Cytosolic Nuclease TREX1 Regulates Oligosaccharyltransferase Activity Independent of Nuclease Activity to Suppress Immune Activation

Abstract

TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frameshift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found that the TREX1 C terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation and autoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1(-/-) mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune defects associated with Trex1 deficiency or fs mutation. This function of the TREX1 C terminus suggests a potential therapeutic option for TREX1-fs mutant-associated diseases.

Figure 1. TREX1 C-terminus plays a critical role in suppressing immune activation

(A) Quantitative RT-PCR analysis of Cxcl10 mRNA (an ISG) in lymphoblasts from TREX1 patients and healthy controls. Three healthy controls (pooled in ‘HC’) and six RVCL patients (V235fs) are shown. (B) Immunoblot analysis of full length and C terminal truncated TREX1 in control and patient cells. HMGB1 is used as a loading control. (C) Diagrams of human TREX1 rescue constructs used in D–H and how the rescue lines are generated. (D) Fluorescent microscopy of the TREX1 constructs. Trex1^−/− MEFs reconstituted with the indicated rescuing construct were fixed and stained with an anti-FLAG antibody (green) or unstained (GFP-TM) and an ER marker anti-Calnexin (red). (E) Quantitative RT-PCR analysis of Ifit1 mRNA (an ISG) in the indicated Trex1^−/− rescue MEFs. Gene expression value was normalized to the housekeeping gene, Gapdh. (F, G) Quantitative RT-PCR analysis of indicated gene groups in Trex1^−/− rescue MEFs. Panel F shows KO-GFP cells comparing to WT cells to demonstrate the ISG signature caused by Trex1^−/− as shown previously(Hasan et al., 2013). The ‘IFN’ group contains Ifnb1, Ifna4, Ifng; the ‘ISG’ group contains Cxcl10, Ifit1, Oasl2, Isg15, Irf7; the ‘Inflammatory gene’ group contains Il6, Il17a, Tnf, Ccl3, Il23a; and the ‘Control gene’ group contains Hprt, Gapdh, Actb. Each data point represents one gene. Gene expression values were normalized to the housekeeping gene, Gapdh. Fold increase was then determined by normalizing to WT to aid group signature analysis. Panel G shows Trex1^−/− rescue cells. Only the ISG group genes are shown. No difference was found in other gene groups. To aid comparison, the same KO-GFP ISG data set is presented in F and G. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant (same throughout). Data are representative of at least three independent experiments. Error bars, SEM (same throughout). Unpaired t-test (A, E). Mann-Whitney test (F, G). See also Figure S1.

Figure 2. TREX1 C-terminus regulates hydrolysis of lipid-linked oligosaccharides

(A) FACE analysis of vesicular free glycans from SLO-permeabilized WT and Trex1^−/− MEFs. Similar results were seen with whole cells. L, glucose oligomer glycan ladder (same throughout). Quantification shown in the bar graph on the right. WT normalized to 1. (B) Quantification of total free glycans from WT and Trex1^−/− mouse BMDMs, kidneys and serum. FACE gels are shown in Figure S2. Each data point is the average value from one individual mouse. (C) FACE analysis of total free glycans from WT and Trex1^−/− MEFs reconstituted with the indicated rescuing construct. Data are representative of at least three independent experiments. Unpaired t-test (A, B). See also Figure S2.

Figure 3. Immunological and biochemical consequences of OST dysregulation

(A) Quantitative RT-PCR array analysis of immune gene activation in wild type BMDMs treated with indicated glycans. Free glycans from WT and Trex1^−/− MEFs were untreated or treated with a cocktail of DNase, RNase and protease before being added to BMDMs for 20 h. Each data point represents one gene as in Figure 1F. (B–C) Quantitative RT-PCR analysis of immune gene activation in wild type BMDMs treated with indicated glycans. Glycan source in B is free glycans isolated from Trex1^−/− MEFs (pooled glycans) or from FACE gels (gel-purified glycans). Glycan source in C is N-glycan isolated from increasing amount of RNase B protein (Sigma Cat#R1153, PNGase digest) or from buffer alone. Ifit1 and Cxcl10 mRNA were analyzed by qRT-PCR. Right, FACE analysis of glycans used in B and C. (D) ‘In-cell’ N-glycosylation assay. WT and Trex1^−/− MEFs were permeabilized by SLO and incubated with reaction buffer (see Method) and control or acceptor peptide. Only the acceptor peptide contains one N-glycysolation site. Transferred N-glycans were then cleaved by PNGase and analyzed by FACE (top gel). Free glycans were also analyzed from the same experiment (bottom gel). Free glycans quantified in this assay are from LLO hydrolysis during the assay, not the pre-existing free glycans in the cell (much smaller in size). Data are representative of at least two independent experiments. Error bars, SEM (same throughout). Mann-Whitney test (A). Unpaired t-test (B). See also Figure S3.

