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) Screening strategy used to identify positive regulators of WNT signaling (see text for details). (B) Volcano plot of CRISPR/Cas9 screen results. Each circle is one gene, the x-axis shows its enrichment or depletion calculated as the mean of all four sgRNAs targeting the gene in the bottom 10% sorted population relative to the unsorted population, and the y-axis shows statistical significance as measured by p -value. Genes that are the focus of this study are highlighted in color. CTNNB1 , which encodes β-catenin, is highlighted as a positive control. Full screen results are provided in Data S1 . (C) WNT-EGFP reporter fluorescence (+/- WNT3A conditioned media treatment) in RKO (left) or HEK293T cells (right) expressing a non-targeting control sgRNA (NTC) or sgRNAs (different from those used for the CRISPR/Cas9 screen in 1B ) targeting selected screen hits from ( B ). Each data point represents median WNT-EGFP reporter fluorescence from one population and bars show the mean of three populations. Statistical significance was determined by one-way ANOVA Sidak’s multiple comparisons test; **** p<0.0001 ( n =3, ∼3300 cells each). (D, E) Active (non-phosphorylated) β-catenin abundance (a metric of WNT signaling strength) was measured (+/- WNT3A) using immunoblots in wild-type cells or clonal cell lines expressing a control (NTC) sgRNA or an sgRNA targeting CCDC134 . The graph in ( E ) shows active β-catenin abundance normalized to α-tubulin abundance in three independently derived clonal cell lines (C1-C3, shown in D ), with the bar representing the mean. Statistical significance was determined by one-way ANOVA Dunnett’s multiple comparisons test; * p<0.05. (F) Active β-catenin abundance (+/- WNT3A) in clonally derived control (NTC) and CCDC134 -/- cell lines stably expressing near endogenous levels of 3xHA-CCDC134 (see ) carrying an N-terminal ER signal sequence (+SS) or, as a control, lacking a signal sequence (-SS) to prevent ER targeting. Numbers below the lanes show the WNT3A-induced fold-increase in active β-catenin abundance normalized to α-Tubulin abundance. (G) LRP6 and LRP5 abundances in total lysate (6% input) or the plasma membrane (cell surface biotinylation and streptavidin immunoprecipitation, 33% elution) from three independently derived (C1- C3) control (NTC) or CCDC134 -/- clonal cell lines. The cell surface protein Na/K ATPase and cytoplasmic protein α-tubulin serve as controls, both for loading and for the specificity of cell surface biotinylation. The ER population and the cell surface population of LRP6 are visible as bands of slightly different mobilities on immunoblots (see 1H ). (H) Glycosidase sensitivity in conjunction with mobility on SDS-PAGE gels was used to measure the ER or cell-surface pools of LRP6 in control or CCDC134 -/- cells. Endoglycosidase H (Endo H) can remove glycans added in the ER but not the complex glycan modifications added in the Golgi; Peptide- N - Glycosidase F (PNGase F) can remove glycans on both ER and cell-surface proteins. See also fig.S1.

Article Snippet: The following primary antibodies were used: mouse anti- CCDC134 (E-5, Santa Cruz Biotechnology, 1:500); mouse anti-HSP90B1 (H-10, Santa Cruz Biotechnology, 1:2000); mouse anti-LRP5 (B-9, Santa Cruz Biotechnology, 1:500); rabbit anti-LRP6 (C5C7, Cell Signaling Technology, 1:1000 for immunoblot); mouse anti-LRP6 ectodomain (clone A59, MilliporeSigma, 2ug/sample for cell surface staining); Alexa Fluor® 488 rabbit anti-Giantin (A488-114L, Covance, 1:500 for immunofluorescence); mouse anti-ɑ-Tubulin (Clone DM1A, MilliporeSigma, 1:10000); rabbit anti-active (non-phosphorylated) β-catenin (D13A1, Cell Signaling Technology, 1:500); rabbit anti- Na/K ATPase (3010S, Cell Signaling Technology, 1:1000); rabbit anti-MESD (10958-1-AP, Proteintech, 1:1000); mouse anti-FLAG (clone M2, MilliporeSigma, 1:2000); rabbit anti-FLAG (F7425, Sigma-Aldrich, 1:2000); rabbit anti-PSAP (GTX101064, GeneTex, 1:1000); rabbit anti-STT3A (12034-1-AP, Proteintech, 1:1000); rabbit anti-STT3B (15323-1-AP, Proteintech, 1:1000); rabbit anti-OSTC (PA5-34060, Invitrogen, 1:1000); mouse anti-RPL17 (C-8, Santa Cruz Biotechnology, 1:2000); rabbit anti-Sec61b (15087-1-AP, Proteintech, 1:1000).

Techniques: CRISPR, Positive Control, Fluorescence, Expressing, Control, Western Blot, Derivative Assay, Stable Transfection, Sequencing, Clinical Proteomics, Membrane, Immunoprecipitation, SDS Page, Glycoproteomics