Targeted protein degradation systems to enhance Wnt signaling
- Surrozen, Inc, United States
Figures
Figure 1 with 1 supplement
Biolayer interferometry (BLI) profiles of antigen carbohydrate recognition domains (CRDs) to antibodies.
(A) Binding of hASGR1 to 8M24-IgG1; (B) non-binding of mASGR1, hASGR2, and mASGR2 to 8M24-IgG1; (C) binding of hASGR1 to 8G8-IgG1; (D) binding of hASGR2 to 8G8-IgG1; (E) binding of mASGR1 to …
-
Figure 1—source data 1
Excel file contains raw binding data in Figure 1.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
8M24 is specific to human ASGR1, and 8G8 binds to both ASGR1 and ASGR2 with mouse cross-reactivity.
(A) Interaction of 8M24-Fab with human and mouse ASGR1 and ASGR2 carbohydrate recognition domains (CRDs). The left-shifted peak, corresponding to complex formation, is observed only with hASGR1-CRD. …
-
Figure 1—figure supplement 1—source data 1
Source data files include raw size-exclusion chromatography (SEC)–high performance liquid chromatography (HPLC) data in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig1-figsupp1-data1-v1.zip
Figure 2
Structure of the hASGR1:8M24 complex.
(A) A cartoon representation of the overall structure of the hASGR1CRD:8M24-Fab complex. The heavy and light chains of 8M24 are colored orange and olive, respectively, with their CDR loops marked as …
Figure 3
Comparison of hASGR1:8M24 and hASGR2:8G8 complex structures.
(A) An alignment, made with MultAlin (Corpet, 1988) and ESPript (Gouet et al., 2003) of human, mouse, and rat ASGR1 and ASGR2 sequences with the secondary structure elements of hASGR1CRD (PDB code: …
Figure 4
Structure of the hASGR2:8G8 complex.
(A) A cartoon representation of the overall structure of the hASGR2CRD:8G8-Fab complex. The heavy and light chains of 8G8 are colored in purple and teal, respectively, with their CDR loops marked as …
Figure 5
Both 8M24 and 8G8 RSPO2RA SWEETS molecules enhance Wnt signaling.
(A) Diagrams of the SWEETS molecules. RSPO2RA is fused at the N-terminus of the heavy chain of IgG. (B) Both 8M24 and 8G8 RSPO2RA SWEETS molecules enhance Wnt signaling in HuH-7 STF cells, which has …
-
Figure 5—source data 1
Excel file contains STF raw reading in Figure 5B–D.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
SWEETS induce degradation of ASGR1 in HuH-7 cells.
(A) Dose-dependent ASGR1 degradation promoted by different concentrations of 4F3-, 8M24-, and 8G8-RSPO2RA SWEETS for 24 hr. (B) Time-course of ASGR1 degradation upon treatment with 10 nM 4F3-, …
-
Figure 6—source data 1
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 6A.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-data1-v1.zip
-
Figure 6—source data 2
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 6B.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-data2-v1.zip
-
Figure 6—source data 3
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 6C.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-data3-v1.zip
-
Figure 6—source data 4
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 6D.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-data4-v1.zip
-
Figure 6—source data 5
Excel file contains the quantification of relative ASGR1 levels in Figure 6A right panel, Figure 6B bottom panel, and Figure 6C right panel.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-data5-v1.xlsx
Figure 6—figure supplement 1
ASGR1 degradation promoted by SWEETS was determined in an additional hepatocyte cell line, HepG2 cells.
(A) ASGR1 degradation induced by different concentrations of 4F3-, 8M24-, and 8G8-RSPO2RA showing the dose-response curves of SWEETS. (B) Time-course of ASGR1 degradation treated with 10 nM of 4F3-, …
-
Figure 6—figure supplement 1—source data 1
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 6—figure supplement 1A.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-figsupp1-data1-v1.zip
-
Figure 6—figure supplement 1—source data 2
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 6—figure supplement 1B.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-figsupp1-data2-v1.zip
-
Figure 6—figure supplement 1—source data 3
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 6—figure supplement 1C.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-figsupp1-data3-v1.zip
-
Figure 6—figure supplement 1—source data 4
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 6—figure supplement 1D.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-figsupp1-data4-v1.zip
-
Figure 6—figure supplement 1—source data 5
Excel file contains the quantification of relative ASGR1 levels in Figure 6—figure supplement 1A right panel, Figure 6—figure supplement 1B bottom panel, and Figure 6—figure supplement 1C right panel.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig6-figsupp1-data5-v1.xlsx
Figure 7
SWEETS induce degradation of ASGR1 by recruitment of E3 ubiquitin ligase activity.
