Author response: Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD (2021)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible. Editor's evaluation This study demonstrates the details of mannose trimming in the ER by EDEM1 and EDEM3, filling an important gap in how mannose trimming creates an ERAD signal and shunts glycoproteins to the ERAD pathway. https://doi.org/10.7554/eLife.70357.sa0 Decision letter eLife's review process Introduction Eukaryotic cells are equipped with a sophisticated system to handle proteins which are unfolded or misfolded in the endoplasmic reticulum (ER), where newly synthesized secretory and transmembrane proteins destined for the secretory pathway gain their own three-dimensional structure (Bukau et al., 2006). These functionally inappropriate proteins are somehow detected in the ER lumen and then transferred to the retrotranslocational channel embedded within the ER membrane, termed the retrotranslocon, followed by ubiquitin-dependent degradation by the proteasome in the cytoplasm. This series of processes is collectively referred to as ER-associated degradation (ERAD-L; L for lumen) (Ninagawa et al., 2021). Extensive analysis of the mechanism of glycoprotein ERAD-L (gpERAD) has revealed that particular structures of the carbohydrate moiety become a signal for degradation (Ninagawa et al., 2021). An oligosaccharide of 14 sugar units, consisting of 3 glucose, 9 mannose, and 2 N-acetylglucosamine molecules (Glc3Man9GlcNAc2, abbreviated as G3M9 hereafter), is transferred to an Asn residue in the consensus sequence (Asn-X-Ser/Thr; X: any amino acid except Pro) present in newly synthesized protein in yeast and mammalian cells. G3M9 is converted to M9 by the sequential actions of Gls1 and Gls2-Gtb1 in yeast and glucosidases I and II in mammalian cells. Productive folding of the protein moiety is facilitated during this period by the calnexin/calreticulin cycle in mammalian cells, which relies on G1M9-specific lectin-type chaperones (calnexin and calreticulin) associated with the oxidoreductase ERp57, and UDP-glucose:glycoprotein glucosyltransferases 1 and 2, which are capable of re-adding glucose to M9 if the protein moiety is not yet folded. It should be noted that the Cne1 (an orthologue of calnexin and calreticulin) cycle is not effective in yeast because a functional orthologue of UGGTs is not present in yeast (Saccharomyces cerevisiae). After completion of folding, the glycoprotein is transported to the next compartment in the secretory pathway, the Golgi apparatus. However, if the protein moiety is not folded within a certain threshold time, the glycoprotein undergoes gpERAD via the conversion of M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue, namely M7A, M6, and M5. α1,6-Linked mannosyl residue-specific lectins OS9 and XTP3B transfer the glycoprotein to the retrotranslocon for subsequent degradation in the cytoplasm in mammalian cells (Ninagawa et al., 2021). In yeast, the α1,2-mannosidases Mns1 and Htm1 carry out the first and second mannose trimming steps, respectively (Ninagawa et al., 2021). We have constructed knockout (KO) cells for α1,2-mannosidase candidate genes, namely EDEM1, EDEM2, and EDEM3 (Htm1 orthologues), in human HCT116 diploid cells, and have shown that EDEM2 is required for the conversion of M9 to M8B and that EDEM3 (mainly) and EDEM1 (partly) are required for the conversion of M8B to oligosaccharides, which exposes the α1,6-linked mannosyl residue (Ninagawa et al., 2014). Further, our recent biochemical analysis has clarified that although EDEM2 alone exhibits