Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID) - PubMed (original) (raw)

. 2007 Jun;117(6):1550-7.

doi: 10.1172/JCI31023. Epub 2007 May 10.

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Dominant-negative effect of the heterozygous C104R TACI mutation in common variable immunodeficiency (CVID)

Lilit Garibyan et al. J Clin Invest. 2007 Jun.

Abstract

B cells from patients with common variable immunodeficiency (CVID) who are heterozygous for transmembrane activator and CAML interactor (TACI) mutation C104R, which abolishes ligand binding, fail to produce Igs in response to TACI ligand. It is not known whether this is due to haploinsufficiency or dominant interference. Using in vitro transfection assays, here we demonstrate that C104R and the corresponding murine TACI mutant, C76R, which also does not bind ligand, dominantly interfere with TACI signaling. This effect was dependent on preassociation of the mutants with WT TACI in the absence of ligand. The mutants did not interfere with ligand binding by WT TACI, suggesting that they may act by disrupting ligand-induced receptor rearrangement and signaling. This work demonstrates that TACI preassembles as an oligomeric complex prior to ligand binding and provides a mechanistic insight into how the heterozygous C104R TACI mutation can potentially lead to CVID.

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Figures

Figure 1

Figure 1. Aligned aa sequences of human and murine TACI.

Cysteine residues that make up the TNFR motif have an asterisk above them; the CRD1 and CRD2 domains are underlined. The vertical lines indicate identical residues and the dots, similar residues. The open triangle denotes the human C104R mutation found in CVID and its corresponding mutation C76R in mTACI.

Figure 2

Figure 2. Self association of mTACI monomers in transfectants.

(A and B) 293T cells were cotransfected with the indicated plasmids. IP was performed 48 hours later, followed by SDS-PAGE and Western blotting (WB). Lysates were probed for expression of tagged TACI. Results are representative of 3 experiments. The anti-GFP mAb used, JL-8, recognizes the CFP variant of GFP.

Figure 3

Figure 3. snIP analysis of TACI receptor assembly in ER microsomes.

(A and B) Assembly of differentially tagged TACI monomers was examined in an in vitro translation system using ER microsomes. Membranes were solubilized with digitonin, and complexes were isolated by snIP using the protein C (PC) and SA-binding protein (SBP) affinity tags on the C terminus of the TACI chains. SBP-tagged complexes were captured by SA beads and PC tagged complexes were captured by anti-PC mAb. Asterisk (lane 6) represents mixing control in which TACI-SBP and TACI-PC monomers were translated separately and mixed prior to solubilization and IP.

Figure 4

Figure 4. TACI self-associates through CRD1 in living cells.

(A) Flow cytometric analysis of 293T cells cotransfected with CFP- or YFP-tagged mTACI and CFP- or YFP-tagged CD40 control. The dual-expressing cells were electronically gated, and more than 20,000 events were acquired into the FRET gate and used for subsequent analysis. (B) Overlay of FRET plots derived as in A for interactions between indicated TNFR molecules.

Figure 5

Figure 5. Preassociation of endogenous mTACI in B cells.

Mouse lymphoma A20 B cells were treated as indicated. Lysates were run on SDS-PAGE under nonreducing (NR) or reducing (R) conditions and probed with anti-TACI mAb. The ladder visible in the reduced samples represents nonspecific bands and is visible, albeit less strongly, under nonreducing conditions without DTSSP.

Figure 6

Figure 6. C76R mTACI associates with WT TACI.

(A) Surface expression and recombinant human BAFF (rhBAFF) binding of WT and mutant mTACI in transfected 293T cells. (B) snIP analysis of the association of WT and mutant mTACI in ER microsomes. Asterisk (lane 6) represents mixing control in which TACI-SBP, TACI-PC, and TACI-YFP monomers were translated separately and mixed prior to solubilization and IP. (C) FRET analysis of the association of WT and mutant mTACI in 293T cells.

Figure 7

Figure 7. C76R mTACI dominantly interferes with signaling, but not ligand binding, by WT mTACI.

(A) 293T cells were transfected with 0.1 μg WT mTACI alone, 0.1 μg C76R mTACI, or 0.1 μg of each along with 0.1 μg of NF-κB–luc reporter plasmid and 10 ng control pRL-TK plasmid. Equal amounts of DNA were used for each transfection by using empty vector DNA when needed. After 4 hours, recombinant mBAFF (0.1 μg/ml) was added, and reporter gene activity was determined 20 hours later. NF-κB luciferase induction was normalized to Renilla luciferase intensity and total protein of each sample. Fold induction was calculated by dividing the normalized NF-κB luciferase result of BAFF-treated samples by the NF-κB luciferase result of untreated samples. n = 7. (B) The same assay as described in A was performed, with C76R mTACI that lacked the CRD1 domain (DM) substituting for C76R mTACI. Values represent the fold increase by BAFF stimulation compared with unstimulated sample and are the mean ± SEM from 6 independent experiments. (C) TACI surface expression and rhBAFF binding by 293T cells transfected with 1 μg of WT mTACI, 1 μg of C76R mTACI alone, or 1 μg of each.

Figure 8

Figure 8. C104R hTACI associates with WT hTACI.

(A and B) 293T cells were cotransfected with the indicated hTACI plasmids. IP was performed 48 hours later followed by SDS-PAGE and Western blotting. Lysates were probed for expression of tagged TACI. Results are representative of 3 experiments.

Figure 9

Figure 9. C104R mutant does not interfere with surface expression of WT hTACI.

293T cells were cotransfected with the indicated hTACI plasmids, and surface expression of Flag-tagged WT hTACI and Myc-tagged C104R hTACI was analyzed 24 hours after transfection using anti-Myc–FITC and anti-Flag–PE antibodies. Mock, untransfected.

Figure 10

Figure 10. C104R hTACI dominantly interferes with signaling, but not ligand binding, by WT hTACI.

hTACI surface expression (A) and rhBAFF binding (B) by 293T cells transfected with 1 μg of WT hTACI, 1 μg of C104R hTACI alone, or 1 μg of each together. (C) 293T cells were transfected with 10 ng WT mTACI alone, 10 ng C104R mTACI alone, or 10 ng of each together along with 0.5 mg of NF-κB–luc reporter plasmid and 10 ng control pRL-null plasmid. Equal amounts of DNA were used for each transfection by using empty vector DNA when needed. After 4 hours, recombinant hBAFF (0.1 μg/ml) was added, and reporter gene activity was determined 20 hours later. NF-κB luciferase induction was normalized to Renilla luciferase intensity. Values represent the fold increase by BAFF stimulation compared with unstimulated sample and are the mean ± SEM from 6 independent experiments.

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