(A) Levels of total and immunoprecipitated ubiquitin and ASGR1 in HepG2 cells subjected to ubiquitin immunoprecipitation (IP) following treatment with 10 nM 4F3-RSPO2RA for the indicated time (1, 2, …
-
Figure 7—source data 1
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 7A.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig7-data1-v1.zip
-
Figure 7—source data 2
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 7B.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig7-data2-v1.zip
-
Figure 7—source data 3
Source data files include raw unedited and uncropped blots with the relevant bands clearly labeled shown in Figure 7C.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig7-data3-v1.zip
-
Figure 7—source data 4
Excel file contains STF raw reading in Figure 7D.
- https://cdn.elifesciences.org/articles/93908/elife-93908-fig7-data4-v1.xlsx
Tables
Table 1
Kinetic parameters of ASGR1 and ASGR2 binding to 8M24 and 8G8 antibodies.
| Load sample | Analyte | KD (nM) | KD (M) | kon (1/Ms) | kdis (1/s) |
|---|---|---|---|---|---|
| 8M24-IgG | hASGR1CRD | <1.0E−3 | <1.0E−12 | 7.39E+05 | <1.0E−07 |
| 8G8-IgG | hASGR1CRD | 1.69 | 1.69E−09 | 5.48E+05 | 9.25E−04 |
| 8G8-IgG | mASGR1CRD | 340 | 3.40E−07 | 5.34E+05 | 1.81E−01 |
| 8G8-IgG | hASGR2CRD | 331 | 3.31E−07 | 2.59E+05 | 8.56E−02 |
| 8G8-IgG | mASGR2CRD | 51,500 | 5.01E−05 | 6.91E+03 | 3.46E−01 |
-
Note: 8M24-IgG does not bind to mASGR1, hASGR2, and mASGR2 (Figure 1B).
Table 2
Crystallography structure determination statistics.
| Data collection | hASGR1CRD:8M24 | hASGR2CRD:8G8 |
|---|---|---|
| PDB code | 8TS0 | 8URF |
| Beamline | ALS BCSB 5.0.2 | ALS BCSB 5.0.2 |
| Wavelength (Å) | 0.9999 | 0.9999 |
| Space group | P212121 | H32 |
| Unit-cell dimensions (Å) | a = 38.9, b = 90.3, c = 167.8 | a = b = 102.41, c = 358.98 |
| Matthew’s coefficient (Å3/Da) | 2.17 | 2.71 |
| Solvent content (%) | 43.20 | 54.56 |
| Resolution (Å) | 38.91–1.70 (1.73–1.70) | 38.90–1.85 (1.94–1.90) |
| Number of unique reflections | 66,288 (3439) | 57,655 (3681) |
| Completeness (%) | 100 (100) | 100 (100) |
| CC1/2 (%) | 99.9 (40.7) | 99.9 (44.0) |
| I/σ(I) | 14.7 (0.9) | 16.3 (1.2) |
| Rmeas | 0.107 (2.923) | 0.109 (3.273) |
| Rpim | 0.030 (0.789) | 0.025 (0.718) |
| Multiplicity | 13.0 (13.6) | 19.8 (20.7) |
| Refinement | ||
| Resolution (Å) | 38.05–1.70 (1.73–1.70) | 33.54–1.90 (1.97–1.90) |
| Number of unique reflections | 66,188 (2660) | 57,636 (5730) |
| Number of reflections for Rfree | 3233 (152) | |
| Rcryst (%) | 17.3 | 16.6 |
| Rfree (%) | 20.5 | 20.35 |
| r.m.s.d.’s from ideal values: | ||
| Bond length (Å) | 0.007 | 0.008 |
| Bond angles (°) | 0.870 | 0.940 |
| Average B-factors (Å2): | ||
| Protein | 32.8 | 48.6 |
| Water molecules | 41.0 | 51.6 |
| Ligands | 47.7 | 57.3 |
| Ramachandran plot: | ||
| MolProbity residues in | ||
| Favored region (%) | 97.63 | 98.15 |
| Allowed region (%) | 2.37 | 0.41 |
