A Novel Protein Domain Required for Apoptosis MUTATIONAL ANALYSIS OF HUMAN Fas ANTIGEN*

The Fas antigen is a cell surface protein that can mediate apoptosis and that belongs to the tumor necrosis factor receptor family. Murine fibroblast L929 cells or T-cell lymphoma WR19L cells expressing the human Fas antigen were killed within 4-6 h by anti-human Fas antibody in a concentration-dependent manner. Human Fas antigen cDNAs with various mutations in the cytoplasmic region were constructed and expressed in L929 cells. A deletion of 15 amino acids from the C terminus of the Fas antigen enhanced the Fas antibody-induced killing activity, whereas a further deletion abolished its activity. This suggests the presence of an inhibitory as well as a signal-transducing domain in the cytoplasmic region of the Fas antigen. A 68-amino acid portion of the signal-transducing domain significantly conserved in the Fas antigen as well as in the type I tumor necrosis factor receptor was considered to be the novel protein domain required for apoptotic signal transduction. JT Inc.) and D. V. Goeddel (Genentech), respectively. The cell-killing assay was carried out in 96-well microtiter plates as described (5). In brief, 2.5 X lo4 transformed cells (100 pl) were cultured for 24 h and incubated at 37 “C for 16 h with various concentrations (3 ng/ml-3 pg/ml) of anti-Fas antibody in the presence (for L929 cell transformants) or in the absence (for WR19L cell transformants) of 0.5 pg/ml actinomycin D. Viable L929 cell transformants were stained with crystal violet, and the dye uptake was quantified by measuring the A64onrn using an automated micro-en-zyme-linked immunosorbent assay autoreader. Dead and viable WR19L cell transformants were distinguished by staining with trypan blue (5).


A Novel Protein Domain Required for Apoptosis
MUTATIONAL ANALYSIS OF HUMAN Fas ANTIGEN* (Received for publication, December 8,1992, and in revised form, February 15, 1993)

Naoto Itoh and Shigekazu Nagata
From the Osaka Bioscience Institute, 6-2-4, Furuedai, Suita-shi, Osaka 565, Japan The Fas antigen is a cell surface protein that can mediate apoptosis and that belongs to the tumor necrosis factor receptor family. Murine fibroblast L929 cells or T-cell lymphoma WR19L cells expressing the human Fas antigen were killed within 4-6 h by anti-human Fas antibody in a concentration-dependent manner. Human Fas antigen cDNAs with various mutations in the cytoplasmic region were constructed and expressed in L929 cells. A deletion of 1 5 amino acids from the C terminus of the Fas antigen enhanced the Fas antibodyinduced killing activity, whereas a further deletion abolished its activity. This suggests the presence of an inhibitory as well as a signal-transducing domain in the cytoplasmic region of the Fas antigen. A 68-amino acid portion of the signal-transducing domain significantly conserved in the Fas antigen as well as in the type I tumor necrosis factor receptor was considered to be the novel protein domain required for apoptotic signal transduction.
During embryonic and postembryonic development, many cells die by programmed cell death, which plays a major role in determining morphologic and functional maturity in a variety of systems, including neural network formation and clonal deletion of autoreactive T-cells (1)(2)(3). Programmed cell death generally occurs by a process called apoptosis. Apoptosis is accompanied by condensation of cytoplasm, loss of plasma membrane microvilli, and extensive degradation of chromosomal DNA into oligomers of about 180 base pairs (1)(2)(3).
The Fas antigen is expressed on the surface of various cell lines, including activated Tand B-cells. The mouse monoclonal anti-human Fas antibody is cytocidal to some cells expressing the Fas antigen (4). We isolated a cDNA encoding the human Fas antigen by expression cloning (5). The human Fas antigen consists of 325 amino acids with a single transmembrane domain. The cytoplasmic region (145 amino acids) does not contain domains for kinases, phosphatases, or any other apparent enzymes. However, the extracellular domain was similar to that of the tumor necrosis factor (TNF)' receptor family, which includes the low affinity nerve growth factor receptor (6), T N F receptors (7)(8)(9), and lymphocyte antigens such as CD27 (lo), CD30 (ll), CD40 (12), and OX40 (13). Trauth et al. (14) also have identified a similar monoclonal antibody called Apo-1. Recent molecular cloning of the *This work was supported, in part, by grants-in-aid from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are: TNF, tumor necrosis factor; PCR, polymerase chain reaction.
Apo-1 antigen cDNA indicated that the Apo-1 antigen is identical to the Fas antigen (15). After expression of human Fas antigen cDNA in mouse fibroblast L929 and T-cell lymphoma WR19L cells, the anti-human Fas antibody kills these cells by apoptosis, which indicates that the Fas antigen mediates an apoptotic signal into the cells (5).
Recently, we have identified a mouse Fas antigen gene (16) as the structural gene for mouse lymphoproliferation (Ipr) mutation (17). The lpr mutation is a recessive and autosomal mutation, and mice homozygous at this locus produce multiple autoantibodies, accumulate nonmalignant CD4-CD8-T-lymphocytes, and develop an autoimmune disease resembling systemic lupus erythematosus (18). These findings indicate that the Fas antigen plays an important role in the programmed cell death of T-lymphocytes in the thymus. Here, we identified a domain in the cytoplasmic region of the Fas antigen, which is required to transduce the death signal. A significantly conserved sequence of this domain was found in the cytoplasmic region of the TNF type I receptor, which also transduces the apoptotic signal into cells (19).

