Figures and data
Effect of UGGT1/2-KO on degradation of ATF6α
(A) Schematic presentation of N-glycan processing and gpERAD.
(B) Cycloheximide chase (50 μg/ml) to determine degradation rate of endogenous ATF6α in WT, EDEM-TKO and SEL1L-KO cells treated with or without 0.5 mM DNJ treatment. DNJ was added 2 hours before the addition of CHX. Endogenous ATF6α was detected by immunoblotting using anti-ATF6α antibody. The means from three independent experiments with standard deviations (error bars) are plotted against the chase period (n = 3). P value: *<0.05, **<0.01.
(C) Cycloheximide chase (50 μg/ml) to determine degradation rate of endogenous ATF6α in WT, UGGT1-KO, UGGT2-KO and UGGT-DKO cells (n=3) as in (B). # denotes a non-specific band. P value: *<0.05, **<0.01.
(D) Cycloheximide chase (50 μg/ml) to determine degradation rate of endogenous ATF6α in WT and UGGT-DKO cells with or without 10 μg/ml kifunensine (Kif) treatment (n=3), as in (B). Kifunensine was added 1 hour before the addition of CHX. P value: *<0.05, **<0.01.
(E) Cycloheximide chase (50 μg/ml) to determine degradation rate of endogenous ATF6α in WT and UGGT-DKO treated with or without 0.5 mM DNJ (n = 3), as in (B). DNJ was added 2 hours before the addition of CHX. P value: *<0.05, **<0.01.
Effect of UGGT1/2-KO on degradation of soluble ERAD substrates
(A) Pulse-chase and subsequent immunoprecipitation experiments using anti-α1-PI antibody to determine degradation rate of NHK in WT, UGGT1-KO, UGGT2-KO and UGGT-DKO cells transfected with plasmid to express NHK (n=3). The radioactivity of each band was determined and normalized with the value at chase period 0 h. The means from three independent experiments with standard deviations (error bars) are plotted against the chase period (n = 3). P value: *<0.05, **<0.01.
(B) Rescue experiments using WT and UGGT1-KO cells transfected with plasmid to express NHK together with or without plasmid to express Myc-UGGT1 or Myc-UGGT1-D1358A (n = 3), as in (A). P value: *<0.05, **<0.01.
(C) Pulse-chase and subsequent immunoprecipitation experiments to determine degradation rate of NHK-QQQ in WT, UGGT1-KO and UGGT2-KO cells transfected with plasmid to express NHK-QQQ using anti-α1-PI antibody (n=3), as in (A).
(D) Pulse-chase and subsequent immunoprecipitation experiments using anti-HA antibody to determine degradation rate of CD3-δ-ΔTM-HA in WT, UGGT1-KO, UGGT2-KO and UGGT-DKO cells transfected with plasmid to express CD3-δ-ΔTM-HA (n =3), as in (A). P value: *<0.05.
(E) Pulse-chase and subsequent immunoprecipitation experiments using anti-HA antibody to determine the degradation rate of CD3-δ-ΔTM-<33-7aa-34>-HA in WT, UGGT1-KO, UGGT2-KO and UGGT-DKO cells transfected with plasmid to express CD3-δ-ΔTM-<33-7aa-34>-HA (n =3), as in (A). P value: *<0.05, **<0.01.
Effect of UGGT1/2-KO on functional proteins in the secretory pathway and on their unstable mutants
(A)–(C) Cycloheximide chase (50 μg/ml) and subsequent immunoblotting experiments to determine the degradation rate of endogenous Grp170 (A), Sil1 (B) and CRT (C) in WT and UGGT-DKO cells using the respective antibody (n = 3).
(D) [left] Cycloheximide chase (50 μg/ml) and subsequent immunoblotting experiments to determine the degradation rate of endogenous Ribophorin I in WT and UGGT-DKO cells using anti-Ribophorin antibody (n=3), as in (A). [right] Pulse chase and subsequent immunoprecipitation experiments using anti-Flag antibody to determine the degradation rate of rRI332-Flag in WT, UGGT1-KO, UGGT2-KO and UGGT-DKO cells transfected with plasmid to express rRI332-Flag (n =3), as in Fig. 3A. P value: *<0.05, **<0.01.
(E) Determination of GnT-V activity in cell lysate of WT, UGGT1-KO, UGGT2-KO and UGGT-DKO cells (n=3).