no mannosidase activity toward M9, as reported previously (Mast et al., 2005; Shenkman et al., 2018), EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 catalyzes the first mannose trimming step from M9 to M8B in vitro (George et al., 2020). Intramolecular disulfide bond formation in the mannosidase homology domain (MHD) is considered essential for EDEM2 to exhibit α1,2-mannosidase activity because mutation of either C65 or C408 inactivated EDEM2 in gpERAD (George et al., 2020). Here, we examined whether the disulfide bonding between C65 and C408 is indeed formed only in the presence of TXNDC11. We then examined whether disulfide bond formation in the MHD is essential for EDEM1 or EDEM3 to exhibit α1,2-mannosidase activity, and whether TXNDC11 is disulfide-bonded to EDEM1 or EDEM3. Finally, we examined whether purified EDEM3 or purified EDEM1 can catalyze the second mannose trimming step from M8B to oligosaccharides exposing the α1,6-linked mannosyl residue. Results Formation of essential disulfide bonding in EDEM2 in the presence of TXNDC11 As we reported previously, analysis by non-reducing SDS-PAGE followed by immunoblotting showed that introduction of 3xFlag-tagged EDEM2 into EDEM2-KO cells produced its monomer, and probably dimer and aggregated forms, whereas simultaneous introduction of 3xFlag-tagged TXNDC11 and 3xFlag-tagged EDEM2 into EDEM2-KO cells produced a high molecular weight complex of EDEM2 and TXNDC11, designated EDEM2§–3xFlag (Figure 1A). It should be noted that both TXNDC11 and the EDEM2-TXNDC11 complex are detected as a doublet band due to alternative translational initiation of TXNDC11 at M1 or M58 (George et al., 2020). Figure 1 with 2 supplements see all Download asset Open asset 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. To determine disulfide bonding status in the MHD, we intended to purify noncomplexed EDEM2 and EDEM2-TXNDC11 complex separately and subject them to liquid chromatography (LC)/mass spectrometry (MS) analysis. For this purpose, TXNDC11-KO cells were transfected with plasmid to express tandem affinity purification (TAP)-tagged EDEM2 to purify noncomplexed EDEM2; this TAP consists of 2× immunoglobulin G-binding domain of protein A, 2× TEV protease recognition site, and 6xMyc (Figure 1—figure supplement 1A). Also, EDEM2-KO cells were transfected with plasmid to express TAP-tagged EDEM2 plus plasmid to express TXNDC11(M1A). It should be noted that TXNDC11(M1A) was used to purify EDEM2-TXNDC11 complex; TXNDC11(M58A) is expressed only as a transmembrane protein, whereas TXNDC11(M1A) is expressed as both soluble and transmembrane proteins because its N-terminal hydrophobic region functions by chance as either a signal sequence or a transmembrane domain (TMD) (Figure 1B), allowing us to purify a soluble complex of EDEM2 and TXNDC11. 6xMyc-tagged EDEM2 noncomplexed or complexed with TXNDC11(M1A) was purified as depicted in Figure 1—figure supplement 1B, and silver-staining showed expected bands (Figure 1C). These proteins purified at a larger scale (Figure 1D (a) ) were digested with Asp-N and the resulting peptides were analyzed by LC/MS. The results of peptides derived from EDEM2-TXNDC11 complex showed that 'Peptide 1' was eluted at 77.38 min in LC (Figure 1D (b)) and produced an ion peak at m/z 778.3999, which contained only 12C as C, plus six other isotopic (13C-containing) ion peaks in MS (Figure 1D (c)). This 'Peptide 1' was not detected in peptides derived from noncomplexed EDEM2 (data not shown). Importantly, the measured accurate mass of the ion peak at m/z 778.3999 matched the