MATERIALS AND METHODS
Plasmid Construction-The deletion mutants, FD2, FD4, and FD5, were constructed by digesting the 2.6-kilobase XhoI DNA fragment of the plasmid pF58 (5) carrying the full-length human Fas antigen cDNA with TaqI (at 942), BglII (at 1083), or SpeI (at 1150). After treatment with the Klenow fragment of Escherichia coli DNA polymerase, an XbaI linker (CTAGTCTAGACTAG, New England Biolabs) was added to the DNA ends, which were then digested with XbaI. The XbaI fragments containing the Fas antigen sequence were then transferred to the pEF-BOS expression vector (20), and the plasmids carrying the Fas antigen sequence in the correct orientation were designated as pEF-FD2, pEF-FD4, and pEF-FD5, respectively. The other two deletion mutants, FD7 and FD8, were constructed by PCR (21). In brief, oligonucleotides HFD2 (GGAATTCTAGACTAGGT-ACAAAGATTGGCTTTTTTGAGAT. nucleotides 1084-1108 of the human Fas antigen cDNA; the extra sequence not present in human Fas cDNA is underlined) and HFD3 (GGAATTCTAGACTAGGTC-TGAATTTTCTCTGCAAGAGTAC, nucleotides 1104-1128) carrying an in-frame termination codon, as well as XbaI and EcoRI sites, were synthesized and used as reverse primers for construction of FD7 and FD8 deletion mutants, respectively. The oligonucleotide HF1 (GAAGGATCCAGATCTAACTT) carrying the nucleotide sequence from 696 to 715 of human Fas antigen cDNA was the forward primer for both constructions. PCR proceeded in 10 mM Tris-HC1 (pH 8.3), 50 mM KCl, 2.5 mM MgC12, 0.1 mg/ml gelatin, 20 PM of each dNTP, 200 nM of each primer, 2 ng of the 1.8-kilobase XhoI-EcoRI fragment of pF58 (5), and 2.5 units of Taq polymerase in a total volume of 50 pl. The conditions for PCR were 1 min at 94 "C, 2 min at 55 "C, and 3 min at 72 "C for 30 cycles. The products were digested with BglII and EcoRI, and then the BglII-EcoRI DNA fragment carrying the truncated Fas antigen cDNA replaced the C-terminal BglII-XbaI fragment of pEFF58 (5).
of the Fas antigen was also constructed by PCR. Primers HF9 The mutant carrying a point mutation in the cytoplasmic region (TGTAACTCTACTGTATGT, nucleotides from 597 to 614 of the human Fas antigen cDNA), HFlO (GCAGTATTTACAGCCAGC, ~~~ ~ 940-949), and HFP2 complementary to HFPl were synthesized. The HFpl and HFP2 primers contain two mutated nucleotides that convert Val-238 (GTC) to Asn-238 (AAC) of the human Fas antigen. During primary PCR, a portion of the human Fas antigen cDNA was amplified either with HF9 and HFP2 or with HFPl and HFlO primers. After treatment with T4 polymerase, the amplified products were isolated by agarose gel electrophoresis, mixed at a ratio of 1:1, and secondary PCR was performed using HF9 and HFlO primers. The product was digested with BamHI and XbaI, and the 0.4-kilobase DNA fragment replaced the 0.4-kilobase C-terminal BarnHI-XbaI fragment of pEFF58. The resulting plasmid was designated as pEF-FP1.
All mutant constructions were verified by DNA sequencing using dideoxy chain termination.
Cell Lines and Transfection-Mouse fibroblast L929 and mouse Tcell lymphoma WR19L were maintained in Dulbecco's modified Eagle's medium containing 5% fetal calf serum (HyClone) or in RPMI 1640 medium supplemented with 10% fetal calf serum, respectively. L929 cells were transfected with the expression plasmids by calcium phosphate coprecipitation, whereas the WR19L cells were transfected by electroporation as described previously (5). After selection with G-418 (0.4 mg/ml for L929 cells, 0.9 mg/ml for WR19L cells), transformants expressing the mutated Fas antigen were identified by fluorescence-activated cell sorting analysis using mouse anti-Fas monoclonal antibody and fluorescein isothiocyanate-conjugated antimouse Ig antibody as described previously (5).
The cell-killing assay was carried out in 96-well microtiter plates as described (5). In brief, 2.5 X lo4 transformed cells (100 pl) were cultured for 24 h and incubated at 37 "C for 16 h with various concentrations (3 ng/ml-3 pg/ml) of anti-Fas antibody in the presence (for L929 cell transformants) or in the absence (for WR19L cell transformants) of 0.5 pg/ml actinomycin D. Viable L929 cell transformants were stained with crystal violet, and the dye uptake was quantified by measuring the A64onrn using an automated micro-enzyme-linked immunosorbent assay autoreader. Dead and viable WR19L cell transformants were distinguished by staining with trypan blue (5).