Figure 4. TREX1 interacts with the OST complex through the C-terminus

(A, B) Immunoblot analyses of TREX1 interaction with subunits of the OST complex. 293T cells were co-transfected with TREX1-V5 and one subunit of the OST complex as indicated (all Myc-tagged). Immunoprecipitation (IP) was performed with anti-V5 antibody, and precipitated immune complexes were analyzed by immunoblot with anti-V5 antibody (to detect primary IP of TREX1) and anti-Myc (to detect co-IP of individual OST subunits). The absolute requirement for TREX1 is emphasized in B. (C) Immunoblot analysis of TREX1 interactions with RPN1 and DDOST. 293T cells were transfected with plasmids indicated on top. Immunoprecipitation (IP) was performed with anti-Myc antibody. (D) The TREX1 C-terminus is required for interaction with RPN1. 293T cells were co-transfected with Myc-RPN1 and the indicated TREX1 (Flag-tagged) plasmids on top. Co-IP was performed with anti-Flag antibody. (E) Immunofluorescent microscopy analyses of TREX1 and RPN1 colocalization. GFP-TREX1, GFP-TREX1_TM or GFP-TM were co-transfected with Myc-RPN1, and immunostained with anti-Myc (red) 24 h later. (F) Rpn1 and Stt3a are required for immune activation in Trex1^−/− cells. Trex1^−/− MEFs were transduced with lentiviruses expressing specific shRNAs as indicated. Expression of Ifit1 mRNA was measured by RT-qPCR. Specific knockdown of individual genes was verified by RT-qPCR in Figure S4. Data are representative of at least three independent experiments. Unpaired t-test (F). See also Figure S4.

Figure 5. Inhibiting OST activity by aclacinomycin (Acm) suppresses free glycan release, immune activation and disease in Trex1^−/− mice

(A) Acm inhibits both OST activities in Trex1^−/− cells. Representative images of FACE gels are shown in Figure S5. (B, C) FACE analysis of free glycans (B) and quantitative RT-PCR analysis of immune gene activation (C) or in WT and Trex1^−/− MEFs treated with Acm. WT and Trex1^−/− MEFs were treated with increasing concentration of Acm or DMSO for 24 h. Ifit1 mRNA were measured by qPCR. Cells treated with 2 micromolar Acm were used for free glycan analysis in C. (D–F) In vivo treatment with Acm. Trex1^+/− and Trex1^−/− mice were treated with DMSO or Acm (5 mg/kg) for 8 weeks. Quantitative RT-PCR analysis of Ifit1 mRNA in mouse heart and spleen after 1 week of treatment are shown in D (n=4). Representative images of spleen and liver are shown in E. Survival curve after 8-week of treatment is shown in F (n=7). Unpaired t-test (A, C, D). Logrank test (F). See also Figure S5.

Figure 6. TREX1 RVCL patient lymphoblasts show elevated free glycans and immune gene expression

(A, B) FACE analysis of free glycans in lymphoblasts from RVCL patients and healthy controls. Three healthy controls, three RVCL patients (V235fs) are shown individually in A. Duplicate measurements of total free glycans in each individual are shown in B. (C, D) Free glycan and ISG analysis of lymphoblasts from RVCL patients and healthy controls treated with 1 micromolar Acm for 24 hrs. Data are representative of at least three independent experiments. Unpaired t-test (B, D).

Figure 7. TREX1-V235fs knock-in (KI) mouse display elevated free glycan, ISG signature and autoantibodies

(A) Immunoblot analysis of TREX1 in BMDMs isolated from two independent WT or V235fs-KI homozygous mice. (B) Quantitative RT-PCR analysis of ISGs in BMDMs of indicated genotype. (C) FACE analysis of free glycans in BMDMs of indicated genotype. (D, E) Autoantibody array analysis of serum isolated from WT, V235fs heterozygous or homozygous mice (8-month old). Representative autoantibodies are shown in E. n=3. Data are representative of at least two independent experiments. Unpaired t-test (B, C, E). See also Figure S6.