Mice immunizations were performed using the recombinantly expressed and purified extracellular domains of human ASGR1 (hASGR1, residues 62–291) and human ASGR2 (hASGR2, residues 66–292). Hybridoma lines were screened by ELISA against immunogens and two ASGR CRDs, hASGR1CRD (residues 154–291) and hASGR2CRD (residues 177–311). The selected hybridoma lines 8M24 and 8G8 were sequenced, and recombinant Fabs or IgGs were cloned, expressed, and purified for further characterization. Analytical size-exclusion chromatography (SEC) was used to investigate complex formation with both human and mouse ASGR1CRD and ASGR2CRD. Compared with the antigens or 8M24-Fab, an early eluting peak (9.10 min), corresponding to the antigen–Fab complex was observed between 8M24-Fab and hASGR1CRD, but not between 8M24-Fab and mASGR1, hASGR2, or mASGR2 CRDs (Figure 1—figure supplement 1A). In contrast, early eluting peaks at 9.03, 9.50, 9.35, and 9.50 min were observed for 8G8-Fab with all four antigens (Figure 1—figure supplement 1B). These observations suggest that 8M24 binds specifically to hASGR1, while 8G8 is dual specific and binds both ASGR1 and ASGR2.

Furthermore, biolayer interferometry (BLI) was performed to corroborate the SEC results. For BLI, 8M24 and 8G8 IgG1 antibodies were captured on an anti-Human IgG Fc capture (AHC) sensor, and the CRDs of the antigens were used as analytes to determine monovalent affinity. As shown in Figure 1A, 8M24 antibody shows tight binding and specific interaction to hASGR1 with K_D in the sub-picomolar range (Table 1) and does not bind to mASGR1, hASGR2, and mASGR2 (Figure 1B). In contrast, 8G8 antibody binds to all four antigens (Figure 1C–F) with the interaction being strongest for hASGR1, followed by those for hASGR2 and mASGR1, and weakest for mASGR2 (Table 1). Overall, the BLI results agree with those of SEC, establishing that 8M24 antibody is specific to human ASGR1, while 8G8 binds to both human ASGR1 and ASGR2 and is cross-reactive to their mouse counterparts. Subsequently, we pursued crystal structure determination of hASGR1:8M24 and hASGR2:8G8 complexes to gain molecular insights into their specificities.

The hASGR1CRD:8M24 complex crystallizes in the P2₁2₁2₁ space group, with one complex molecule per asymmetric unit. The refined structure of hASGR1CRD:8M24 complex, determined at a resolution of 1.7 Å, shows good stereochemistry with R_cryst and R_free factors of 17.3% and 20.5%, respectively (Table 2). The overall structure of hASGR1CRD:8M24 is shown in Figure 2A. The structure of ASGR1CRD from its 8M24 complex can be superimposed onto apo-hASGR1CRD (PDB code: 1DV8; Meier et al., 2000) with a root mean square deviation (r.m.s.d.) of 0.44 Å over 128 C^∝ atoms, revealing that the binding of 8M24 to hASGR1 does not induce any significant conformational changes of the antigen. The binding site of 8M24 on hASGR1 is distant from the substrate-binding site marked by glycerol, used as a cryoprotectant while freezing crystals, bound to the Ca²⁺ ion (Figure 2A, B). Upon complex formation, both hASGR1 and 8M24 bury an average surface area of 792 Ų, and epitope residues of 8M24 on hASGR1 that are within 4.5 Å from the antibody form a close-knit cluster (Figure 2B).

The strong interaction between hASGR1 and 8M24, which has sub-picomolar affinity, is predominantly contributed to by residues from the helix ∝1 and those flanking ∝1 (Figure 2C), with residues from the β6 strand determining the specificity of 8M24 for hASGR1 over ASGR2 (Figure 3A). Lys173 of hASGR1, near the N-terminus of the helix ∝1, forms a hydrogen bond with the main-chain carbonyl oxygen of Thr105 from the HCDR3 loop of 8M24 (Figure 2D). Lys173 also interacts strongly with Asp177 within the helix ∝1 of hASGR1, positioning later for hydrogen bonding interaction with Asn32 from the LCDR1 of 8M24. Furthermore, this Asn32 (LCDR1) also interacts with the sidechain of Ser106 from HCDR3. hASGR1 Lys173 is conserved in hASGR2 and substituted with Arg and Leu in mASGR1 and mASGR2, respectively (Figure 3A). Introduction of the longer-polar residue Arg or hydrophobic Leu would likely disrupt the structural conformation of closely juxtaposed interacting residues near the N-terminus of the ∝1 helix (Figure 2D) and thus might explain lack of interaction between 8M24 and mASGR1 and mASGR2.

