Journal: Molecular Metabolism

Article Title: TALK-1 reduces delta-cell endoplasmic reticulum and cytoplasmic calcium levels limiting somatostatin secretion

doi: 10.1016/j.molmet.2018.01.016

Figure Lengend Snippet: TALK-1 channels are expressed in mouse and human δ-cells. (A) Mouse pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 mice). (B) Mouse pancreas section stained for TALK-1 (green), ER (GRP94, red), and SST (cyan). (C) Human pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 pancreata). (D) Human pancreas section stained for TALK-1 (green), ER (GRP94, red), and somatostatin (E) K2P currents recorded from WT and TALK-1 KO δ-cells ( N = 3 mice per genotype). (F) K2P currents recorded from human δ-cells expressing TALK-1 DN or control mCherry. ( N = 3 islet preparations); * P < 0.05, ** P < 0.005.

Article Snippet: Sections were stained using primary antibodies against somatostatin (Santa Cruz Biotechnology sc-7819: 1:250), TALK-1 (Novus Biologicals #NBP1-83071; 1:175) or TALK-1a (Antibody Verify AAS72353C; 1:250), glucagon (Proteintech #15954-I-AP: 1:500), and the endoplasmic reticulum marker GRP94 (Novus Biologicals #NB300-619; 1:100); secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit (Jackson Immunoresearch #711-546-152; 1:300), DyLight 650-conjugated donkey anti-goat (Thermo Fisher #SA5-10089; 1:250), and Cy3-conjugated donkey anti-mouse (Jackson Immunoresearch #715-166-150; 1:500).

Techniques: Staining, Expressing, Control

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc increase S protein specific IgG responses in vivo. a) C57Bl6 mice were vaccinated at day 0 and 14 with different concentrations of cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S and OX40L-Fc or with AD100 or PBS (controls). b) Mice were vaccinated at day 0 and 14 with 1μg/ml of ZVX-55 and ZVX-60 or with AD100 and PBS (controls). Serum was collected 5 days after last vaccination, and S protein specific IgG response was analyzed by ELISA. c) Mice were vaccinated at day 0 and 14 with 1 μg/ml ZVX-55 or ZVX-60 and S protein specific IgG response in serum was analyzed at day 5, 14 and 19. Data represent 3 independent biological replicates per group and mean ± standard error. To compare control (ZVX55) with experimental (ZVX60) group (alpha level of 0.05) unpaired t-test (two-tailed) was applied, *p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc induce B cells responses. C57Bl6 mice were vaccinated at day 0 and 14 with a cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S (ZVX-55, 1ug gp96-Ig) and OX40L-Fc (ZVX-60,1 ug gp96-Ig) or with AD100 or PBS (controls). a) Spleen cells (SPL) were isolated from vaccinated and control mice 5 days after last vaccination, stained for CD45, CD3, CD19, IgM, CD21, CD23, CD49, CD93. Frequency of CD19+IgM+ (activated B cells) and CD21+CD23- (marginal zone, MZ), CD21+CD23+ (follicular, FO) and CD21-CD23- (double negative or ABC cells) CD19+IgM+ cells was determined by flow cytometry. b) SPL were isolated from unvaccinated mice and co-cultured with vaccine cells (ZVX55 or ZVX60) and control cells AD100 at 5:1 ratio for 96h. Frequency of activated B cells (CD19+IgM+) within total CD45+ T cells and frequency of FO (CD21+CD23+) within CD19+IgM+ cells was determined by flow cytometry. Data represent 3 independent biological replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Isolation, Staining, Flow Cytometry, Cell Culture