Rapid Cell Death Induced by Anti-Fas Antibody or TNF-
We previously showed that mouse L929 cell transformants expressing the human Fas antigen are killed within 16 h by anti-human Fas antibody in a dose-dependent manner in the presence of 0.5 pg/ml actinomycin D (5). In  (Fig. 2). These mutated cDNAs were placed under the promoter of the human elongation factor l a (EFla) and introduced into mouse L929 cells together with the neomycin resistance gene.
Fluorescence-activated cell sorting analysis of mutated Fas antigen expression in stable transformants using anti-Fas monoclonal antibody indicated that more than 80% G-418resistant transformant clones expressed the human Fas antigen on the cell surface. The expression levels of the Fas antigen in the positive clones were similar among different mutant proteins. Fig. 3 shows representative results. Western blotting analysis of the cell lysates with anti-human Fas antibody also indicated that all of the transformed clones expressed similar amounts of the mutated Fas antigen (data not shown).
Signal-transducing Domain in the Fas Antigen-The ability of the mutated Fas antigen to transduce the apoptotic signal was then examined with the transformants established above.

FD5
To eliminate clonal variations, four to six independent transformant clones were chosen for each mutant. Fig. 4 shows the anti-Fas antibody-dependent cell death of the transformants expressing the wild-type Fas antigen and the mutants. Clones expressing the wild-type Fas antigen and FD5 clones expressing the mutant lacking the C-terminal 15 amino acids were killed by the anti-Fas antibody (Fig. 4, A and B ) . FD5 clones were more sensitive to the anti-Fas antibody than the clones of wild type. On the other hand, clones expressing mutants carrying a further deletion (FD8, FD7, FD4, and FD2) were unaffected by the anti-Fas antibody. Fig. 4C shows typical results using FD8 clones. These findings indicate that the 130-amino acid portion from 175 to 304 in the cytoplasmic region of the human Fas antigen is essential for the Fas antibody-triggered apoptotic signal transduction in L929 cells. Similar results were obtained with WR19L cell transformants expressing the mutated Fas antigen (data not shown).
Mice with the allelic lpfg defect carry a point mutation in the cytoplasmic region of the mouse Fas antigen, which converts Ile-225 to Asn-225. To confirm that this mutation abolishes the apoptotic signal transduction of the Fas antigen, the corresponding valine residue of the human Fas antigen at amino acid 238 was mutated to asparagine, and the mutant human Fas antigen was expressed in mouse L929 cells. As shown in Fig. 40, the transformants expressing the Fas antigen with this point mutation were unaffected by anti-Fas antibody.
The C-terminal Domain of the Fas Antigen Is an Inhibitory Domain-As described above, the deletion of the C-terminal 15 amino acids from human Fas antigen up-regulated the ability of the Fas antigen to transduce the apoptotic signal. As shown in Fig. 4, A and B, the half-maximal response of anti-Fas antibody with transformant clones expressing the deletion mutant occurred at a concentration of 5-30 ng/ml, whereas 80-300 ng/ml anti-Fas antibody was required for the half-maximal response with transformant clones expressing  incubated with various concentrations (0-3 pglml) of anti-Fas antibody in the presence of 0.5 pg/ml actinomycin D at 37 "C for 16 h and stained with crystal violet. The percentage of viable cells was calculated as described in Fig. 1.

