Fig 1: TXNDC11 is a disulphide reductase involved in glycoprotein ERAD.(a,b) Predicted architecture of the TXNDC11 protein. In (b), potential N-glycosylation sites are indicated with asterisks. (c,d) Epitope-tagged TXNDC11 predominantly localizes to the ER as assessed by co-localization with calnexin by immunofluorescence (c), supported biochemically by sensitivity to digestion with EndoH (d). (c) Scale bar, 20 μm. (e,f) Genetic reconstitution of TXNDC11 knockout cells. Expression of wild-type TXNDC11 rescues GFP-HLA-A2 degradation in TXNDC11 knockout cells. Mutation of the active site cysteine residues of the Trx5 domain, but not the Trx1 domain, abolishes the function of TXNDC11. (g) Redox titration of the TXNDC11 Trx5 domain with lipoic acid (DHLA/LA). The redox state of the purified Trx5 domain was assessed using an AMS shift assay in the presence of increasing ratios of DHLA/LA. Controls of the reduced (10 mM TCEP, left lane) and oxidized (untreated protein, right lane) forms of the protein are included. See Supplementary Fig. 6 for further discussion. (h–j) Depletion of TXNDC11 impairs the degradation of model glycoprotein ERAD substrates. (h) Immunoblot validation of efficient shRNA-mediated depletion of TXNDC11 in KBM7 cells. (i) KBM7 cell lines stably expressing fluorescently tagged CD3d (top panel), TCRa (middle panel) and NHK (bottom panel) were transduced with shRNA expression vectors targeting TXNDC11, and protein levels of the ERAD substrates measured by flow cytometry. (j) Depletion of TXNDC11 impairs the degradation of a NHK mutant lacking any cysteine residues. KBM7 cell lines were established stably expressing mCherry-tagged NHK (top panel), NHK(QQQ), which cannot be N-glycosylated (middle panel) or NHK(C/S), which lacks cysteine residues (bottom panel). The three cell lines were transduced with shRNA expression vectors targeting TXNDC11, and stabilization of NHK protein levels was measured by flow cytometry. Depletion of TXNDC11 impairs the ERAD of the NHK(C/S) mutant to a similar extent to that of wild-type NHK, but does not have a significant effect on the degradation of the non-glycosylated NHK(QQQ) variant.
Fig 2: Disulfide bond formation between EDEM2 and TXNDC11.(A) Cell lysates were prepared from WT and EDEM2-KO cells, subjected to SDS-PAGE under reducing and non-reducing conditions, and analyzed by immunoblotting using anti-EDEM2 (a), anti-PDI (b) and anti-TXNDC11 (c) antibodies. § denotes high molecular weight forms of EDEM2 and TXNDC11. Open triangle indicates a non-specific band. (B) Cell lysates were prepared from EDEM2-KO cells expressing WT or one of the three cysteine mutants of 3x Flag-tagged EDEM2 by transfection, and subjected to immunoprecipitation using anti-Flag antibody. An aliquot of cell lysates (Input) and immunoprecipitates {IP(Flag)} were subjected to SDS-PAGE under reducing and non-reducing conditions, and analyzed by immunoblotting using anti-TXNDC11, anti-PDI, anti-ERp72, and anti-Flag antibodies. (C) Structure of human TXNDC11 containing the TMD, five Trx domains, and coiled coil domain is schematically shown. ¶ denote potential N-glycosylation sites. The positions of two initiation methionines are also shown.
Fig 3: Identification of a disulfide-linked peptide in EDEM2-TXNDC11 complex by liquid chromatography (LC)/mass spectrometry (MS).(A) Cell lysates were prepared from untransfected wild-type (WT) cells and EDEM2-knockout (KO) cells untransfected or transfected with (+) or without (-) plasmid to express EDEM2-3xFlag or TXNDC11-3xFlag, subjected to SDS-PAGE under non-reducing and reducing conditions, and analyzed by immunoblotting using anti-EDEM2 antibody. EDEM2§ denotes EDEM2 stably disulfide-bonded to TXNDC11. (B) Structures of the M58A and M1A mutants of human TXNDC11 containing the transmembrane domain (TMD), five Trx domains, and coiled coil domain are shown schematically. ¶ denotes potential N-glycosylation sites. (C) Eluates were obtained from EDEM2-KO cells overexpressing TAP-EDEM2 plus TXNDC11(M1A) and from TXNDC11-KO cells overexpressing TAP-EDEM2, subjected to SDS-PAGE under reducing conditions, and silver-stained. The positions of TXNDC11(M1A)–3xFlag and 6xMyc-EDEM2 are indicated. (D) (a) EDEM2 stably disulfide-bonded to TXNDC11 was purified at a larger scale and silver-stained after reducing SDS-PAGE. The eluate was analyzed sequentially by LC/MS, MS/MS, and MS/MS/MS as indicated. This experiment was conducted once. (b) Extracted ion chromatogram of the ion at m/z 778.40 (±0.01) from EDEM2-TXNDC11 complex is shown. (c) MS spectrum of Peptide 1 observed in (b) is shown. The six peaks other than m/z 778.3999 are isotopic (13C-containing) ion peaks. (d) Electron-transfer/higher-energy collisional dissociation-tandem mass spectrometry (EThcD-MS/MS) spectrum of the ion at m/z 778.6505 ± 1 (m/z 778.3999–779.4015) derived from Peptide 1 (c) is shown.
Fig 4: TXNDC11 interacts with EDEM2/3 and the alpha-glucosidase complex.(a) Overview of the experiment designed to identify TXNDC11-interacting partners by MS. (b) Schematic representation of the role of TXNDC11 and its binding partners in the ERAD pathway. (c) Overlap between the hits from the genetic screens with the TXNDC11 binding partners identified by MS. (d) Loss of TXNDC11 increases the protein levels of EDEM2 and EDEM3. The protein levels of the TXNDC11-interacting partners were analyzed by immunoblot in two independent TXNDC11-deficient clones.
Fig 5: Effect of TXNDC11 knockout on EDEM2.(A) Quantitative RT-PCR was conducted to determine the levels of endogenous EDEM2 mRNA (a) using the two primer sets indicated as well as spliced XBP1 mRNA (b) relative to the level of GAPDH mRNA in WT and two TXNDC11-KO cells (n = 3). (B) Cell lysates were prepared from WT, EDEM2-KO, and two TXNDC11-KO cells, subjected to SDS-PAGE under reducing and non-reducing conditions, and analyzed by immunoblotting using anti-EDEM2 and anti-TXNDC11 antibodies. (C) Cell lysates were prepared from WT, EDEM2-KO, and TXNDC11-KO#1 cells expressing 3x Flag-tagged TXNDC11 or both 3x Flag-tagged TXNDC11 and Myc-tagged EDEM2 by transfection, subjected to SDS-PAGE under non-reducing conditions, and analyzed by immunoblotting using anti-EDEM2 and anti-TXNDC11 antibodies. (D) Cell lysates were prepared from WT and TXNDC11-KO#1 cells, treated with the indicated amount of trypsin at 4°C for 15 min, subjected to SDS-PAGE under reducing and non-reducing conditions, and analyzed by immunoblotting using anti-EDEM2 antibody. The band with open triangle denotes a non-specific protein which serves as a control for trypsin digestion. Quantified data are shown at the bottom.
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