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Gp96-Ig and OX40L-Fc induce T follicular helper (TFH) cell responses. C57Bl6 mice were vaccinated at day 0 and 14 with a cell-based gp96-Ig vaccine that expressed SARS-CoV-2 glycoprotein S (ZVX-55, 1ug gp96-Ig) and OX40L-Fc (ZVX-60,1 ug gp96-Ig) or with AD100 or PBS (controls). a) Spleen cells (SPL) were isolated from vaccinated and control mice 5 days after last vaccination, stained for CD45, CD3, CD4, PD1 and CXCR5. Frequency of PD1+CXCR5+ (TFH cells) within CD4+ T cells was determined by flow cytometry. b) SPL were isolated from unvaccinated mice and co-cultured with vaccine cells (ZVX55 or ZVX60) and control cells AD100 at 5:1 ratio for 96h. Frequency of TFH cells (PD1+CXCR5+) within total CD4+ T cells was determined by flow cytometry. Data represent 3 independent biological replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Isolation, Staining, Flow Cytometry, Cell Culture

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Characteristics of the cell line expressing gp96-Ig, SARS-CoV-2 Spike (S) protein and OX40L-Fc. Cell line (AD100) was transfected with plasmids encoding a) gp96-Ig and full length protein S and b) gp96-Ig, full length protein S and OX40L-Fc. c) Secreted gp96-Ig was measured in the cell supernatant by ELISA. One million cells were plated in 1 ml for 24 h. Purified gp96-Ig was used as standard d) Secreted OX40L-Fc was measured in the cell supernatant by ELISA. One million cells were plated in 1 ml for 24 h. Purified OX40L-Fc was used as standard e) SARS-CoV2 protein S expression was analyzed by immunofluorescence f) SARS-CoV2 protein S expression in supernatant was measured by ELISA. One million cells were plated in 1 ml for 48 h and purified SARS-CoV2 protein S was used as standard.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Purification, Immunofluorescence

Journal: Vaccine: X

Article Title: Secreted heat shock protein gp96-Ig and OX40L-Fc combination vaccine enhances SARS-CoV-2 Spike (S) protein-specific B and T cell immune responses

doi: 10.1016/j.jvacx.2022.100202

Figure Lengend Snippet: Enhancement of S1- specific CD8+ T cell responses by Gp96-Ig-S-OX40L-Fc in the spleen, lung tissue, and BAL . a) 5 days after the vaccination of HLA-A2 transgenic mice (n=3-5) with the ZVX-55, ZVX-60 vaccine cells (1ug secreted gp96-Ig) or AD100 or PBS (controls), splenocytes (SPL), lung cells and bronchioalveolar lavage (BAL) cells were isolated from vaccinated and control mice (PBS). Cells were stained with HLA-A2 02-01 pentamer containing YLQPRTFLL peptides, followed by surface staining for CD45, CD3, CD4, CD8, CD69, CXCR6. Bar graphs represent percentage of the pentamer positive cells within CD8+ T cells. b) 5 days after the vaccination of C57Bl6 mice (n=3), splenocytes and lung cells were isolated from vaccinated and control mice (PBS and AD100) and in vitro restimulated with S1 and S2 overlapping peptides in the presence of protein transport inhibitor, brefeldin A for the last 5 hours of culture. After 20 hours of culture, intracellular cytokine (IFNg, TNFa and IL-2) staining was preformed to quantify protein S-specific CD8+ T-cell responses. Cytokine expression in the presence of no peptides was considered background and it was subtracted from the responses measured from peptide pool stimulated samples for each individual mouse. Data represent at least 2 technical replicates with 3–5 independent biologic replicates per group and mean ± standard error. To compare >2 experimental groups, 2-way analysis of variance (ANOVA) test with Holm-Sidak multiple-comparison test were applied, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: One million cells were plated in 1 mL of growth medium for 24 hours and secreted gp96-Ig production was determined by ELISA using sheep anti-human gp96 antibody (HSP90B1-R&D Systems, Cat. No. AF7606) as coating antibody, peroxidase conjugated goat anti-human IgG antibody as a secondary antibody (Abcam Ab7499), and purified gp96-Ig IgG1 as a standard.