calculated exact mass of the two peptides (P1: D58~C65 of EDEM2 and P2: D395~K414 of EDEM2) covalently connected by disulfide bonding between C65 and C408 of EDEM2 (Figure 1D (c)). Indeed, subsequent electron-transfer/higher-energy collisional dissociation (EThcD)-MS/MS analysis of Peptide 1 (m/z 778.6505 ± 1) produced P1 at m/z 946.5 and P2 at m/z 1084.8 by preferential cleavage of disulfide bonds (Figure 1D (d)). Further, higher-energy collisional dissociation (HCD)-MS/MS/MS analysis of P1 (m/z 946.5 ± 1) and P2 (m/z 1084.8 ± 1), during which peptide bonds are cleaved, confirmed that P1 corresponded to D58ELRPLTC65, whereas P2 corresponded to D395AVESIEKISKVEC408GFATIK414 (Figure 1—figure supplement 2). We concluded that TXNDC11 helps EDEM2 to form the disulfide bond between C65 and C408, which is essential for EDEM2 to exhibit α1,2-mannosidase activity (George et al., 2020). Characterization of EDEM1 and EDEM3 C65 of human EDEM2 is conserved as C160 of human EDEM1 and C82 of human EDEM3, whereas C408 of human EDEM2 is conserved as C529 of human EDEM1 and C441 of human EDEM3 (Figure 2A). To determine their importance, we mutated all cysteine residues present in EDEM1 and EDEM3. To evaluate their functionality in gpERAD, we constructed EDEM1, 3-double KO (DKO) cells (two independent clones #1 and #2, Figure 2—figure supplement 1A and S1B), which expressed neither EDEM1 mRNA nor EDEM3 mRNA (Figure 2B), and grew slightly more slowly than wild-type (WT) cells (Figure 2—figure supplement 1C), as we previously described for the construction of EDEM1/2/3-triple KO (TKO) cells (Ninagawa et al., 2015). We used #1 as EDEM1, 3-DKO cells hereafter. M9 was accumulated in EDEM-TKO cells as we described previously (Ninagawa et al., 2015), whereas M8B was accumulated in EDEM1, 3-DKO cells (Figure 2—figure supplement 1D), as we expected. Accordingly, degradation of human ATF6α, a transmembrane-type gpERAD substrate, and mCD3-δ-ΔTM-HA, a soluble gpERAD substrate, was markedly delayed in EDEM1, 3-DKO cells (Figure 2—figure supplement 1E and 1F), similarly to the case of EDEM2-KO cells (Ninagawa et al., 2014). Figure 2 with 1 supplement see all Download asset Open asset Effect of mutation of various cysteine residues in EDEM1 and EDEM3 on endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). (A) Structures of human EDEM1, EDEM2, and EDEM3 are schematically shown with cysteine residues (C) highlighted together with their positions (black bars underneath C indicate conserved cysteine residues, whereas white bars over C indicate non-conserved cysteine residues). The purple and yellow boxes denote the signal sequence and mannosidase homology domain (MHD), respectively. Sequence comparison around the three cysteine residues (C65, C191, and C408 of EDEM2) is shown below (asterisk and colon indicate identical and similar amino acids, respectively). (B) RT-PCR to amplify cDNA corresponding to full-length open reading frame in EDEM1/2/3 mRNA in wild-type (WT) and EDEM1, 3-double knockout (DKO) cells (two independent clones #1 and #2) is shown. (C) Cell lysates were prepared from EDEM1, 3-DKO cells expressing WT or one of various cysteine mutants of 3× Flag-tagged EDEM1 together with mCD3-δ-ΔTM-HA by transfection, and analyzed by immunoblotting using anti-HA and anti-EDEM1 antibodies. (D) Cell lysates were prepared from EDEM1, 3-DKO cells expressing WT or one of various cysteine mutants of 3× Flag-tagged EDEM3 together with mCD3-δ-ΔTM-HA by transfection, and analyzed by immunoblotting using anti-HA and anti-Flag antibodies. We employed a gpERAD substrate