Toward the C-terminus of hASGR1 ∝1 helix, conserved or similar residues Leu184, Glu185, and Asp186 are involved in strong hydrogen bonding interactions with 8M24 (Figures 2E and 3A). Conserved Glu185 forms ionic and hydrogen bonds with Arg101 and His107 from the HCDR3 of 8M24, and hASGR1 Asp186 forms similar interactions with Arg54 and Asn57 of HCDR2 (Figure 2E).

Detailed structural analyses reveal that, in addition to the above mentioned Lys173, Asn180 may also contribute to the specificity of 8M24 for human ASGR1 (Figure 3B). The sidechain amide-nitrogen Asn180 of hASGR1 forms a strong hydrogen bond with the mainchain peptide carbonyl-oxygen of Phe91 from the LCDR3 of 8M24. Asn180 also forms a C–H=O hydrogen bond with the C^∝ atom of Trp92 from the LCDR3 of 8M24. Furthermore, the sidechain of Asn180 fits snugly into a cavity formed by the Phe91–Trp92–Gly93 (LCDR3 loop) and Asn32 (LCDR1 loop) of 8M24, as can be seen from the tight overlap of atomic surfaces formed by these residues (Figure 3B). hASGR1 Asn180 is replaced by residues with longer sidechains, such as lysine in mASGR1 and hASGR2, and glutamine in mASGR2. It is likely that residues with longer sidechains are not well accommodated at this position given the tight juxtaposition of Asn180 against the Phe91–Trp92–Gly93 (LCDR3 loop) and Asn32 (LCDR1 loop) of 8M24.

Other critical sequence differences in ASGR1 and ASGR2 are noted near the C-terminus of their CRDs (Figure 3A). The Thr279 and Leu281 residues, conserved in ASGR1, are replaced in ASGR2 by residues with longer, positively charged sidechains, such as arginine and lysine. The sidechain hydroxyl of hASGR1 Thr279 forms a hydrogen bond with the Arg54 from the HCDR2 of 8M24 (Figure 2E). It is likely that the positively charged residues of hASGR2 and mASGR2 near the C-terminus of their CRDs are detrimental for their interaction with 8M24 and may thus contribute toward the specificity of 8M24 for the human ASGR1.

The hASGR2CRD:8G8 complex crystallizes in the H32 space group, with one complex molecule per asymmetric unit. The structure of the hASGR2CRD:8G8 complex was determined by the molecular replacement method at a resolution of 1.9 Å, and refined to R_cryst and R_free factors of 16.6% and 20.4%, respectively, with good stereochemistry (Table 2). The overall structure of hASGR2CRD:8G8 is shown in Figure 4A. The structures of hASGR2CRD (determined here) and apo hASGR1CRD (PDB code: 1DV8; Meier et al., 2000) could be superimposed with an r.m.s.d. of 0.74 Å over 125 C^∝ atoms and a sequence identify of 65%. Thus, the overall fold of ASGR2CRD – made up of six β-strands flanked by two ∝-helices on either side – and three Ca²⁺ ions, which are important for structural stability, are both conserved between the apo hASGR1 and hASGR2 CRDs. This suggests that the binding of 8G8 to ASGR2 does not induce any significant conformational changes in the antigen. Furthermore, the overall structures of the hASGR1 and hASGR2 CRDs from the respective 8M24 and 8G8 complexes can be superimposed on each other with r.m.s.d. values of 0.85 Å over 126 C^∝ atoms. Such a superposition reveals that both the 8G8 and 8M24 binds away from the ASGR2 and ASGR1 substrate-binding sites with non-overlapping interaction surfaces, on opposite sides of the antigens, around the helices ∝2 and ∝1, respectively (Figure 3C).