(F) Pulse-chase and subsequent immunoprecipitation experiments using anti-EMC1 antibody to determine degradation rate of endogenous EMC1 and EMC1-ΔPQQ-Flag in WT, UGGT1-KO, UGGT2-KO and UGGT-DKO cells transfected with plasmid to express EMC1-ΔPQQ-Flag (n =3), as in Fig. 3A. P value: *<0.05, **<0.01.
Effects of UGGTs on unfolded protein response via ATF6α
(A) Immunoblotting to determine the protein level of ATF6α(P) in WT, UGGT1-KO, UGGT2-KO, UGGT-DKO, ATF6α-KO cells using anti-ATF6α antibody (n=3). P value: *<0.05.
(B–D) HCT116 cells cultured in 35-mm dishes were incubated in the presence of 300 nM thapsigargin (Tg) for the indicated periods. Endogenous ATF6α(P) and ATF6α(N) in WT was detected together with UGGT1-KO (B), UGGT2-KO (C), and UGGT-DKO (D), respectively. # indicates non-specific band.
(E-G) HCT116 cells transiently transfected with ERSE (E), UPRE (F) or ATF4 (G) reporters. Twenty-four hours after the transfection, cells were treated with 300 nM Tg for 6 h and harvested to determine luciferase activity (n=3). Relative activity of Luciferase and Renilla, and fold induction of Tg + and Tg – are shown.
Model of behavior of glycoproteins in the ER
(A). In WT cells, glycoproteins with M9 or M8 are recognized by UGGTs to produce GM9 or GM8 to promote their folding by CNX or CRT, or by EDEMs to trim mannoses of N-glycan to expose the α1,6-bond of N-glycan in the C chain for gpERAD. Glycoproteins are caught in a tug-of-war between the fate of structure formation by UGGTs and degradation by EDEMs. (B). In WT cells with DNJ, glycoproteins are degraded by EDEMs-mediated gpERAD despite inefficiency of mannose trimming due to the presence of glucoses at A chain. (C). In UGGT-DKO cells, glycoproteins are subjected to premature degradation by EDEMs-mediated gpERAD. (D). In UGGT-DKO cells with DNJ, the almost the same situation as in (B) exists. (E–F). In EDEM-TKO cells, glycoproteins are not degraded via gpERAD.
Generation of UGGT1/2-KO HCT116 cells
(A) Strategy for obtaining UGGT1-KO, UGGT2-KO and UGGT-DKO cells from HCT116 cells. UGGT1-KO #2, UGGT2-KO #2 and UGGT-DKO #2 were mainly used in this report.
(B) Schematic presentation of UGGT1 and UGGT2 loci.
(C) Strategy of the CRISPR/Cas9-mediated targeting of exon 2 of the UGGT1 gene.
(D) Strategy of the CRISPR/Cas9-mediated targeting of exon 4 of the UGGT2 gene.
(E) Genomic PCR to confirm recombination of UGGT1 locus.
(F) Genomic PCR to confirm recombination of UGGT2 locus.
(G) RT-PCR to amplify cDNA corresponding to full length UGGT1 from WT and two independent UGGT1-KO cell lines (#1 and #2).
(H) RT-PCR to amplify cDNA corresponding to full length UGGT2 from WT and two independent UGGT2-KO cell lines (#1 and #2).
(I) Schematic presentation of UGGT1 mRNA expressed in two independent UGGT1-KO cell lines (#1 and #2) and their translational products. The sequence of the amplified cDNA in (G) was determined.
(J) Schematic presentation of UGGT2 mRNA expressed from the deleted and targeted alleles in UGGT2-KO cell lines and their translational products. The sequence of the amplified cDNA in (H) was determined
(K) Immunoblotting to determine endogenous protein expression of UGGT1 and UGGT2 in UGGT1-KO, UGGT2-KO and UGGT-DKO HCT116 cells using anti-UGGT1, anti-UGGT2 and anti-GAPDH antibodies.
(L) Doubling time of WT, UGGT1-KO, UGGT2-KO and UGGT-DKO HCT116 cells (n = 3).
Generation and characterization of UGGT1/2-KO HCT116 cells
(A) Immunoblotting of cell lysates prepared from WT HCT116 and HeLa cells transiently expressing UGGT1-Myc3 or UGGT2-Myc3 using anti-Myc, anti-UGGT1, anti-UGGT2 and anti-GAPDH antibodies. The intensity of the lower left band in each panel is set to 1.00, and relative intensities are shown below. Anti-Myc antibody was used to estimate the ratio of the amounts of UGGT1-Myc3 and UGGT2-Myc3. Anti-UGGT1 antibody was used to estimate that of endogenous UGGT1 and UGGT1-Myc3. Anti-UGGT2 antibody was used to estimate that of endogenous UGGT2 and UGGT2-Myc3. These values can be used to calculate the relative amounts of endogenous UGGT1 and UGGT2. Approximate expression level of UGGT2 relative to UGGT1 in HCT116 and HeLa cells shown as % in bottom panel.