Techniques: Transgenic Assay, Isolation, Staining, In Vitro, Expressing

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: (A) Abundance and glycosylation status of HSP90B1 on SDS-PAGE gels in lysates from three (C1-C3) independent control (NTC) and CCDC134 -/- clonal cell lines. (B) HSP90B1 protein abundance in wild-type (WT), CCDC134 -/- , HSP90B1 -/- RKO cell lines measured by mass spectrometry, normalized to WT cells. Bars represent the mean abundance +/- SEM from six independent mass spectrometry runs, represented as individual data points. (C) Glycosidase sensitivity (Endo H or PNGase F see ) of HSP90B1 in lysates of control (NTC) or CCDC134 -/- cells. HSP90B1 is glycosylated at either a single constitutive site (monoglycosylated band) or at multiple facultative sites (hyperglycosylated bands) (see and text for details). (D) Abundance and glycosylation status of HSP90B1 (hyperglycosylated form is denoted by a red arrowhead) in lysates from control (NTC) or CCDC134 -/- cell lines with or without CCDC134 expression from a doxycycline-inducible, stably integrated transgene (see ). (E) Domain architecture of HSP90B1 as defined in. The constitutive and facultative N-glycosylation sites are labeled in green and purple, respectively. The point mutations used to disrupt the one constitutive sequon (1N mutant) and the five facultative sequons (5N mutant) are listed. The N-terminal domain (NTD) binds ATP, the Middle domain (MD) participates in ATP hydrolysis and client recognition, and the C-terminal domain (CTD) in dimerization. (F) Structure (PDB 5ULS) highlighting one protomer of a HSP90B1 dimer, with the pre-N, NTD, MD and CTD domains colored as in ( E ) . The ATP bound to the NTD and the asparagine side chains of the constitutive sequon (green) and the facultative sequons (purple) are shown in space filling representation. The inset shows proximity of two facultative sequons to the ATP binding site. (G) Abundance of glycosylated peptides in CCDC134 -/- cells measured using global, unbiased N- glycoprotemics. Each data point (top graph) represents the fold change in abundance of a distinct glycopeptide (defined by sequence and glycan structure) in CCDC134 -/- compared to wild-type cells. Glycopeptides that include the constitutive and facultative sequons in HSP90B1 are colored green and purple, respectively. Amongst the ten glycopeptides (in the entire proteome) that show the greatest fold- increase in CCDC134 -/- cells, nine are from regions of HSP90B1 that include the facultative sites (bottom Table). Full dataset is provided in Data S2 . (H, I) Abundances of cell-surface LRP6 ( H ) or active β-catenin abundance ( I ) in clonally derived control (NTC), HSP90B1 -/- , and CCDC134 -/- ; HSP90B1 -/- cell lines stably expressing wild-type (WT) FLAG- HSP90B1 or variants carrying mutations in the one constitutive (1N) or all five facultative (5N) sites (see ). Abundances of stably expressed HSP90B1 variants were comparable . (J-M) LRP6 and HSP90B1 abundances ( J ), WNT3A dose-response curves ( L ), WNT signaling strength ( M ), and cell-surface LRP6 levels measured by flow-cytometry ( K ) in primary fibroblasts isolated from two OI patients carrying homozygous loss-of-function mutations in CCDC134 (c.2T>C) (P1, P2) and two age-matched healthy control individuals (C1 and C2, respectively). ( L,M ) The abundance of AXIN2 mRNA, encoded by an immediate-early WNT target gene, was measured by quantitative reverse transcription PCR (qRT-PCR) as a metric of WNT signaling strength. In K , L and M error bars show the mean +/- SEM from three independent experiments. Statistical significance was determined by two-way ANOVA Dunnett’s multiple comparisons test; * p<0.05, ** p<0.01 ( M ) or two-way ANOVA Tukey’s multiple comparisons test; **** p<0.0001 ( K ). See also fig.S2.