8G8 binds to hASGR2CRD between the β1, β2, and β6 strands and the helix ∝2, away from the substrate-binding site marked by glycerol bound to the Ca²⁺ ion (Figure 4A, B). Upon complex formation, both hASGR2 and 8G8 bury an average surface area of 663 Ų, and epitope residues of 8G8 on hASGR2 that are within 4.5 Å from the antibody form a close-knit cluster (Figure 4B). Among the 17 epitope residues, 5 out of 6 from the β1 strand, 2 out of 3 from the β2 strand, 3 out of 7 from the ∝2 helix, and the Arg297 from the β6 strand are conserved or similar between human, mouse, and rat ASGR1 and ASGR2 (Figure 3A). The hASGR2CRD epitope residues Glu183, Lys222, Gln226, His227, Asn229, and Arg297 are involved in hydrogen bonding/ionic interactions with paratope residues from the LCDR3, HCDR2, and HCDR3 of 8G8 (Figure 4C). Among these, Glu183, His227, and Arg297 are conserved in human and mouse ASGR1 and ASGR2 (Figure 3A), and they are therefore unlikely to contribute toward wide-ranging affinities with the 8G8 antibody (Table 1). Asn229, a glycine in h/mASGR1 and a serine in mASGR2, interacts with the heavy-chain Ser57 only through its main-chain peptide carbonyl oxygen (Figures 3A and 4E). The side-chain nitrogen of Gln226 is involved in hydrogen bonding interactions with Gln50 (HCDR2) and Thr94 (LCDR3; Figure 4E). Gln226 of ASGR2 is diversely substituted by His, Arg, and Lys, respectively, in hASGR1, mASGR1, and mASGR2 (Figure 3A). The low-affinity (Table 1) binding to mASGR2 may be explained by interaction with Lys222 of hASGR2, which is conserved in hASRG1 (residue Lys200) but substituted by Asn and Asp residues in mASGR1 and mASGR2, respectively (Figure 3A). In addition, Lys222 forms strong ionic and hydrogen bonds with Asp93 of LCDR3 (Figure 4D). Therefore, an Asp substitution will likely reduce the affinity of mASGR2:8G8 interaction significantly.

Thus, the high-resolution crystal structures have provided molecular insights into the specificities of 8M24 and 8G8 antibodies as well as revealing that their epitopes on ASGR are non-overlapping and away from the glycoprotein-binding site on the receptor.

RSPO proteins bind to ZNRF3/RNF43 and LGRs via their Furin Fu1 and Fu2 domains, respectively. We have previously engineered a F105R/F109A mutant of human RSPO2 (RSPO2RA), which shows abolished binding to LGRs (Zhang et al., 2020). Fusion of RSPO2RA to the anti-ASGR1 antibody 4F3 (4F3-RSPO2RA, also termed 4F3-SWEETS), which binds to the stalk region of ASGR1 outside of the CRD, leads to hepatocyte-specific RSPO mimetic activity (Zhang et al., 2020).

Since the binding sites of 8M24 and 8G8 located on the CRD of ASGR are different from that of 4F3, RSPO2RA fusions of 8M24 and 8G8 were constructed to examine the effectiveness of these new ASGR epitopes on RSPO mimetic activity. RSPO2RA is fused to the N-terminus of 8M24 and 8G8 heavy chains using a 15-mer (GGGGS)x3 linker, resulting in 8M24-RSPO2RA and 8G8-RSPO2RA (Figure 5A). These two new 8M24- and 8G8-SWEETS molecules also showed robust enhancement of Wnt signaling activity in the presence of a synthetic Wnt source, comparable to that of 4F3-RSPO2RA SWEETS in human hepatocyte HuH-7 cells (Figure 5B).

To demonstrate that the RSPO mimetic activity depends on ASGR expression, 8M24-RSPO2RA and 8G8-RSPO2RA were then tested in HEK293 cells, which do not express ASGR. As shown in Figure 5C, parental HEK293 cells without ASGR1 overexpression, transfected with an empty negative control vector, respond weakly to the different protein treatments, and no Wnt signaling enhancement is observed by 4F3-RSPO2RA, 8M24-RSPO2RA, or 8G8-RSPO2RA above the negative control protein, αGFP-RSPO2RA. In contrast, when HEK293 cells transfected with the ASGR1 expression vector, all three ASGR1-targeted SWEETS molecules show much higher potency than αGFP-SWEETS in enhancing Wnt signaling (Figure 5D). These results revealed that the two new epitopes on the CRD of the ASGR receptor, bound by 8M24 and 8G8, also serve as effective epitopes to facilitate clearance of E3 ligase from the cell surface, leading to enhanced Wnt signaling.

