{"gene":"EDEM3","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2006,"finding":"EDEM3 has alpha1,2-mannosidase activity in vivo, accelerating mannose trimming from misfolded glycoproteins and total glycoproteins. Mutation of the conserved active-site residue E147Q abolishes mannose trimming and greatly reduces ERAD stimulation, demonstrating that mannosidase activity is required for EDEM3's ERAD function.","method":"Transfection of HEK293 cells with wild-type and E147Q catalytic mutant EDEM3; degradation assays of misfolded alpha1-antitrypsin NHK and TCRα; mannose trimming assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — active-site mutagenesis combined with functional ERAD and mannose-trimming assays in a single focused study, clear mechanistic dissection","pmids":["16431915"],"is_preprint":false},{"year":2018,"finding":"ERp46, an ER-resident oxidoreductase, stably associates with EDEM3 via a disulfide bond between ERp46 redox-active cysteine residues and the EDEM3 alpha-mannosidase domain. This covalent interaction is required for EDEM3 mannose-trimming activity toward misfolded TCRα substrate, as reconstituted in a defined in vitro system with purified recombinant proteins.","method":"Co-immunoprecipitation, in vitro reconstitution with purified recombinant proteins from HEK293 cells, disulfide bond analysis, mannose-trimming activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins plus disulfide bond mapping, single lab but multiple orthogonal methods","pmids":["29784879"],"is_preprint":false},{"year":2021,"finding":"Purified EDEM3 alone is the major alpha1,2-mannosidase responsible for the second step of N-glycan trimming in gpERAD, converting M8B to M7 (M7A and M7C), M6, and M5 oligosaccharides. EDEM3 also efficiently trims M8B from intact glycoproteins.","method":"In vitro mannosidase assay with purified recombinant EDEM3 and pyridylamine-labeled M8B glycan substrates; glycoprotein substrate trimming assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro enzymatic activity with purified protein and defined substrates, functional glycoprotein assay, peer-reviewed","pmids":["34698634"],"is_preprint":false},{"year":2021,"finding":"EDEM3 consists of four functional domains: GH47 (mannosidase), intermediate (IMD), protease-associated (PA), and intrinsically disordered (IDD). The GH47 domain mediates substrate binding even without mannose trimming; IMD is required for GH47 folding; PA domain positively modulates ERAD of specific substrates; IDD domain negatively modulates substrate turnover, providing unique ERAD timing.","method":"EDEM3 knockout cell line complemented with domain-deletion mutants; ERAD substrate degradation assays for NHK and soluble tyrosinase mutant; LC/MS interactome analysis","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO rescue with domain mutants and defined phenotypic readouts, single lab, multiple mutants tested","pmids":["33671632"],"is_preprint":false},{"year":2021,"finding":"Loss-of-function EDEM3 variants in humans cause a congenital disorder of glycosylation (EDEM3-CDG) characterized by decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, and impaired Man5GlcNAc2-to-Man4GlcNAc2 conversion with accumulation of Glc1Man5GlcNAc2, confirming EDEM3's enzymatic role in vivo. Loss of EDEM3 also impairs the unfolded protein response (reduced PERK/EIF2AK3 induction upon ER stress).","method":"Exome sequencing; glycan profiling in patient fibroblasts, human plasma, and mouse plasma/brain tissue; tunicamycin-induced UPR assay in human fibroblasts","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cell and plasma glycan profiling with multiple independent families, functional UPR assay, but no in vitro reconstitution","pmids":["34143952"],"is_preprint":false},{"year":2022,"finding":"EDEM3 exhibits in vitro alpha1,2-mannosidase activity toward asparagine-linked (N-linked) oligomannose glycans, converting M9 to M8 and M7, but not toward glycine-linked M9 glycan. ERp46 co-incubation enhances this activity.","method":"In vitro mannosidase assay with purified EDEM3 and synthetic asparagine-linked vs. glycine-linked M9 glycan substrates; activity measured with and without ERp46","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical assay with defined substrates, single lab, single method, no mutagenesis validation","pmids":["35500441"],"is_preprint":false},{"year":2017,"finding":"EDEM3 interacts with ER-resident proteins including components of the ERAD cargo recognition and targeting machinery, as well as UBA1 and UBA2 