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: Glycoproteomics, SDS Page, Control, Quantitative Proteomics, Mass Spectrometry, Expressing, Stable Transfection, Labeling, Mutagenesis, Binding Assay, Sequencing, Derivative Assay, Flow Cytometry, Isolation, Reverse Transcription, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: (A) Abundance and glycosylation status of HSP90B1 on SDS-PAGE in lysates from WT RKO cells, cells expressing a control (NTC) sgRNA or sgRNAs targeting the indicated genes. The hyperglycosylated HSP90B1 band is indicated with a red arrowhead. (B) Impact of CCDC134 co-expression on HSP90B1 hyperglycosylation (red arrowhead) when the indicated proteins are transiently expressed in HEK293T cells. (C) Effects of low and high 3xHA-CCDC134 expression (from a stably integrated, doxycycline-inducible transgene) on HSP90B1 hyperglycosylation (red arrowhead) in CCDC134 -/- RKO cells treated with 0.1 uM thapsigargin for 24 hrs. Low (near-endogenous, see ) and high CCDC134 expression was achieved by supplementing media with 5 nM or 25 nM doxycycline, respectively. (D) Abundances and glycosylation status of HSP90B1 and LRP6 in lysates from clonally derived control (NTC) and CCDC134 -/- RKO cells treated (18 hours) with small molecule inhibitors of lysosomal acidification (50 nM Bafilomycin A1), the proteasome (1 μM bortezomib), or the ERAD pathway (5 μg/mL kifunensine or 5 μM NMS-873). As a control, media was also supplemented with an equivalent concentration of the solvent (DMSO) contributed by the inhibitor stock solutions. The hyperglycosylated HSP90B1 band is indicated with a red arrowhead. Numbers below the lanes show the percentage of HSP90B1 that is hyperglycosylated; note the increase in bortezomib, kifunensine, and NMS-873 treatments. (E) Abundances of endogenous HSP90B1 in control (NTC) cells compared to stably integrated 3xFLAG- HSP90B1 variants in HSP90B1 -/- and CCDC134 -/- ; HSP90B1 -/- cell lines (see ). (F) LRP6 abundance and HSP90B1 hyperglycosylation (red arrowhead) in a panel of human (HEK293T, HAP1) and mouse (MC3T3, C3H10T1/2) cell lines expressing sgRNAs targeting the indicated genes (NTC=non-targeting control sgRNA). (G) Expression of the WNT target gene TNFRSF19 (mean +/- SEM of TNFRSF19 mRNA normalized to GAPDH mRNA measured by qRT-PCR in three independent experiments) was quantified in primary patient fibroblasts treated with 0, 3.125, 6.25, 12.5, 25, 50% WNT3A conditioned media. See for the same comparison using a different WNT target gene ( AXIN2 ). (H) Expression of the WNT target gene TNFRSF19 (mean +/- SEM of TNFRSF19 mRNA normalized to GAPDH mRNA, measured by qRT-PCR in three independent experiments) in primary patient fibroblasts stably expressing 3xHA-CCDC134 treated with 25% WNT3A conditioned media. Statistical significance was determined by two-way ANOVA Dunnett’s multiple comparisons test; * p<0.05, **** p<0.0001. See for the same comparison using a different WNT target gene ( AXIN2 ). (I) Abundances of LRP6 and HSP90B1 in primary fibroblasts isolated from a CCDC134 -/- patient (P2, ) with or without stable re-expression of 3xHA-CCDC134 .

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: Glycoproteomics, SDS Page, Expressing, Control, Stable Transfection, Derivative Assay, Concentration Assay, Solvent, Quantitative RT-PCR, Comparison, Isolation