While our initial goal was to develop liver-specific RSPO mimetic SWEETS molecules to enhance Wnt signaling by ASGR-mediated elimination of E3 ligases, we also wanted to understand whether these bispecific SWEETS molecules have any effect on ASGR1 protein levels. In particular, we wondered if juxtaposing E3 ligases to ASGR by SWEETS binding may induce ubiquitination and subsequent degradation of ASGR proteins as an unintended consequence. Whole-cell extracts from HuH-7 and another hepatocyte cell line, HepG2, treated with various concentrations of the three SWEETS molecules were subjected to western blot analysis with a commercially available anti-ASGR1 antibody (Figure 6A and Figure 6—figure supplement 1A). The total amount of ASGR1 was reduced in a dose dependent manner after treatment with SWEETS molecules, with 4F3-RSPO2RA and 8M24- RSPO2RA being more potent than 8G8-RSPO2RA. ASGR1 degradation induced by SWEETS molecules was very effective, with only ∼20% of the western blot signal remaining at higher concentrations (>1 nM) compared with the untreated samples (Figure 6A and Figure 6—figure supplement 1A).

Next, we evaluated the kinetics of the SWEETS-mediated ASGR1 degradation. As shown in Figure 6B and Figure 6—figure supplement 1B, the onset of the degradation is fairly rapid, with ~40% reduction being observed just 4 hr post-treatment (for 4F3- and 8M24-SWEETS), and maximal reduction achieved by 8 hr. Treatments with the anti-ASGR1 antibodies lacking the RSPO2RA domain did not result in the same level of ASGR1 reduction, suggesting that the SWEETS-induced proximity of E3 ligase to ASGR1 indeed facilitates ASGR1 degradation (Figure 6C and Figure 6—figure supplement 1C).

To determine the route of ASGR1 degradation, we assessed the effect of the autophagy–lysosomal pathway inhibitor bafilomycin A1 and the proteasomal pathway inhibitor MG132 on ASGR1 degradation promoted by SWEETS. As shown in Figure 6D and Figure 6—figure supplement 1D, either bafilomycin A1 or MG132 treatment significantly impairs ASGR1 degradation, suggesting that SWEETS promotes ASGR1 degradation through both lysosomal and proteasomal pathways. Since these two main degradation pathways have been shown to be linked (because blocking one activates the other) (Ding et al., 2007; Korolchuk et al., 2009), in addition to assessing the ASGR1 degradation, we examined whole protein ubiquitination and LC3B lipidation to monitor whether each pathway was compromised by inhibitors. Only the MG132-treated group shows a significant increase in whole protein ubiquitination, whereas only the bafilomycin-A1-treated group shows accumulation of autophagosome marker LC3B-II, demonstrating that the inhibitors have little effect on the other degradation pathway (Figure 6D and Figure 6—figure supplement 1D). Moreover, treatment with the ubiquitin-activating enzyme inhibitor (E1 ligase inhibitor) TAK-243 results in a reduction of ASGR1 degradation, implying that the catalytic function of E3 ligases is involved in SWEETS-mediated ASGR1 degradation (Figure 6D and Figure 6—figure supplement 1D).

To determine whether ASGR1 is indeed ubiquitinated with SWEETS treatment, we next examined the ubiquitination of ASGR1 by ubiquitin immunoprecipitation (IP). To determine the best timepoint to illustrate ASGR1 ubiquitination, a time-course ubiquitination assay was conducted following 4F3-RSPO2RA incubation for 1, 2, 4, and 6 hr. As shown in Figure 7A, 4F3-RSPO2RA treatment results in enhanced ASGR1 ubiquitination in HepG2 cells, and ASGR1 ubiquitination increases with treatment time, reaching its maximum at 2 hr.

