dna sequence encoding copb2 1–304  (GenScript corporation)

Journal: Science Advances

Article Title: Identification of the Wnt signal peptide that directs secretion on extracellular vesicles

doi: 10.1126/sciadv.ado5914

Figure Lengend Snippet: ( A ) SP is not required for EVs-Wnt7a secretion in transfected HEK293T cells. ( B ) Mutant Wnt7a_S206A, lacking the palmitoylation site, is secreted on EVs in transfected HEK293T cells. ( C ) Drug inhibition of PORCN does not affect secretion of Wnt7a on EVs. ( D ) Knockdown of WLS in siRNA partially affects secretion of Wnt7a on EVs but does abolish secretion of non-EV Wnt7a. ( E ) BirA constructs for BioID analysis. ( F ) Heatmap displaying fold change (log 2 scale) of enriched proteins in mass spectrometry (ESP_BirA:BirA and Wnt7a_BirA:BirA). Shown are enrichment of >50% [log 2 (FC) > 0.5849] on EBP and a positive enrichment [log 2 (FC) > 0] on Wnt7a. ( G ) Wnt7a: COPA PLA (red) performed in murine primary myotubes either expressing Wnt7a-BirA or BirA. GM310 in green and 4′,6-diamidino-2-phenylindole (DAPI) in blue. Scale bars, 10 μm. ( H ) Wnt7a:COPA PLA (orange) performed in RPTEC- hTERT1 cells. Wheat Germ Agglutinin (WGA) in green and DAPI in blue. CTR-neg is control without Wnt7a antibody. Scale bars, 10 μm. ( I ) Wnt7a is secreted on EVs derived from RPTEC-hTERT1 cells. HEK293T cells lacking Wnt7a were used as a negative control. ( J ) Wnt7a:COPA PLA (red) performed in HEK293T cells expressing Wnt7a-FL, Wnt7a_ΔEBP*GSGS, or Wnt7a_ΔSP. GM310 in green and DAPI in blue. Scale bars, 10 μm. CTR-neg is control without Wnt7a antibody. ( K ) Wnt7a-HA interacts with COPA and COPB2. HEK293T cells overexpressing Wnt7a-HA were immunoprecipitated with COPB2 antibody or ( L ) immunoprecipitated with HA antibody. ( M ) Immunoblot EVs secretion analysis of Wnt7a after siRNA knockdown of COPA and COPB2 shows disruption of Wnt7a-EV secretion in transfected HEK293T cells. n = 3 biological replicates. IgG, immunoglobulin G; WGA, Wheat Germ Agglutinin.

Article Snippet: The DNA sequence encoding COPB2 1–304 was custom-synthesized with codon optimization for expression in Escherichia coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with an N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Transfection, Mutagenesis, Inhibition, Knockdown, Construct, Mass Spectrometry, Expressing, Control, Derivative Assay, Negative Control, Immunoprecipitation, Western Blot, Disruption

Journal: Science Advances

Article Title: Identification of the Wnt signal peptide that directs secretion on extracellular vesicles

doi: 10.1126/sciadv.ado5914

Figure Lengend Snippet: ( A ) Predicted Wnt7a structure in AlphaFold (blue) with the EBP region highlighted in orange and the three positively charged motifs (red) within the EBP. Note that EBP is a solvent-exposed region. ( B ) Wnt7a-ESP*Scramble mutant maintains the dilysine motif and exhibits no impairment in EV secretion. ( C ) ITC measurements for COPB2 1–304 binding to potential dilysine/arginine motifs within the EBP. WT COPB2 1–304 binds to the LKIKKP subregion. ( D to F ) views of the KxKx motif of Wnt7a bound to COPB2 1–304 . (D) Top view of the WD-repeat domain of COPB 1–304 (green) with the LKIKKP peptide (orange) in ribbon representation. (E) Close-up view of the LKIKKP peptide with a difference electron density map calculated by omitting the peptide and contoured at 3σ (blue mesh). COPB2 surface is colored by electrostatic potential ranging from −5 kT/e (red) to 5 kT/e (blue). (F) Lateral view of the binding motif with hydrogen bonds and distances. ( G ) Structure-based point mutations confirm the molecular recognition of the KxKx motif in ITC assays. ( H ) Double lysine mutation of K253 and K255 by alanine disrupts Wnt7a-EV secretion. ( I ) Replacement of Wnt7a-EBP by either Wnt10a-EBP or Wnt16 EBP containing KR and RR (right). Replacement with Wnt10a-EBP or Wnt16 EBP rescues Wnt7a-EV secretion. ( J ) EV secretion analysis of Wnt10b after EBP removal or double arginine mutation within its EBP (right). Double arginine mutation disrupts secretion of Wnt10b on EVs to the same extent as removal of the entire Wnt10b EBP sequence. ( K ) Secretion analysis of Wnt3a after EBP removal or mutation of the entire positively charged motifs RPR, KHR, and KH within its EBP (right). Only concomitant mutation of RPR and KHR motifs disrupts secretion of Wnt3a on EVS to the same extent as removal of the entire Wnt3a EBP sequence. n = 3 biologic replicates.