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: (A) DepMap co-essentiality relationships between CCDC134, STT3A, HSP90B1, and OSTC visualized using Fireworks . The bi-directional edges between these four genes (bold orange lines) indicate concordant effects on cell growth across >400 cell lines. (B) Abundances and glycosylation status of the indicated proteins (labeled to the right of each immunoblot segment) in lysates from cells expressing a control (NTC) sgRNA or sgRNA’s against various genes. (C) Abundance of glycosylated peptides in STT3A -/- cells compared to WT cells (represented as ). Glycopeptides that include the constitutive and facultative sequons in HSP90B1 are colored green and purple, respectively. Amongst the ten glycopeptides in the entire proteome that show the greatest fold- increase in STT3A -/- cells, five are from regions of HSP90B1 that include the facultative sites (table on right). Full dataset is provided in Data S2 . (D) Enrichment of glycopeptides (normalized to total HSP90B1 protein abundance from mass spectrometry) that include each of the facultative and constitutive sequons of HSP90B1 in CCDC134 -/- or STT3A -/- cells compared to WT cells. Bars show the mean +/- SEM from six independent mass spectrometry runs, represented as individual data points. The abundances of all peptides that include each HSP90B1 sequon were integrated using individual peptide data summarized in and 3C ). ( E, F ) Abundances of LRP6 ( E ) and active β-catenin ( F ) in WT or clonal STT3B -/- and STT3A -/- cells, with or without stable expression of a variant of HSP90B1 (HSP90B1 5N , ) carrying disrupting mutations in all five facultative sequons. ( G ) Abundance and glycosylation status of HSP90B1 in STT3B -/- and STT3A -/- cells expressing different levels of CCDC134: No CCDC134 (-), endogenous CCDC134( + ), stably overexpressed 3xHA-CCDC134 ( +++ ) on top of endogenous CCDC134. ( H,I ) Abundances of LRP6 and HSP90B1 ( H ) or active β-catenin ( I ) in WT or STT3A -/- cells stably expressing FLAG-STT3A variants carrying mutations in various sites involved in catalytic transfer of the glycan from the lipid-linked oligosaccharide to the asparagine in sequons. Variants (shown on a structure in ) carry mutations in residues involved in active site (AS) chemistry, lipid-linked oligosaccharide binding (LLO), sequon binding (WWD) or N-glycosylation of STT3A itself (N and NN). Glycosylation of PSAP ( I ) was used to assess OST-A activity in cells. CCDC134 serves as a loading control in ( H ). ( J ) A provisional pathway diagram constructed based on genetic interactions (dotted lines) uncovered in and and physical interactions (solid lines) described in the literature. See also fig.S3.

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: Glycoproteomics, Labeling, Western Blot, Expressing, Control, Quantitative Proteomics, Mass Spectrometry, Variant Assay, Stable Transfection, Binding Assay, Activity Assay, Construct

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: (A) Cryo-EM structure of the OST-A complex (PDB 6S7O ) with STT3A highlighted in yellow. Residues that were mutated to generate the STT3A variants tested in and 3I are shown on the structure and listed in the table below. Variants carry mutations in residues involved in active site (AS, blue) chemistry, lipid-linked oligosaccharide binding (LLO, green), sequon binding (WWD, magenta) or N-glycosylation of STT3A itself (N and NN, orange). (B) Abundance and glycosylation status (red arrowhead) of HSP90B1 in STT3A -/- cells stably expressing catalytically inactive FLAG-STT3A carrying mutations in its active site (AS, see A ) and sgRNAs targeting CCDC134 or OSTC. NTC=non-targeting control sgRNA. See also .

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: Cryo-EM Sample Prep, Binding Assay, Glycoproteomics, Stable Transfection, Expressing, Control

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: (A) Glycosylation status of full-length FLAG-HSP90B1 variants co-expressed in STT3B -/- HEK293T cells with functional CCDC134 ( + ) carrying a native ER signal sequence or non-functional CCDC134 (-) lacking an ER signal sequence. WT refers to HSP90B1 carrying all native sequons while 5N refers to a variant that lacks the five facultative sequons (shown in ). Bur3N and Exp3N refer to mutations that introduce three additional artificial sequons predicted to be buried (Bur) or exposed (Exp) based on the PDB 5ULS HSP90B1 structure. Red arrowheads indicate hyperglycosylated forms of each variant. (B) Glycosylation status of the indicated deletion mutants of FLAG-HSP90B1 lacking its CTD and containing only three sequons in the M domain (ΔCTD, see ). The deletion series starts at amino acid 337 and extends sequentially into the NTD and pre-N segments. The minimum construct that still retains regulation of glycosylation is boxed in red, this is also named the 1-93M construct. (C) Triplet alanine scanning mutagenesis was used to identify amino acid residues within the pre-N domain of HSP90B1 that regulates its own glycosylation. Sets of three consecutive residues (A1-A24, as shown in the pre-N sequence above the immunoblots) were mutated to Ala-Ala-Ala in the 1-93M variant of HSP90B1 (see for domain structure) and glycosylation tested by transiently transfecting the encoding constructs into HEK293T cells. See also .