DISCUSSION
We showed that the Fas antigenlantibody system induces rapid cell death. All WR19L cell transformants expressing the Fas antigen were killed within 4 h, which is much faster than the death caused by the depletion of growth factors from dependent cell lines (usually more than 10 h) (23)(24)(25). This suggests that the Fas antigen actively transduces the death signal, while the cell death caused by factor depletion is an indirect process. Previously, we found that the Fas antigen gene is the structural gene for lpr and suggested an important role of the Fas antigen in the development of T-cells in the thymus (17). During the thymic development of T-cells, more than 90% of the T-cells die by apoptosis at the CD4+ CD8+ stage, which is the major fraction of thymocytes (26). Nevertheless, only a small percentage of the cells were positive for apoptotic bodies, indicating that only a small percentage of the thymocytes is at the death stage. This might be due to the rapid dying process. That is, the CD4+ CD8+ T-cell precursors stay in the thymus for 3.5 days before they become single positive cells (27). If the dying process requires only 4 h, as does Fas antibody-triggered cell death, a rough estima-tion suggests that only 4.7% (4184) cells are at the dying stage.
Previously, we observed that a domain in the cytoplasmic region of the Fas antigen is homologous to the part of the cytoplasmic region of the TNF type I receptor (5). Between two TNF receptors, type I (55 kDa) and type I1 (75 kDa), the former but not the latter is responsible for the cytotoxic activity of TNF (19). Deletion analysis of the human Fas antigen from the C terminus indicated that the region homologous to the TNF type I receptor is essential for the Fas antigen to transduce the death signal into cells. As shown in   (A) or the FD5 mutant ( B ) were incubated with various concentrations (0-3 rg/ml) of anti-Fas antibody in the absence of actinomycin D at 37 "C for 16 h and stained with crystal violet. The percentage of viable cells was calculated as described in Fig. 1. Three independent clones for the wild-type transformants (0, 0, U) or four independent clones for the mutant transformants (0,Q U, A) were analyzed.  (16), and human Fas antigen (amino acids 217-319) (5) are shown. The amino acid numbers starting from the putative N-terminal amino acid in mature proteins are indicated on both sides. Amino acids are bored when the residues are conserved in more than three members. The C-terminal ends of deletion mutants (FD2, FD4, FD7, FD8, and FD5) are indicated by arrows. The arrowhead indicates the position of the mutation in the Fas antigen of Ipfg mice.
platelet-derived growth factor and epidermal growth factor receptors, contain a tyrosine residue near the C terminus of the molecule (28). The tyrosine phosphorylation of this residue inhibits the tyrosine kinase activity of the receptor, whereas G-protein-coupled receptors carrying seven transmembrane regions such as adrenergic receptors contain a phosphorylation site for CAMP-dependent protein kinase in the last cytoplasmic domain (29). Phosphorylation of this residue desensitizes the receptor function. The C-terminal region of the Fas antigen may regulate the Fas antigenmediated apoptotic signal transduction by a similar mechanism. In this respect, it is noteworthy that human and mouse Fas antigens have a sequence (XSXXEX in human Fas, XSXXDX for mouse Fas antigen) similar to the consensus phosphorylation site motif for casein kinase I1 (30) near the C terminus of the molecule. More detailed analyses of the Fas antigen-mediated apoptotic signal transduction may clarify the mechanism for apoptosis.