The molecular basis for apoptotic defects in patients with CD95 (Fas/Apo-1) mutations
Akshay K. Vaishnaw, … , Moses V. Chao, Keith B. Elkon
Akshay K. Vaishnaw, … , Moses V. Chao, Keith B. Elkon
Published February 1, 1999
Citation Information: J Clin Invest. 1999;103(3):355-363. https://doi.org/10.1172/JCI5121.
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The molecular basis for apoptotic defects in patients with CD95 (Fas/Apo-1) mutations

Abstract

Heterozygous mutations of the receptor CD95 (Fas/Apo-1) are associated with defective lymphocyte apoptosis and a clinical disease characterized by lymphadenopathy, splenomegaly, and systemic autoimmunity. From our cohort of 11 families, we studied eight patients to define the mechanisms responsible for defective CD95-mediated apoptosis. Mutations in and around the death domain of CD95 had a dominant–negative effect that was explained by interference with the recruitment of the signal adapter protein, FADD, to the death domain. The intracellular domain (ICD) mutations were associated with a highly penetrant Canale-Smith syndrome (CSS) phenotype and an autosomal dominant inheritance pattern. In contrast, mutations affecting the CD95 extracellular domain (ECD) resulted in failure of extracellular expression of the mutant protein or impaired binding to CD95 ligand. They did not have a dominant–negative effect. In each of the families with an ECD mutation, only a single individual was affected. These observations were consistent with differing mechanisms of action and modes of inheritance of ICD and ECD mutations, suggesting that individuals with an ECD mutation may require additional defect(s) for expression of CSS.

Authors

Akshay K. Vaishnaw, Jason R. Orlinick, Jia-Li Chu, Peter H. Krammer, Moses V. Chao, Keith B. Elkon

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Figure 7

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The ECD mutants, P4 and P6, do not exert a dominant–negative effect and ...
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.