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: Glycoproteomics, Functional Assay, Sequencing, Variant Assay, Introduce, Construct, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: ( A ) Variants of HSP90B1 used for cell-based and in vitro assays in and 5 , diagrammed following the scheme shown in . Key features include the ERss, ER signal sequence; FLAG, 3xFLAG tag; pre-N, unstructured segment; NTD, N-terminal domain; MD, middle domain; CTD, C-terminal domain. The N-glycosylation sites in pre-N and NTD were eliminated to allow easy assessment of the glycan modification of all three sequons in the M domain by gel shifts (see C and text). ( B , C ) Glycosylation status of HSP90B1 variants shown in ( A ) was assessed using gel shifts and Endo H sensitivity (see ) after transient co-expression in HEK293T cells with WT CCDC134 ( + ) or a non- functional variant (-) lacking its ER signal sequence (see ). The four predicted N-glycoforms (carrying 0, 1, 2 or 3 glycans) are labeled in ( C ) as 0N-3N in red lettering. ( D ) Glycosylation status of the 1-93M variant (see A ) of HSP90B1 carrying the indicated mutations in the “SRT” motif (underlined in the sequence of the pre-N segment of HSP90B1 shown above). Each construct was co-expressed with functional ( + ) or non-functional (-) CCDC134. See fig.S4 for deletion analysis and alanine scanning mutagenesis of the pre-N segment. ( E ) Glycosylation status of full-length (see A ) FLAG-HSP90B1 carrying a T44A mutation in the “SRT” pseudosubstrate site identified in D . Bur3N and Exp3N refer to variant proteins containing three additional artificial sequons predicted to be buried (Bur) or exposed (Exp) based on the PDB 5ULS HSP90B1 structure. ( F ) Glycosylation status of chimeric proteins constructed by fusing the 1-93 segment of HSP90B1 (or mutants within the “SRT” pseudosubstrate site) to the obligate OST-A substrate PSAP, which contains five N-glycosylation sites shown in the domain diagram. T44A changes “SRT” to “SRA” and A8 changes “RTD” ro “AAA” (see and ). All chimeras carry the ERss of HSP90B1. Note that in D , E and F , all constructs were transiently expressed in STT3B -/- HEK293T cells to exclude any contribution from OST-B glycosylation. See also fig.S4 .

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: In Vitro, Sequencing, Glycoproteomics, Modification, Expressing, Functional Assay, Variant Assay, Labeling, Construct, Mutagenesis

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: (A) Constructs used for in vitro translation experiments. For the 1-93M (stall) constructs, the dark gray indicates the region predicted to be fully in the ER lumen and light gray indicates the region predicted to be in the translocon and ribosome exit tunnel. (B) Glycosylation status of the 1-93M variant of HSP90B1 (see ) translated in rabbit reticulocyte lysate (RRL) in the presence of rough microsomal membranes generated from wild-type or clonally- derived CCDC134 -/- , STT3A -/- , or STT3B -/- HEK293T cells and concentrated by immunoprecipitation on anti-FLAG beads. The four N-glycoforms of 1-93M are labeled (compare to ) and show sensitivity to Endo H treatment. (C) Association of endogenous CCDC134 with truncation variants of 1-93M lacking a STOP codon translated in the presence of rough microsomal membranes generated from wild-type HEK293T cells. The stalled nascent chain was immunoprecipitated (IP) using anti-FLAG beads and association with STT3A, CCDC134, and the ribosome (RPL17) assessed by immunoblotting. To clearly visualize the sizes of the truncation variants, each sample was also treated with RNase A to release tRNA and Endo H to collapse glycoforms (top right immunoblot). (D) Association of endogenous CCDC134 with stalled 1-93M, M domain alone or a 1-93M variant carrying a T44A mutation in the “SRT” pseudosubstrate site, see ), assessed as in B . NC-tRNA: nascent chain-tRNA conjugates. (E) Association of endogenous CCDC134 with a stalled 1-93M nascent chain translated in the presence of microsomes isolated from wild-type (WT), CCDC134 -/- , STT3A -/- , or STT3B -/- HEK293T cells, assessed is in B .