| Outliers (%) | 0.00 | 0.00 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | Expi293F cells | Thermo Fisher Scientific | A14527 | |
| Cell line (H. sapiens) | HEK293 STF | https://doi.org/10.1371/journal.pone.0009370 | Cells containing a luciferase gene controlled by a WNT-responsive promoter | |
| Cell line (H. sapiens) | Huh7 STF | https://doi.org/10.1038/s41598-020-70912-3 | Cells containing a luciferase gene controlled by a WNT-responsive promoter | |
| Cell line (H. sapiens) | HuH-7 | JCRB Cell Bank | JCRB0403 | |
| Cell line (H. sapiens) | Hep G2 | ATCC | HB-8065 | |
| Antibody | Rabbit polyclonal anti-Human ASGR1 antibody | Thermo Fisher | PA5-80356 | RRID:AB_2787681 1:1000 |
| Antibody | Rabbit polyclonal anti-LC3B antibody | Novus | NB100-2220 | RRID:AB_10003146 1:1000 |
| Antibody | Mouse monoclonal anti-Ubiquitin (eBioP4D1) antibody | Thermo Fisher | 14-6078-82 | RRID:AB_837154 1:500 |
| Antibody | Mouse monoclonal anti-Human Vinculin (V284) antibody | Bio-Rad | MCA465GA | RRID:AB_2214389 1:1000 |
| Antibody | Goat polyclonal anti-rabbit IgG H&L (HRP) | Abcam | ab205718 | RRID:AB_2819160 1:20,000 |
| Antibody | Goat polyclonal anti-mouse IgG H&L (HRP) | Abcam | ab205719 | RRID:AB_2755049 1:20,000 |
| Peptide, recombinant protein | Fc-R-spondin 2 | https://doi.org/10.1038/s41598-020-70912-3 | Produced in Expi293F cells | |
| Commercial assay or kit | Luciferase Assay System | Promega | E1501 | |
| Commercial assay or kit | Mem-PER Plus Membrane Protein Extraction Kit | Thermo Fisher | 89842 | |
| Commercial assay or kit | Pierce Classic Magnetic IP/Co-IP Kit | Thermo Fisher | 88804 | |
| Commercial assay or kit | Rapid Gold BCA Protein Assay Kit | Thermo Fisher | A53225/A53226 | Discontinued at Thermo Fisher, Use alternatives. |
| Recombinant DNA reagent | ASGR1 | GenScript | OHU03658D | Accession No. NM_001671 |
| Recombinant DNA reagent | pAmCyan1-N1 | Takara Bio | 632442 | |
| Chemical compound, drug | IWP2 | Tocris Bioscience | 3533 | Porcupine inhibitor |
| Software, algorithm | Octet Data Analysis 9.0 | Sartorius | https://www.sartorius.com/en/products/biolayer-interferometry/octet-systems-software | RRID:SCR_023267 |
| Software, algorithm | Prism | GraphPad | https://www.graphpad.com/scientific-software/prism/ | RRID:SCR_002798 |
| Software, algorithm | MOE | Chemical Computing Group | https://www.chemcomp.com/ | RRID:SCR_014882 |
| Software, algorithm | PyMol | Schrödinger | https://pymol.org/ | RRID:SCR_000305 |
| Other | cOmplete His-Tag purification resin | Sigma-Aldrich | 5893801001 | Used for protein purification (methods) |
| Other | CaptivA Protein A affinity resin | Repligen | CA-PRI-0100 | Used for protein purification (methods) |
| Other | Superdex 200 Increase 10/300 GL | Cytiva | 28990944 | Used for protein purification (methods) |
| Other | Anti-hIgG Fc Capture (AHC) biosensors | Sartorius | 18-5060 | Used for protein-binding determination (methods) |