migration assay. mCD3-δ-ΔTM-HA, possessing three N-glycosylation sites, migrates slightly faster due to a mannose trimming-mediated decrease in its molecular weight if cotransfected α1,2-mannosidase is active in gpERAD, and migration position is closely correlated with degradation rate (George et al., 2020). We describe this effect on migration as a downward shift hereafter. Results showed that the mutations C160A and C529A of EDEM1 as well as C82A and C441A of EDEM3 indeed inactivated EDEM1 and EDEM3, respectively, in gpERAD (Figure 2C and D). Thus, the conserved cysteine residues, at which intramolecular disulfide bonding was confirmed in EDEM2, turned out to be critical for EDEM1 and EDEM3 to exhibit α1,2-mannosidase activities, as expected. Results also showed that the mutations C410A and C457A of EDEM1 inactivated the α1,2-mannosidase activity of EDEM1 (Figure 2C), suggesting that C410 and C457 of EDEM1 might form a local disulfide bond to stabilize the MHD of EDEM1. In this connection, it was previously suggested that four of eight cysteine residues present in EDEM1 can act as free thiols and may be involved in thiol-dependent interaction with substrates, whereas the other four cysteine residues are in sufficient proximity to form two disulfide bonds (C160-C529 and C410-C457) (Lamriben et al., 2018). In contrast, cysteine residues outside of the MHD of EDEM1 and EDEM3 are not important for their α1,2-mannosidase activity (Figure 2C and D), unlike C558 of EDEM2, which is stably disulfide-bonded to C692 of TXNDC11 (George et al., 2020). Subcellular localization of MAN1B1 Our previous genetic analysis revealed that the α1,2-mannosidase MAN1B1 (a sole Mns1 orthologue) appears to play only a very minor role in gpERAD (Ninagawa et al., 2014), contrary to the original proposals (Avezov et al., 2008; Gonzalez et al., 1999; Hosokawa et al., 2003). To further evaluate the contribution of MAN1B1 to the mannose trimming step in gpERAD, we determined the subcellular localization of MAN1B1 fused to mCherry which was expressed by transfection in HCT116 cells or HeLa cells under the control of the CMV promoter or its truncated version, which we termed CMVshort promoter; the CMVshort promoter is significantly weaker than the CMV promoter in driving gene expression (Nadanaka et al., 2004). MAN1B1-mCherry driven by the CMV promoter or CMVshort promoter was not colocalized with mEGFP-KDEL, an ER marker, but was colocalized with mEGFP-Giantin, a Golgi marker, in HCT116 cells (Figure 3). Because signaling from Lamp1-mGFP, a marker of lysosome, was weak in HCT116 cells, we also checked their colocalization in HeLa cells. Again, MAN1B1-mCherry driven by the CMV promoter or CMVshort promoter was not colocalized with mEGFP-KDEL but rather with mEGFP-Giantin in HeLa cells (Figure 3—figure supplement 1). Interestingly, some MAN1B1-mCherry highly expressed from the CMV promoter overlapped with Lamp1-mGFP (Figure 3—figure supplement 1F), whereas MAN1B1-mCherry never overlapped with mEGFP-KDEL, even after higher expression (Figure 3—figure supplement 1D). Thus, MAN1B1-mediated mannose trimming cannot participate in gpERAD, the events in the ER. Figure 3 with 1 supplement see all Download asset Open asset Localization of MAN1B1-mCherry in HCT116 cells. (A, B) HCT116 cells were transfected with plasmid to express MAN1B1-mCherry under the control of the CMVshort promoter together with plasmid to express mEGFP-KDEL (A) or mEGFP-Giantin (B), and analyzed by confocal microscopy (Airyscan). Scale bar: 10 μm. (C, D) HCT116 cells were transfected with plasmid