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: Construct, In Vitro, Glycoproteomics, Variant Assay, Generated, Derivative Assay, Immunoprecipitation, Labeling, Western Blot, Mutagenesis, Isolation

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: ( A, B ) Translation time course of 1-93M ( A ) or 1-93M carrying the T44A mutation in the “SRT” pseudosubstrate site ( B ) in rabbit reticulocyte lysate (RRL) supplemented with rough microsomes isolated from wild-type or CCDC134 -/- HEK293T cells. The sizes and Endo H sensitivities of the translation products were analyzed after anti-FLAG immunoprecipitation. Glycosylated translational intermediates (highlighted by a red line) were identified by their size (shorter than the full-length protein seen at 15 min) and sensitivity to Endo H. (C) Pulse-labeling of HSP90B1 in clonal HSP90B1 -/- ; CCDC134 -/- double knock-out cells stably expressing 3xFLAG-HSP90B1. Culture media was supplemented with 35 S-Methionine and 35 S-Cysteine for 3, 5 or 7 minutes, followed by lysis and immunoprecipitation of HSP90B1 on anti-FLAG beads. The Endo H-treated sample is equivalent to 33% of the undigested sample. (D) Pulse labeling (5 min) of HSP90B1 with 35 S-Methionine/ 35 S-Cysteine in HSP90B1 -/- or HSP90B1 -/- ; CCDC134 -/- cells stably expressing 3xFLAG-HSP90B1 was followed by a chase in unlabelled media for 5, 10 or 30 minutes. The ratio of the hyperglycosylated (Hg) to monoglycosylated (Mg) HSP90B1 band intensity is indicated from three independent experiments.

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: Mutagenesis, Isolation, Immunoprecipitation, Labeling, Knock-Out, Stable Transfection, Expressing, Lysis

Journal: bioRxiv

Article Title: Substrate-directed control of N-glycosylation in the endoplasmic reticulum calibrates signal reception at the cell-surface

doi: 10.1101/2024.04.25.591210

Figure Lengend Snippet: (A) Conservation of CCDC134, non-catalytic OST subunits, STT3A/B, HSP90 and endoplasmin (which includes HSP90B1 in mammals) across evolution. Shading indicates the presence of the protein in a specific sub-clade, and a black dot in the HSP90 column indicates the presence of a member of the endoplasmins. The conservation pattern of the pre-N segment in each sub-clade is shown as a sequence logo to the left. The logos are anchored to the start of the HSP90 NTD (the ATP-binding GHKL domain) and the residues are scaled according to the conservation bit score. The concomitant loss of CCDC134, STT3A-specific subunits OSTC and KRTCAP2 that anchor OST-A to the SEC61 translocon, and HSP90B1 in fungi is highlighted with a red arrow. The complete HSP90 phylogenetic tree is shown in fig.S6 . (B) A model for the regulation of HSP90B1 hyperglycosylation (see text for details).

Article Snippet: HSP90B1 constructs: pDONR223_HSP90B1_WT was purchased from Addgene (Plasmid #82130) and Gibson assembly was used to insert a 3xFLAG tag just after the ER signal sequence.

Techniques: Sequencing, Binding Assay