Next, to examine if ASGR1 is ubiquitinated by all three SWEETS treatments, we incubated HepG2 cells with 4F3-, 8M24- or 8G8-RSPO2RA for 2 hr. As shown in Figure 7B, all three SWEETS treatments promote ASGR1 ubiquitination over the negative control groups. To further demonstrate that ubiquitination contributes to ASGR1 degradation, we created an ASGR1 mutant (mut ASGR1) in which all the lysine residues in the cytoplasmic domain of ASGR1 are changed to arginine (K3R and K23R) to inhibit potential ubiquitination of ASGR1. Comparing the degradation of mut ASGR1 and wild-type ASGR1 following SWEETS treatment, mutating lysine residues in the cytoplasmic domain of ASGR1 significantly impair the degradation of ASGR1 (Figure 7C). Importantly, mutating lysine residues in the cytoplasmic domain of ASGR1 does not disrupt E3 ligase clearance and Wnt signaling enhancement activity of SWEETS molecules (Figure 7D).

Collectively, our data demonstrate that all three SWEETS molecules can act as a TPD platform to degrade not only E3 ligases but also to degrade ASGR through both lysosomal and proteasomal pathways.

Expi293F cells were grown in Expi293 Expression Medium (Thermo Fisher Scientific) at 37°C with humidified atmosphere of 8% CO₂ in air on an orbital shaker. HEK293 STF cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Fisher Scientific) at 37°C in a 5% CO₂ environment. Both HuH-7 STF and HuH-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum and non-essential amino acids (Fisher Scientific) at 37°C in a 5% CO₂ environment. HepG2 cells were maintained in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum at 37°C in a 5% CO₂ environment. Cell line authentications were performed at American Type Culture Collection (ATCC) by STR profiling, mycoplasma testing by PCR Mycoplasma detection kit.

All proteins were cloned into pcDNA3.1(+) vector with the human kappa light-chain secretion signal sequence at the N-terminus and transiently expressed in Expi293 cells using the FectoPro transfection agent (Polyplus Transfection, NY, USA). In 4F3-RSPO2RA, 8M24-RSPO2RA, and 8G8-RSPO2RA SWEETS molecules, the F105R/F109A mutant of human RSPO2 furin domain was fused at the N-terminus of the heavy chain of the respective antibodies via a 15-mer (G4S) × 3 linker. hASGR1, mASGR1, hASGR2, mASGR2, 8M24-Fab, and 8G8-Fab were expressed with His-tag and purified using cOmplete His-Tag purification resin (Roche, USA) following standard protocols. 4F3, αGFP, 8M24, and 8G8 antibodies and 4F3-RSPO2RA, 8M24-RSPO2RA, and 8G8-RSPO2RA SWEETS were purified using either CaptivA resin (Repligen, USA) under gravity-flow or using a prepacked MabSelect SuRe (Cytiva, USA) column attached to an AKTA FPLC. All proteins were further polished on a Superdex 200 size-exclusion chromatography column equilibrated with 2× HBS (HEPES-Buffered Saline) (40 mM HEPES pH 7.5, 300 mM sodium chloride) buffer.

For the BLI assays, the 8M24 and 8G8 antibodies were prepared at 50 nM in 1× phosphate buffer saline + (0.05%) (PBST) + 0.5 mg/ml bovine serum albumin (assay buffer) (Teknova P1192, Fisher BP1600) and captured on Anti-hIgG Fc Capture (AHC) biosensors (Sartorius 18-5060). Capture was recorded for 40 s. Association was measured by transferring the IgG-loaded sensors to wells containing CRDs of hASGR1, mASGR1, hASGR2, and mASGR2 at a threefold dilution series from 1 µM to 4 nM for 120 s. Dissociation was measured by placing sensors in wells containing only assay buffer for 150 s. Assays were performed in duplicate. Kinetic parameters were determined using instrument analysis software to fit a global 1:1 kinetic model with Rmax linked across all concentrations.