to express MAN1B1-mCherry under the control of the CMV promoter together with plasmid to express mEGFP-KDEL (C) or mEGFP-Giantin (D), and analyzed by confocal microscopy (Airyscan). Sale bar: 10 μm. Purification of EDEM3 and EDEM1 We next examined whether TXNDC11 is disulfide-bonded to EDEM1 or EDEM3. Simultaneous introduction of 3xFlag-tagged TXNDC11 and 3xFlag-tagged EDEM1 into EDEM1-KO cells or simultaneous introduction of 3xFlag-tagged TXNDC11 and 3xFlag-tagged EDEM3 into EDEM3-KO cells did not change band patterns compared with single introduction of 3xFlag-tagged EDEM1 into EDEM1-KO cells or single introduction of 3xFlag-tagged EDEM3 into EDEM3-KO cells (Figure 4A (a, b)). When any of 3xFlag-tagged EDEM1, EDEM2, and EDEM3 was introduced into WT HCT116 cells, followed by immunoprecipitation with anti-Flag antibody and subsequent immunoblotting using anti-TXNDC11 antibody under reducing and non-reducing conditions, endogenous TXNDC11 was clearly co-immunoprecipitated from cells overexpressing 3xFlag-tagged EDEM2, indicating that EDEM2 is the main partner of TXNDC11 (Figure 4B). Accordingly, we intended to purify noncomplexed EDEM1 and EDEM3 from HCT116 cells overexpressing respective TAP-tagged protein. Figure 4 Download asset Open asset Effect of coexpression of TXNDC11 on EDEM1 and EDEM3. (A) (a) Cell lysates were prepared from untransfected wild-type (WT) cells and EDEM1-knockout (KO) cells untransfected or transfected with (+) or without (-) plasmid to express EDEM1-3xFlag or TXNDC11-3xFlag, subjected to SDS-PAGE under non-reducing and reducing conditions, and analyzed by immunoblotting using anti-EDEM1 antibody. (b) Cell lysates were prepared from untransfected WT cells and EDEM3-KO cells untransfected or transfected with (+) or without (-) plasmid to express EDEM3-3xFlag or TXNDC11-3xFlag, subjected to SDS-PAGE under non-reducing and reducing conditions, and analyzed by immunoblotting using anti-EDEM3 antibody. (B) Cell lysates were prepared from WT cells untransfected or transfected with plasmid to express EDEM1-3xFlag, EDEM2-3xFlag, or EDEM3-3xFlag, and subjected to immunoprecipitation using anti-Flag antibody. Aliquots of cell lysates (Input) and immunoprecipitates (IP[Flag]) were subjected to SDS-PAGE under reducing (a) and non-reducing (b) conditions, and analyzed by immunoblotting using anti-TXNDC11 and anti-Flag antibodies. TXNDC11§ denotes TXNDC11 stably disulfide-bonded to EDEM2. (C) (a) Structures of MAN1B1 and MAN1B1(Δ105) are schematically shown. TMD denotes the transmembrane domain. (b) EDEM2-KO cells were transfected with plasmid to express mCD3-δ-ΔTM-HA together with or without plasmid to express MAN1B1-3xFlag, TAP-MAN1B1(Δ105), or TAP-MAN1B1. Cell lysates were then prepared and analyzed by immunoblotting using anti-HA and anti-Myc antibodies. (D) (a) Location of potential TEV cleavage site in EDEM3 is shown. Its consensus sequence is E-X-X-Y-X-Q-G/S. (b) EDEM1, 3-DKO cells expressing TAP-EDEM3 or TAP-EDEM3(Q543N) by transfection were subjected to small-scale purification as in Figure 1—figure supplement 1B. Eluates were analyzed by immunoblotting using anti-Myc antibody. We employed MAN1B1 as a control of an extensive α1,2-mannosidase, based on a report that it can produce M6 and M5 from M9 in vitro (Aikawa et al., 2012); we confirmed this observation (see Figure 5C). Because MAN1B1 is a transmembrane protein, we deleted its TMD-containing N-terminal 105 aa to obtain MAN1B1(Δ105) for efficient purification (Figure 4C (a)). Both TAP-tagged MAN1B1 and MAN1B1(Δ105) are active mannosidases, as their