The molecular basis for apoptotic defects in patients with CD95 (Fas/Apo-1) mutations (original) (raw)
Clinical features of CSS patients. Patients 1–3 have been described elsewhere (16). CSS was diagnosed in P4–P11 on the basis of a chronic disease of early childhood characterized by lymphoaccumulation and systemic autoimmunity (15). The clinical features of the eight probands and other affected family members are summarized in Table 1. All developed disease within the first four years of life. Family histories of CSS index cases revealed that P8, P9, and P10 had additional affected members, suggesting an autosomal dominant pattern of inheritance (see below). A detailed clinical description of these patients is presented elsewhere (Vaishnaw, A.K., et al, manuscript in preparation).
Clinical features of CSS index cases
CD95/CD95L mutational studies. All patients had heterozygous CD95 mutations as summarized in Table 2 and shown schematically in Fig. 1. Most patients had mutations of the CD95 ICD, which were either frameshift-associated with premature termination (P5 S214X, P10 S209X) or missense mutations (P7 D244G, P8 R234P, P9 T254I). For both P5 and P10, the frameshifts were associated with consensus splice-donor or acceptor mutations. PCR amplification and sequencing of exon 9 and flanking sequences revealed that P5 had an intron 8 mutation that created a premature splice acceptor site (ctatttttagATGTT →ctagttttagATGTT). Similar exon 7 studies confirmed that P10 had an intron 7 splice-donor mutation (TCCTgtaggt→TCCTgaaggt). P4, P6, and P11 each had a mutation within the CD95 extracellular domain. P4 had an exon 3 point mutation, resulting in a missense codon (C66R). P6 had an exon 2 insertion that caused a frameshift and premature termination at codon 47 (C47X); P11 had a dinucleotide exon 2 deletion followed by an in-frame stop codon, corresponding to position 1 of the mature CD95 protein (R1X). Patients’ cDNA was also analyzed for CD95L mutations, but none were identified.
Schematic of the predicted protein structure encoded by the CD95 mutant alleles from CSS patients, P1–P11. Extracellular cysteine-rich domains (CRD 1–3) and the intracellular death domain are indicated for wild-type CD95. Truncations mutations are indicated by loss of the corresponding domain(s) and missense mutations by a hatched domain. Mutant alleles used for in vitro analyses are boxed. CSS, Canale-Smith syndrome; ICD, intracellular domain; ECD, extracellular domain.
Heterozygous CD95 mutations in patients with the Canale-Smith syndrome
ICD CD95 mutant proteins are expressed and inhibit apoptosis mediated by wild-type CD95. To examine CD95 expression in CSS patients, freshly isolated PBMC were analyzed by flow cytometry using the anti-CD95–specific MAB, UB2. All individuals with an ICD mutation (P1, P2, P3, P5, P7, P8, P9, and P10) had normal CD95 expression, but expression was reduced in patients with the ECD mutations (P4, P6, P11) (Fig. 2). These findings suggested that the ICD, but not the ECD mutant proteins were coexpressed with wild-type CD95 on the cell surface. To verify this finding, a representative spectrum of ICD (P1, P2, P5, P7, P8, P10) and ECD (P4, P6) mutants were subcloned into the expression vector pCDNA3 and transfected into 293T cells. Consistent with findings from patients’ PBMC, the wild-type (WT) and ICD, but not the ECD, mutant alleles were readily detected by flow cytometry with the UB2 MAB (Fig. 3a).
In vivo expression of ECD mutant alleles. PBMC were isolated from patients P4, P6, P10, and P11, and CD95 expression was quantified by flow cytometry using the anti-CD95 MAB, UB2. PBMC staining with an isotype control antibody is indicated by shaded histograms. The percent of CD95 cells for each individual is shown. The corresponding mean channel fluorescence results were as follows: P4 (18.2), P6 (17.1), P10 (34.2), and P11 (16.8). PBMC, peripheral blood mononuclear cells; MAB, monoclonal antibody.