Purified hASGR1CRD and hASGR2CRD were mixed with the 8M24 and 8G8 Fabs, respectively, at 1.1:1 molar ratio and incubated with carboxy-peptidase A and B at a wt/wt ratio of 100:1 overnight at 4°C. Complex formation was confirmed by observation of a single major peak on a SuperdexS200 Increase (10/300 GL) column pre-equilibrated in 20 mM HEPES pH 7.5 and 150 mM sodium chloride, and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Initial crystallization screening (hASGR1CRD:8M24 at 20 mg/ml and hASGR2CRD:8G8 at 28 mg/ml) using commercially available reagents was performed using Mosquito (TTP LabTech) liquid handler and equilibrated at 18°C inside an EchoTherm incubator (Torrey Pines Scientific, USA). Crystallization conditions were further optimized by grid-screens or microseed matrix screens (D’Arcy et al., 2014). Diffraction-quality crystals of the hASGR1CRD:8M24 complex grew in buffer containing 100 mM SPG (succinic acid, sodium phosphate monobasic monohydrate, glycine buffer) at pH 9.0 and 25% wt/vol PEG 1500. Crystals were cryo-protected using 20% glycerol in the well solution. Diffraction-quality crystals of the hASGR2CRD:8G8 complex grew in buffer containing 100 mM sodium HEPES at pH 7.5, 100 mM calcium chloride, and 30% (wt/vol) PEG 400. Crystals were cryo-protected using 16% glycerol in the well solution. X-ray diffraction datasets were collected at the Berkeley Center for Structural Biology at the Advanced Light Source (ALS), Berkeley, CA, and processed with XDS (Kabsch, 2010) and xdsme (Legrand, 2017) programs. Structures were determined by the molecular replacement method using Phaser (McCoy et al., 2007) with a poly-alanine model of ASGR1CRD (PDB code: 5JPV; Sanhueza et al., 2017) and variable and constant domains Fab, followed by refinement using Phenix (Liebschner et al., 2019) and validation by MolProbity (Williams et al., 2018). Crystallography models were manually inspected and built using COOT. Analyses of refined crystal structures and image creation were performed using MOE (CCG, https://www.chemcomp.com/) and PyMol (https://pymol.org/).

Wnt-signaling activity was measured using cell lines containing a luciferase gene controlled by a Wnt-responsive promoter (STF reporter) as previously reported (Janda et al., 2017). In brief, cells were seeded in 96-well plates at a density of 10,000 per well 24 hr prior to treatment, then treated with various proteins overnight in the presence of 100 pM Wnt mimetic FA-L6 (Fowler et al., 2021). Cells were lysed with Luciferase Cell Culture Lysis Reagent (Promega, Madison, WI) and activity was measured with Luciferase Assay System (Promega, Madison, WI) using vendor-suggested procedures. Data were plotted as average −/ + standard deviation of triplicate and fitted by non-linear regression using Prism (GraphPad Software, San Diego, CA). For overexpression of exogenous receptors, cells were transiently transfected with plasmids containing receptors of interest under eukaryotic expression promoters (ASGR1 was clone OHU03658D from GenScript, negative control pAMCyan1 from Takara), then split into 96-well plates (20,000 cells per well) for SuperTop Flash (STF) assay 24 hr post-transfection.

For the ASGR1 degradation assay, HuH-7 and HepG2 cells at a density of 1 × 10⁵ cells per well (24-well plate) were plated a day before the experiment, then treated with 10 nM 4F3-RSPO2RA, 8M24-RSPO2RA, 8G8-RSPO2RA, 4F3-IgG, 8M24-IgG, 8G8-IgG, or the control (αGFP-RSPO2RA) for 24 hr. For the dose–response experiments, indicated concentrations of SWEETS or controls were treated for 24 hr. For the time-course experiments, each SWEETS molecule (10 nM) was incubated for the indicated duration. For the ASGR1 degradation comparison assay between wild-type ASGR1 transfection and mutant ASGR1 transfection, HEK293 STF cells at a density of 1 × 10⁶ cells per well (6-well plate) were plated a day before the experiment. The cells were transfected with vectors containing either wild-type ASGR1 or mutant ASGR1 using the Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000008). Cells were split into five wells (12-well plate) 30 hr after transfection and subjected to different treatment conditions (10 nM 4F3-RSPO2RA, 8M24-RSPO2RA, 8G8-RSPO2RA, or the controls, i.e., fresh cell media or 10 nM αGFP-RSPO2RA) for 24 hr. When lysosomal pathway inhibitor bafilomycin A1 (10 nM, Millipore Sigma 1661), proteasome inhibitor MG132 (1 µM, Selleck Chemicals S2619), E1 ubiquitin ligase inhibitor TAK-243 (50 nM, Selleck Chemicals S8341), or DMSO (as the control vehicle) was treated, inhibitors were pre-treated for 18 hr, 10 nM of SWEETS or control (αGFP-RSPO2RA) was then introduced for 6 hr in the presence of inhibitors or DMSO. Following incubation, cells were washed in ice-cold TBST and lysed in RIPA (Radioimmunoprecipitation Assay Buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS)) buffer (Thermo Fisher, 89900) supplemented with 1% Halt protease inhibitor (Thermo Fisher, 87786), 1% Halt phosphatase inhibitor (Thermo Fisher, 78420), and 0.1% Benzonase (Millipore Sigma, E1014) on ice for 20 min. Whole-cell lysates were prepared by collecting supernatants after centrifugation at 14,000 × g for 20 min at 4°C. Protein concentration was determined by BCA assay (Thermo Fisher, A53225/A53226). Samples were prepared by mixing with 4× Laemmli Sample Buffer (Bio-Rad, 1610747) supplemented with BME (2-mercaptoethanol) and incubated for 5 min at 95°C. Samples prepared with equal amounts of proteins were run on 10% Mini-PROTEAN TGX Precast Gels (Bio-Rad, 4561033) (for ASGR1 blot) or 4–20% Mini-PROTEAN TGX Precast Gels (Bio-Rad, 4568093) (for Ubiquitin and LC3B blot) using Tris/Glycine/SDS running buffer (Bio-Rad, 1610732). Gels were transferred to nitrocellulose membranes (Bio-Rad, 1704158EDU) using the Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked with SuperBlock T20 (TBS) Blocking Buffer (Thermo Fisher, 37536) for 1 hr at room temperature. Following blocking, the membranes were incubated with primary antibodies overnight at 4°C. Blots were washed three times with TBST for 5 min each and subsequently incubated with horseradish peroxidase (HRP) secondary antibodies for 1 hr at room temperature. Blots were then washed three times with TBST for 5 min each, developed using enhanced chemiluminescent (ECL) HRP substrates (Pierce, 34580), and visualized using ChemiDoc Imaging System (Bio-Rad). Image Lab (Bio-Rad) was used to quantify the signal intensities of the bands.