(B) Expression levels of BiP, XBP1(S), spliced form of XBP1, ATF4 and GAPDH determined by immunoblotting of cell lysates, which were prepared from WT, WT treated with 300 nM thapsigargin (Tg) for 6 h, UGGT1-KO, UGGT2-KO and UGGT-DKO cells.
(C) Schematic presentation of UGGT1 and UGGT2 proteins with potential N-glycosylation sites and catalytic domain indicated. Cell lysates were prepared from WT HCT116 cells, treated or not treated with EndoH, and analyzed by immunoblotting using anti-UGGT1 and anti-UGGT2 antibodies.
(D), (E) Secretion of A1AT (D) and EPO-Myc3 (E) in WT, UGGT1-KO, UGGT2-KO and UGGT-DKO HCT116 cells. Cells were transfected with plasmid to express A1AT or EPO-Myc3. Twenty-four hours later, cells were pulse labeled with 35S-methionine and cysteine for 20 min and then chased for the indicated periods, followed by immunoprecipitation of A1AT or EPO-Myc3 from cells and medium using anti-A1AT or anti-Myc antibody, respectively, and then subjected to reducing SDS-PAGE and autoradiography (n=3). The amount of A1AT or EPO-Myc3 in WT cells at 0 min was set as 100 %. The means from three independent experiments with standard deviations (error bars) were plotted against the chase period.
(F) Maturation of HA in WT, UGGT1-KO, UGGT2-KO and UGGT-DKO HCT116 cells. Pulse-chase experiments were conducted using various cells transfected with plasmid to express HA, followed by immunoprecipitation of HA from cells using anti-HA antibody (n=3), as in (D). The immunoprecipitates were digested with EndoH prior to subjection to reducing SDS-PAGE. The amount of HA with high-mannose type N-glycan at time 0 h in WT cells was set as 100 %.
Generation of SEL1L-KO HCT116 cells
(A) Schematic presentation of SEL1L locus
(B) Strategy of the TALEN-mediated targeting of exon 1 of the SEL1L gene.
(C) Genomic PCR to confirm recombination of SEL1L locus.
(D) RT-PCR to amplify cDNA corresponding to full length SEL1L from WT and two independent SEL1L-KO cell lines (#1 and #2).
(E) Immunoblotting of SEL1L protein in WT and two independent SEL1L-KO cell lines (#1 and #2) using anti-SEL1L antibody.
(F) Doubling time of WT and two independent SEL1L-KO cell lines (#1 and #2).
N-glycan profiling of various types of cells treated or not treated with DNJ
(A)–(D) Isomer composition of high mannose-type N-glycans prepared from total cellular glycoproteins of WT, UGGT-DKO and EDEM-TKO cells (A), WT cells treated with or without 0.5 mM DNJ for 16 h (B), UGGT-DKO cells treated with or without 0.5 mM DNJ for 16 h (C), EDEM-TKO cells treated with or without 0.5 mM DNJ for 16 h (D). This experiment was completed once.
Characterization of various proteins present in the secretory pathway
(A) Schematic structures of human Grp170, human Sil1, human CRT, human GnT-V, human Ribophorin I, rat Ribophorin I, human EMC1, and human EMC1-ΔPQQ-Flag with potential N-glycosylation sites indicated. It has not been confirmed whether two N-glycans are attached to human and rat Ribophorin I. Sequence identities between human and rat Ribophorin I are shown as percentages. rRI332-Flag lacks aa 333–606 of rat Ribophorin I.
(B) Immunoblotting with respective antibody of cells lysates prepared from WT cells and cells treated with or without EndoH treatment.
Structural assessment of ATF6α in UGGT1-KO
(A) Trypsin digestion assay to evaluate the folding state of ATF6α(P) in WT and UGGT1-KO. The same amounts of proteins in cell lysate from WT and UGGT1-KO cells were treated with the indicated concentration of trypsin for 15 minutes at room temperature. Enzymatic activity of trypsin was terminated by addition of Laemmli SDS sample buffer containing 100 mM DTT and 10x protease inhibitor cocktail. Immunoblotting was conducted as in Fig. 4A, using anti-ATF6α and anti-Sil1 antibodies. In electrophoresis for the detection of ATF6α, the UGGT1-KO sample is loaded with twice as much protein as the WT sample.