Article Snippet: The DNA sequence encoding COPB2 1–304 was custom-synthesized with codon optimization for expression in Escherichia coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with an N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Solvent, Mutagenesis, Binding Assay, Sequencing

Journal: Science Advances

Article Title: Identification of the Wnt signal peptide that directs secretion on extracellular vesicles

doi: 10.1126/sciadv.ado5914

Figure Lengend Snippet: ( A ) Knockdown of COPA and COPB2 using siRNA disrupts Wnt7a-EV secretion in Wnt7a stably expressing primary myoblasts. ( B ) Knockdown of COPA and COPB2 decreases hypertrophy in Wnt7a-transfected myotubes. Conversely, knockdown did not affect hypertrophy in myotubes not expressing Wnt7a. ( C ) Representative images after simultaneously siRNA knockdown of COPA and COPB2 in myotubes. Scale bars, 50 μm. ( D ) Schematic representation of in vivo workflow. ( E ) Myofiber caliber distribution comparing TA electroporated with Wnt7a (green) versus Wnt7a_ΔEBP*GSGS (gray). ( F ) Average minimal fiber feret comparing TA electroporated with Wnt7a (green) versus Wnt7a_ΔEBP*GSGS (gray). ( G ) Quantification of fiber number comparing TA electroporated with Wnt7a (green) versus Wnt7a_ΔEBP*GSGS (gray). ( H ) Quantification of muscle area comparing TA electroporated with Wnt7a (green) versus Wnt7a_ΔEBP*GSGS (gray). ( I ) Section of TA muscles showing reduced myofiber caliber an increased number of myofibers after electroporation of Wnt7a_ΔEBP*GSGS into TA muscle of mdx mice. Scale bars, 100 μm. TA, tibialis anterior. In vitro experiments are representative of three independent biological replicates performed in murine primary myoblasts. In vivo experiments are representative n = 6 mice, means ± SEM. P value was determined by two-sided Student’s t test (* P < 0.05 and ** P < 0.005).

Article Snippet: The DNA sequence encoding COPB2 1–304 was custom-synthesized with codon optimization for expression in Escherichia coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with an N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Knockdown, Stable Transfection, Expressing, Transfection, In Vivo, Muscles, Electroporation, In Vitro

Journal: bioRxiv

Article Title: Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation

doi: 10.1101/2023.05.30.542914

Figure Lengend Snippet: a , Signal peptide is not required for EVs-Wnt7a secretion in transfected HEK293T cells. b, Mutant Wnt7a_S206A, lacking the palmitoylation site, is secreted on EVs in transfected HEK293T cells. c, Drug inhibition of PORCN does not affect secretion of Wnt7a on EVs in transfected HEK293T cells. d, Knockdown of WLS in siRNA in transfected HEK293T cells does not affect secretin of Wnt7a on EVs but does abolish secretion of free Wnt7a. e, BirA constructs designed for the BioID analysis. f , Heat map displaying fold change (log2 scale) of enriched proteins in mass spectrometry versus control conditions (ESP_BirA:BirA and Wnt7a_BirA:BirA). Shown are proteins that present a minimum enrichment of 50% (log2 (FC)>0.5849) on EBP and a positive enrichment (log2 (FC)>0) on Wnt7a. COPI complex subunits are highlighted in red. g , Wnt7a: COPA PLA (red) performed in murine primary myotubes either expressing Wnt7a-BirA or BirA. PLA signal was counterstained with GM310 (green), a Golgi Apparatus marker, and with DAPI (blue). Scale bar 10 μm. h , Wnt7a:COPA PLA (orange) performed in RPTEC-hTERT1 cells. PLA signal was counterstained with WGA (green), a cell membrane marker and with DAPI (blue), showing interaction in the cytosol. CTR-neg is an internal control without Wnt7a antibody. Scale bar 10 μm. i, Wnt7a is secreted on EVs derived from RPTEC-hTERT1 cells. HEK293T cells were used as a negative control for Wnt7a expression. j, Wnt7a:COPA PLA (red) performed in HEK293T cells either expressing Wnt7a-FL, Wnt7a_ΔEBP*GSGS or Wnt7a_ΔSP. PLA signal was counterstained with GM310 (green), a Golgi Apparatus marker, and with DAPI (blue). Scale bar 10 μm. CTR-neg is an internal control without Wnt7a antibody. k , HEK293T cells overexpressing Wnt7a-HA were immunoprecipitated with COPB2 antibody. Wnt7a-HA interacts with COPA and COPB2. l , HEK293T cells overexpressing Wnt7a-HA were immunoprecipitated with HA antibody. Wnt7a-HA interacts with COPA and COPB2. m , Immunoblot EVs secretion analysis of Wnt7a after siRNA knock down of COPA and COPB2 shows disruption of Wnt7a-EVs secretion. Experiments are representative of three independent biological replicates.