Surface expression and dominant–negative function of ICD mutant CD95 alleles. (a) Expression. Human embryonic kidney 293T cells were transfected with pCDNA3 expression vectors encoding WT or mutant CD95. CD95 expression was analyzed by flow cytometry using the anti-CD95 MAB, UB2. The percent of CD95-positive cells for each transfection is shown. (b) Function. CD95-positive Jurkat T cells were cotransfected with the same panel of expression vectors as in a and with the reporter gene, RSV-Luc. The cells were separated into two aliquots and cultured with either the agonistic anti–CD95 antibody CH11 (50 ng/ml) or an isotype control antibody 24 h after transfection. Luciferase expression was assayed at 48 h and percent viability was calculated by the following formula: (luciferase activity of CH11/luciferase activity of control antibody) × 100. The data shown are the mean results of two independent experiments. WT, wild type; RSV, Rous sarcoma virus.
To assess the functional impact of a heterozygous CD95 mutation, we next examined CD95 function following transfection of the mutant alleles in Jurkat T cells, which express CD95 and undergo apoptosis in response to an agonistic anti-CD95 stimulus (30). Jurkat cells were cotransfected with either WT CD95, mutant CD95, or pCDNA3 vector, together with the constitutively active reporter construct, RSV-Luc. Twenty-four hours after transfection, aliquots of transfected cells were incubated with the agonistic anti-CD95 antibody CH-11 or isotype control for an additional 24 hours, and then assayed for luciferase activity. The mean values of two such Jurkat protection assays are shown in Fig. 3b. All ICD mutants clearly inhibited CD95-mediated apoptosis, as evidenced by the relatively high cell viability in the presence of CD95 agonist. These results suggested that the ICD mutants act as dominant–negative molecules as also shown for the ICD mutants reported by Fisher et al. (19).
ICD CD95 mutants abrogate FADD recruitment to the death domain. In the presence of CD95L, CD95 aggregation results in the generation of a death signal via the cytoplasmic recruitment of FADD to the death domain of CD95 (3, 4). The binding of FADD to WT or ICD mutant CD95 receptors was therefore assessed. 293T cells were cotransfected with Flag-tagged FADD and either WT CD95 or one of the ICD mutants. Cell lysates were prepared and equal amounts of protein analyzed by immunoprecipitation (IP), using the agonistic anti-CD95 MAB anti–Apo-1, followed by Western blot analysis with anti-Flag. As shown in Fig. 4a, despite comparable expression of WT or mutant CD95 and Flag-FADD (upper and middle panels), IP with anti-CD95 coprecipitated FADD in cells transfected with WT CD95 but not in cells transfected with an ICD mutant (lower panel).
Defective protein–protein interactions of ICD CD95 mutant alleles. (a) ICD CD95 mutant alleles fail to recruit FADD to the CD95 death domain. 293T cells were cotransfected with pCDNA3 expression vectors for WT or mutant CD95 and Flag-FADD in pFlag-CMV-2. Transfected cells were lysed and subjected to IP-Western blot using the antibodies indicated in the figure. The trace signals of WT CD95 in lanes P1, P5, and P10 are due to slight spillover from adjacent lanes and were not seen when these mutants were run separately. (b) The dominant–negative action of ICD mutant CD95 is dose-dependent. 293T cells were transiently cotransfected with expression vectors for Flag-FADD and WT or P10 mutant CD95. Transfections were carried out using constant amounts of total DNA (4 μg) using the pCDNA vector DNA, WT CD95 (0.5 μg) and Flag-FADD (0.5 μg) DNA, but the amount of DNA for the ICD mutant P10, was increased between experiments (lanes 4_–_6; + [0.5 μg] to +++ [1.5 μg]). The total amount of DNA transfected was kept constant. Transfected cells were lysed and analyzed by immunoprecipitation followed by Western blotting using the antibodies indicated. IP, immunoprecipitation.
To approximate the in vivo situation more closely, we next examined FADD recruitment after coexpression of an ICD mutant with WT CD95 and Flag-FADD. 293T cells were transfected with constant amounts of the WT CD95 and Flag-FADD expression constructs, along with increasing amounts of the P10 mutant. Lysates were immunoprecipitated with anti–Apo-1 and analyzed by Western blot. As shown in Fig. 4b, the amount of FADD recruited to the complex diminished with increasing P10 expression. At approximately equivalent WT and mutant CD95 expression levels, FADD binding was undetectable, providing a molecular basis for the dominant–negative action of ICD mutant alleles.
