenbaugh, R.J. Noelle, H.D. Ochs, and A. Aruffo. 1995. Identification of residues on CD40 and its ligand which are critical for the receptor-ligand interaction. Biochemistry. 34: 1833-1840 [Medline]. |
17. | Bajorath, J., J.S. Marken, N.J. Chalupny, T.L. Spoon, A.W. Siadak, M. Gordon, R.J. Noelle, D. Hollenbaugh, and A. Aruffo. 1995. Analysis of gp39/CD40 interactions using molecular models and site-directed mutagenesis. Biochemistry. 34: 9884-9892 [Medline]. |
18. | Hollenbaugh, D., L.S. Grosmaire, C.D. Kullas, N.J. Chalupny, S. Braesch-Andersen, R.J. Noelle, I. Stamenkovic, J.A. Ledbetter, and A. Aruffo. 1992. The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO (Eur. Mol. Biol. Organ.) J. 11: 4313-4321 [Abstract]. |
19. | Malik, N., B.W. Greenfield, A.F. Wahl, and P.A. Kiener. 1996. Activation of human monocytes through CD40 induces matrix metalloproteinases. J. Immunol. 156: 3952-3960 [Abstract]. |
20. | Hollenbaugh, D., J. Douthwright, V. McDonald, and A. Aruffo. 1995. Cleavable CD40Ig fusion proteins and the binding to sgp39. J. Immunol. Methods 188: 1-7 [Medline]. |
21. |
Whitney, G.S.,
G.C. Starling,
M.A. Bowen,
B. Modrell,
A.W. Siadak, and
A. Aruffo.
1995.
The membrane-proximal
scavenger receptor cysteine rich domain of CD6 contains the
activated leukocyte cell adhesion molecule binding site.
J. Biol.
Chem.
270:
18187-18190
|
22. |
Marsters, S.A.,
A.D. Frutkin,
N.J. Simpson,
B.M. Fendly, and
A. Ashkenazi.
1992.
Identification of cysteine-rich domains of the type 1 tumor necrosis factor receptor involved in
ligand binding.
J. Biol. Chem.
267:
5747-5750
|
23. | Armitage, R.J., T.A. Sato, B.M. Macduff, K.N. Clifford, A.R. Alpert, C.A. Smith, and W.C. Fanslow. 1992. Identification of a source of biologically active CD40 ligand. Eur. J. Immunol. 22: 2071-2076 [Medline]. |
24. | Alderson, M.R., C.A. Smith, T.W. Tough, T. Davis-Smith, R.J. Armitage, B. Falk, E. Roux, E. Baker, G.R. Sutherland, W.S. Din, et al . 1994. Molecular and biological characterization of human 4-1BB and its ligand. Eur. J. Immunol. 24: 2219-2227 [Medline]. |