Article Snippet: The DNA sequence encoding COPB2 1-304 was custom synthesized with codon optimization for expression in E. coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with a N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Transfection, Mutagenesis, Inhibition, Knockdown, Construct, Mass Spectrometry, Control, Expressing, Marker, Membrane, Derivative Assay, Negative Control, Immunoprecipitation, Western Blot, Disruption

Journal: bioRxiv

Article Title: Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation

doi: 10.1101/2023.05.30.542914

Figure Lengend Snippet: a , Predicted Wnt7a structure in AlphaFold (blue) with the EBP region highlighted in orange and the three positively charged motifs (red) within the EBP. Note that EBP is a solvent-exposed region. b, Wnt7a-ESP*Scramble mutant maintains the dilysine motif and exhibits no impairment in EV secretion. c, Isothermal calorimetry measurements for COPB2 1-304 binding to potential dilysine/arginine motifs within the EBP. Wild-type COPB2 1-304 binds to the LKIKKP sub-region. d-f, views of the KxKx motif of Wnt7a bound to COPB2 1-304 . d , Top view of the WD-repeat domain of COPB 1-304 (green) with the LKIKKP peptide (orange) in ribbon representation. e, Close-up view of the LKIKKP peptide with a difference electron density map calculated by omitting the peptide and contoured at 3σ (blue mesh). COPB2 surface is colored by electrostatic potential ranging from −5kT/e (red) to 5kT/e (blue). f, Lateral view of the binding motif with hydrogen bonds and distances. g, Structure-based point mutations confirm the molecular recognition of the KxKx motif in ITC assays. h , Double lysine mutation of K253 and K255 by alanine disrupts Wnt7a-EVs secretion. i , Replacement of Wnt7a-EBP by either Wnt10a-EBP or Wnt16 EBP containing KR and RR (right panel). Replacement with Wnt10a-EBP or Wnt16 EBP rescues Wnt7a-EVs secretion. j , EV secretion analysis of Wnt10b after EBP removal or double arginine mutation within its EBP (right panel). Double arginine mutation disrupts secretion of Wnt10b on EVs to the same extent as removal of the entire Wnt10b EBP sequence. k , Secretion analysis of Wnt3a after EBP removal or mutation of the entire positively charged motifs RPR, KHR, KH within its EBP (right panel). Only concomitant mutation of RPR and KHR motifs disrupts secretion of Wnt3a on EVS to the same extent as removal of the entire Wnt3a EBP sequence. Experiments are representative of three independent biological replicates performed in HEK293T cells transfected with different Wnt-HA tagged truncates.

Article Snippet: The DNA sequence encoding COPB2 1-304 was custom synthesized with codon optimization for expression in E. coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with a N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Solvent, Mutagenesis, Binding Assay, Sequencing, Transfection

Journal: bioRxiv

Article Title: Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation

doi: 10.1101/2023.05.30.542914

Figure Lengend Snippet: a , Knockdown of COPA, COPB2 using siRNA disrupts Wnt7a-EVs secretion. b , Knock down of COPA, COPB2 decreases hypertrophy in Wnt7a transfected myotubes. Conversely, knock down did not affect hypertrophy in myotubes not expressing Wnt7a. Data shown as fold change of myotube diameter over the siRNA control (%). c , Representative images after simultaneously siRNA knock down of COPA and COPB2 in myotubes. Scale bar 50 μm. d , Schematic representation of in vivo workflow. d , Myofiber caliber distribution comparing TA electroporated with Wnt7a (green) vs Wnt7a_ΔEBP*GSGS (grey). f, Average feret comparing TA electroporated with Wnt7a (green) vs Wnt7a_ΔEBP*GSGS (grey). g, Quantification of fiber number comparing TA electroporated with Wnt7a (green) vs Wnt7a_ΔEBP*GSGS (grey). h, Section of TA muscles showing reduced myofiber caliber after electroporation of Wnt7a_ΔEBP*GSGS into TA muscle of mdx mice. Scale bar 100 μm. Pan myosin heavy chain (pMHC). Tibialis anterior (TA). In vitro experiments are representative of three independent biological replicates performed in murine primary myoblasts. In vivo experiments are representative of six mice per condition. Statistical analysis was performed using two-sided Student’s t-test. Data are mean ± s.e.m. Source (*p<0.05, **p<0.005).

Article Snippet: The DNA sequence encoding COPB2 1-304 was custom synthesized with codon optimization for expression in E. coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with a N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Knockdown, Transfection, Expressing, Control, In Vivo, Muscles, Electroporation, In Vitro