Structural distortions of the death domain secondary to ICD mutations. Nuclear magnetic resonance (NMR) spectroscopy has shown that the CD95 death domain is composed of six α-helices (α1 to α6), which are crucial for homotypic death-domain interactions (31). To understand the impact of the ICD mutations (P2, P7, P8) on the helical structure of the death domain, we undertook a secondary structure analysis using nnpredict and nnssp, as described in Methods. Both nnpredict and nnssp modeled the peptide sequence for WT CD95 (residues 223–273) into three discrete α-helical domains, corresponding closely to α2, α3, and α4 from the studies of Huang et al. (31). When ICD mutants were analyzed, nnpredict and nnsp predicted that P7 D244G caused a break in the α3 helix and that P8 R234P had a similar effect on the α2 helix (Fig. 5). These data provide a structural basis for the failure of the these ICD mutants to bind FADD (Fig. 4). Unlike P7 D244G, the P2 missense mutation D244Y was not predicted to cause an alteration of the α3 helix. Because D244 is a charged surface residue on the surface of the death domain (31), P2 D244Y may have a direct effect on FADD binding.
Death domain missense mutations (P7 and P8) distort its α-helical structure. The peptide sequence for wild-type CD95 (residues 223–273) and the same regions from P2, P7, and P8 mutant alleles were analyzed by secondary structure predictive models (see Methods). The regions of each sequence that were predicted to adopt an α-helical conformation (H) are indicated above the corresponding region of the amino acid sequence. Dashes (–) represent areas predicted not to form an α-helix. The stippled boxes highlight the regions predicted to be altered by the mutations.
Expression of ECD CD95 mutant proteins. In contrast to CD95 expression on PBMC from patients with ICD mutations, CD95 expression was significantly lower on T cells of P4, P6, and P11 when analyzed by the MAB UB2 (Fig. 2) and was undetectable on 293T cells transfected with the corresponding CD95 expression vectors (Fig. 3a). Reduced expression of CD95 in P6 and P11 was expected, because these mutations are predicted to lead to severely truncated peptides (Fig. 1), whereas P4 contains only a single point mutation at residue 66 (Table 2).
To define the mechanisms responsible for alteration in CD95 expression and function in these patients, the mutant cDNAs were transfected into 293T and Jurkat T cells. Northern blot analysis revealed that equivalent levels of CD95 mRNA were expressed in all transfectants (data not shown). None of the anti-CD95 antibodies-UB2, CH11, anti–Apo-1, or DX2- stained 293T cells transfected with P4 or P6 by flow cytometry. However, the MAB CLB-CD95/2 stained P4 transfected cells, albeit less intensely than wild type (Fig. 6a). Because CLB-95/2 binds to CRD2 of CD95 (Van Lopik, T., Vaishnaw, A. K., and Aarden, L., unpublished observations), we conclude that the P4 CRD1 mutation (C66R) substantially distorts the structure of both CRD1 and CRD2.
CD95 expression for ECD mutation patients P4 and P6 293T cells were transfected with expression vectors for P4 or WT CD95. Transfected cells were analyzed by flow cytometry using UB2 or CLB-CD95/2. For each transfectant, the percent of CD95-positive cells is indicated, with shaded histograms representing staining of pCDNA3 (vector control) transfected cells. The results shown are representative of three experiments.
The P6 mutation predicted the synthesis of a 46–amino acid peptide, truncated in the CRD1 of CD95. To determine whether this peptide was secreted, 293T cells were transfected with expression vectors for P6 or TM (WT CD95 lacking the transmembrane domain), or with pCDNA3 alone. Cell supernatants were analyzed by dot–blot using the NH2-terminus–specific antibody N18. While TM was readily detected in this assay, P6 was not (data not shown). Because immunofluorescence revealed intense intracellular staining for WT CD95 but no staining for P6 (not shown), these results suggested that the P6 ECD mutant was degraded intracellularly. A similar result was expected for P11, which is truncated immediately after the signal peptide.