ASGR1 ubiquitination with antibody treatment was evaluated in HepG2 cells. Cells (3 × 10⁶) were plated in 10 cm dishes a day before the experiment. For ubiquitination time-course experiments, cells were treated with 10 nM 4F3-RSPO2RA or the controls (fresh media or 10 nM αGFP-RSPO2RA) at 37°C for the indicated duration. For ASGR1 ubiquitination experiments, cells were treated with 10 nM 4F3-RSPO2RA, 8M24-RSPO2RA, 8G8-RSPO2RA, or the controls (fresh media or 10 nM αGFP-RSPO2RA) at 37°C for 2 hr. Following treatment, membrane proteins were extracted using the appropriate kit (Thermo Fisher, 89842) following the manufacturer’s instructions with adjustment. In brief, cells were scraped and resuspended in growth media, then harvested by centrifugation at 300 × g for 5 min. Cell pellets were washed twice in 1.5 and 0.75 ml of Cell Wash Solution and collected by centrifugation at 300 × g for 5 min. Cytosolic proteins were extracted by incubating the cell pellet with 0.75 ml Permeabilization Buffer for 10 min at 4°C with constant mixing. The supernatant containing cytosolic proteins was removed from the permeabilized cells after centrifuging for 15 min at 16,000 × g. Membrane proteins were extracted by incubating the cell pellet with 0.6 ml Solubilization Buffer for 1 hr at 4°C with constant mixing. The mixture was centrifuged at 16,000 × g for 15 min at 4°C, and the membrane-associated proteins were transferred to a new tube followed by protein concentration determination by BCA assay (Thermo Fisher, A53225/A53226). For ubiquitin IP at 3.4 mg per sample, IP/Co-IP kit (Thermo Fisher, 88804) was used, and the membrane protein lysates were incubated with 10 μg mouse monoclonal anti-ubiquitin (eBioP4D1) antibody (Invitrogen, 14-6078-82) at 4°C overnight. The immune complex samples were incubated with 25 μl prewashed Pierce Protein A/G Magnetic Beads per sample for 1 hr at room temperature. The beads were washed twice with 500 µl IP Lysis/Wash Buffer and once with 500 µl of ultra-pure water following incubation, and proteins were eluted with 28 µl of Laemmli Sample Buffer (Bio-Rad, 1610747). For IP antibody control, 1 µl of each elution sample was supplemented with BME and prepared for western blot analysis. For input samples, 0.34 µg were used from the prepared IP samples prior to IP antibody incubation for western blot analysis.

All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Detailed information about statistical analyses is stated in the figure legends.