overexpression in EDEM2-KO cells caused a downward shift to the transfected mCD3-δ-ΔTM-HA band (Figure 4C (b)). Figure 5 Download asset Open asset Effect of four purified α1,2-mannosidases on PA-M8B. (A) Wild-type (WT) cells were transfected with plasmid to express mCD3-δ-ΔTM-HA. EDEM2-knockout (KO) cells and EDEM1, 3-double knockout (DKO) cells were transfected with plasmid to express mCD3-δ-ΔTM-HA together with or without plasmid to express TAP-EDEM1, TAP-EDEM2, TAP-EDEM3(Q543N), or TAP-MAN1B1(Δ105). Cell lysates were then prepared and analyzed by immunoblotting using anti-HA and anti-Myc antibodies. (B) Eluates obtained from WT cells untransfected (-) or transfected with plasmid to express TAP-EDEM1, TAP-EDEM2 plus TXNDC11(M1A), EDEM3(Q543N), or MAN1B1(Δ105) were subjected to SDS-PAGE under reducing conditions, silver-stained, and then analyzed by immunoblotting using anti-Myc antibody. PA-M8B was incubated with samples in (B) for 24 hr as indicated and then analyzed by HPLC (amide column) for mannose contents. (D) The M7 peak (a), M6 peak (b), and M5 peak (c) obtained in (C) were analyzed by HPLC (ODS column) for isomer identification. (E) Route of oligosaccharide processing in the mammalian endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD) is shown. Our initial purification of 6xMyc-tagged EDEM1, EDEM3, and MAN1B1(Δ105) from HCT116 cells overexpressing TAP-tagged EDEM1, EDEM3, and MAN1B1(Δ105), respectively, followed by silver staining and immunoblotting, revealed that 6xMyc-tagged EDEM1 was purified as a single band, whereas 6xMyc-tagged EDEM3 was purified as two bands designated EDEM3 and EDEM3C (Figure 1—figure supplement 1C, c for cleaved). Sequence revealed one TEV protease recognition site in EDEM3 (Figure and the mutation was sufficient to EDEM3 to with TEV protease (Figure (b)). EDEM3 and EDEM1 produce oligosaccharides exposing the α1,6-linked mannosyl residue from pyridylamine-labeled M8B As M9 is accumulated in EDEM2-KO cells (Ninagawa et al., and M8B is accumulated in EDEM1, 3-DKO cells (Figure 2—figure supplement 1D), transfected mCD3-δ-ΔTM-HA slightly more slowly in EDEM2-KO cells than in WT cells (Figure 1 with or more slowly in EDEM1, 3-DKO cells than in WT cells (Figure with When transfected into EDEM2-KO cells, only TAP-tagged EDEM2 caused a downward shift to the transfected mCD3-δ-ΔTM-HA band TAP-tagged EDEM1, EDEM2, and (Figure whereas TAP-tagged EDEM1 and but not TAP-tagged EDEM2 did transfected into EDEM1, 3-DKO cells (Figure 9 and with the results of our gene KO which showed that EDEM2 is required for the conversion of M9 to M8B and that EDEM3 and EDEM1 are required for the conversion of M8B to oligosaccharides exposing the α1,6-linked mannosyl residue (Ninagawa et al., 2014). 6xMyc-tagged EDEM1 and were purified as a single band on silver staining and immunoblotting in to 6xMyc-tagged EDEM2-TXNDC11 complex (Figure After 24 hr with purified 6xMyc-tagged EDEM3(Q543N), M8B was converted to M6, and M5 (Figure 5C). M7 turned out to be and (Figure indicating no of EDEM3 toward the mannose residue present in the or C of M8B. 6xMyc-tagged EDEM1 similar mannosidase activity toward with much weaker activity than 6xMyc-tagged (Figure 5C). In contrast, M8B was converted to M7 only slightly by purified EDEM2-TXNDC11 and M6 and M5 were detected even after 24 hr (Figure indicating a of EDEM2-TXNDC11 complex toward M9 (George et al., 2020). on we have established the route of oligosaccharide processing in mammalian gpERAD (Figure EDEM3 and EDEM1 produce oligosaccharides exposing the α1,6-linked mannosyl residue from M8B on a protein We examined the mannosidase activity of purified EDEM1, EDEM2-TXNDC11 and EDEM3 toward M8B on a purified gpERAD substrate, as mCD3-δ-ΔTM-HA (Figure and (Figure the region of the unfolded protein response ATF6α, degradation on mannose trimming et al., et al., 2014). 