Apoptosis in ECD mutants. The findings so far indicated that partial loss of CD95 function in P6 and 11 could be explained by lack of expression of the mutant proteins and that defective CD95 function of the P4 mutant resulted from failure to bind CD95 ligand (1). However, the apoptotic defect in the patients’ own cells was greater than 50% (16). To determine whether the ECD mutants exerted a protective effect against authentic CD95 ligand, Jurkat cells were transfected with the two ECD mutants and used as targets for 293T cells expressing CD95L (29) in a coculture experiment. As shown in Fig. 7a, P10 transfected Jurkat T cells were protected from CD95L-mediated apoptosis, but those transfected with P4, P6, or WT CD95 were as susceptible to apoptosis as pCDNA3-transfected Jurkat cells. It has been reported previously that short ECD-truncation mutants could be secreted and inhibit anti-CD95–mediated apoptosis (32). We therefore tested the ability of supernatants from activated T cells obtained from P6 to inhibit apoptosis. As shown in Fig. 7b, the culture supernates failed to inhibit anti-CD95–mediated apoptosis, even when added undiluted. A similar lack of inhibition was observed when supernates from P6 pCDNA3 transfected 293T cells were used (not shown). Finally, because P4 and P6 CD95-Fc fusion proteins also failed to bind and inhibit CD95 ligands (Fig. 7c), the ECD mutants appear to exert their effect solely by loss of function.
The ECD mutants, P4 and P6, do not exert a dominant–negative effect and do not inhibit CD95L function. (a) Direct transfection assay. Jurkat T cells were cotransfected with RSV-Luc and P4, P6, P10, WT CD95, or pCDNA3. Half of the transfected cells were applied to monolayers of 293T cells expressing WT CD95L and half to gld CD95L, where gld represents the inactive CD95L mutant 24 h after transfection (29). At 48 h, the Jurkat cells were harvested, and the ratio of luciferase activity [(WT CD95L/gld CD95L) × 100] was calculated. (b) Supernate transfer assay. T cells from a normal control, P6, or P10 were activated with anti-CD3 and IL-2 for 7 days. The supernates from these cultures were added in the proportions shown on the x-axis to 50,000 Jurkat cells. After the addition of 50 ng/ml of anti-CD95, viability was assessed in triplicate by the Alamar blue assay at 24 h, and the results were expressed by the fluorescence emission at 590 nm. (c) CD95 ligand binding assay. Jurkat T cells were incubated with the CD95-Fc fusion proteins: WT, P4, and P6 (0.1–10 mg/ml) in the presence of recombinant soluble CD95L. The percent specific rescue from apoptosis associated with each protein is shown. The experiments shown in a–c were performed on at least two separate occasions with virtually identical results. Greater than 100% viability is most likely explained by the costimulatory effect of CD95 signaling in the absence of apoptosis (44). IL, interleukin.
ICD mutations are highly penetrant in CSS families. Because ICD mutants exhibited a dominant–negative action, we hypothesized that they may have higher penetrance than ECD mutations. Clinical histories and CD95 genotypes were therefore obtained from the first-degree relatives of patients P1–P11 (except for P1 and P7, which arose spontaneously). Families of ICD-mutant patients P2, P8, P9, and P10 each had additional member(s) with the CSS phenotype (Fig. 8). When the proportion of nonindex cases with an ICD CD95 mutation were determined, 60% (6/10) were affected. In contrast, the index cases P4, P6, and P11 were the only affected members in ECD-mutation families, and none of the first-degree relatives with a CD95 mutation expressed the CSS phenotype (0/5).
Penetrance analysis of CSS families P1–P11. The nuclear families for index cases P1–P11 (arrows) are shown. CSS affected are noted by shaded symbols. CD95 genotypes were determined by SSCP and are represented as +/– (heterozygous mutation) or +/+ (WT CD95). Parents of P1 are not shown, but have been genotyped previously, and both are +/+ (16). Overall, 77% (14/18) individuals with an ICD mutation and 37% (3/8) of ECD mutation carriers had CSS. Considering only nonindex cases, to correct for ascertainment bias, penetrance was 6/10 (ICD) and 0/5 (ECD). SSCP, single-stranded conformational polymorphism.