6xMyc-tagged mCD3-δ-ΔTM-HA and with were purified from EDEM1, 3-DKO cells overexpressing TAP-tagged mCD3-δ-ΔTM-HA and with (see Figure 1—figure supplement respectively, by transfection (Figure (a) and (a)). were expected to of which we confirmed by MS analysis (see of Figure (b) and Figure it should be noted that oligosaccharide was detected as two peaks consisting of a and a Indeed, were to with (Figure (b) and (b)). Because 6xMyc-tagged mCD3-δ-ΔTM-HA was detected as a doublet band by both anti-Myc and anti-HA (Figure we that the and lower bands were produced by cleavage at the first and second TEV cleavage sites, respectively (see Figure 1—figure supplement whereas the doublet band of (C) degradation during Figure Download asset Open asset Effect of purified EDEM1/2/3 on M8B present in mCD3-δ-ΔTM-HA. (A) (a) obtained from EDEM1, 3-double knockout (DKO) cells overexpressing was subjected to SDS-PAGE under reducing conditions, silver-stained, and then analyzed by immunoblotting using anti-HA antibody. (b) in (a) was (-) or (+) with subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using and is identical to antibodies. (B) (a) in (A) was incubated with purified EDEM1, EDEM2-TXNDC11 or for the indicated time, and then analyzed by immunoblotting using anti-Myc antibody. (b) prepared from samples in (a) after 24 hr were analyzed by mass spectrometry (MS). This experiment was conducted once. Figure Download asset Open asset Effect of purified EDEM1/2/3 on M8B present in (A) (a) obtained from EDEM1, 3-double knockout (DKO) cells overexpressing was subjected to SDS-PAGE under reducing conditions, silver-stained, and analyzed by immunoblotting using anti-Myc antibody. (b) in (a) was (-) or (+) with subjected to SDS-PAGE under reducing conditions, and analyzed by immunoblotting using anti-Myc and is identical to antibodies. (B) (a) in (A) was incubated with purified EDEM1, EDEM2-TXNDC11 or for the indicated time, and then analyzed by immunoblotting using anti-Myc antibody. (b) prepared from samples in (a) after 24 hr were analyzed by mass spectrometry (MS). This experiment was conducted once. The band of 6xMyc-tagged mCD3-δ-ΔTM-HA purified from transfected EDEM1, 3-DKO cells a downward shift after with 6xMyc-tagged EDEM1 (Figure (a), and but not with 6xMyc-tagged EDEM2-TXNDC11 complex MS analysis of 6xMyc-tagged mCD3-δ-ΔTM-HA samples after 24 hr with 6xMyc-tagged EDEM1 and revealed that M8B was converted to M5 whereas a major peak was M8B after 24 hr with 6xMyc-tagged EDEM2-TXNDC11 complex (Figure (b)). similar results were obtained with with which was purified from EDEM1, 3-DKO cells (Figure and although somehow a of M5 was produced after 24 hr of with 6xMyc-tagged EDEM2-TXNDC11 complex than was the case with mCD3-δ-ΔTM-HA (Figure (b)). that the peak of Figure (b)) and of and Figure (b)) were oligosaccharides derived from immunoglobulin around a molecular weight marker of Figure (a) and possessing only oligosaccharides, which was from band silver-stained around a molecular weight marker of (Figure (a) and was to be by MS analysis in our previous study et al., which no N-glycan, as by its to with (Figure (b) and (b)). We concluded that EDEM3 and EDEM1 are more active on M8B than on free M8B. Discussion Identification of the exact route of oligosaccharide