ubiquitinating enzymes. This interaction network is sensitive to perturbation of early ER N-glycan processing by kifunensine and NB-DNJ.","method":"Immunoprecipitation coupled with mass spectrometry; Western blot validation; glycan processing inhibitor treatments","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/MS experiment, single lab, partial Western blot validation only","pmids":["28366632"],"is_preprint":false},{"year":2020,"finding":"EDEM3 deficiency increases LRP1 cell-surface expression and VLDL uptake by upregulating surface mannose-containing glycoproteins, thereby reducing plasma triglyceride levels. EDEM3 deletion upregulates RNA and ER protein processing/transport pathways.","method":"EDEM3 knockout cell and mouse models; VLDL uptake assays; LRP1 expression analysis; cell-surface glycoprotein profiling; metabolomics","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell and in vivo KO models with multiple functional readouts (uptake, receptor expression, metabolomics), single lab","pmids":["32213464"],"is_preprint":false},{"year":2025,"finding":"COPB2 physically interacts with EDEM3 and enhances its ER localization and mannose-trimming activity. COPB2 depletion impairs EDEM3 function, causing glycan processing defects and ER stress accumulation.","method":"Co-immunoprecipitation; protein interaction analysis; glycoproteomic analysis; COPB2 knockdown and overexpression in ovarian cancer cells; xenograft models","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP interaction plus functional KD assay, single lab, no in vitro reconstitution","pmids":["40736660"],"is_preprint":false}],"current_model":"EDEM3 is an ER-resident soluble alpha1,2-mannosidase (GH47 family) that acts as the major enzyme for the second step of N-glycan mannose trimming in glycoprotein ERAD, converting M8B to M7/M6/M5 oligosaccharides; its catalytic activity requires formation of a disulfide-bond-dependent complex with the ER oxidoreductase ERp46, is modulated by distinct functional domains (GH47, IMD, PA, IDD), and is further regulated by COPB2-dependent ER localization, with loss-of-function causing a congenital disorder of glycosylation and impaired unfolded protein response in humans."},"narrative":{"mechanistic_narrative":"EDEM3 is an ER-resident alpha1,2-mannosidase that catalyzes the second step of N-glycan mannose trimming during glycoprotein ER-associated degradation (ERAD), and its activity directly drives clearance of misfolded glycoproteins [PMID:16431915, PMID:34698634]. Purified EDEM3 is the major enzyme converting Man8GlcNAc2 isomer B (M8B) to M7 (M7A/M7C), M6, and M5 oligosaccharides, and it also trims intact glycoproteins, with catalysis depending on the conserved active-site glutamate (E147) [PMID:16431915, PMID:34698634]. Full activity requires a covalent partnership with the ER oxidoreductase ERp46, which forms a disulfide bond with the EDEM3 alpha-mannosidase domain and is needed for mannose trimming of substrates such as misfolded TCRalpha [PMID:29784879, PMID:35500441]. The protein is organized into discrete functional domains — a catalytic GH47 domain that binds substrate even without trimming, an intermediate (IMD) domain required for GH47 folding, and protease-associated (PA) and intrinsically disordered (IDD) domains that respectively promote and restrain degradation of specific substrates to set ERAD timing [PMID:33671632]. Human loss-of-function variants cause a congenital disorder of glycosylation (EDEM3-CDG) marked by defective M8B trimming, accumulation of Glc1Man5GlcNAc2, and a blunted unfolded protein response [PMID:34143952].","teleology":[{"year":2006,"claim":"Established that EDEM3 is an active mannosidase whose enzymatic activity, not merely lectin binding, is required for its ERAD-promoting function — resolving whether EDEM proteins act catalytically.","evidence":"Wild-type versus E147Q active-site mutant transfection in HEK293 cells with NHK and TCRalpha degradation and mannose-trimming assays","pmids":["16431915"],"confidence":"High","gaps":["Did not define the precise glycan products generated","In-cell assay could not exclude contributions from associated factors","No purified-protein reconstitution"]},{"year":2018,"claim":"Identified ERp46 as a disulfide-bonded partner essential for EDEM3 catalysis, showing the enzyme requires a redox-active cofactor rather than acting alone.","evidence":"Co-IP, disulfide bond mapping, and in vitro reconstitution with purified recombinant proteins from HEK293 cells","pmids":["29784879"],"confidence":"High","gaps":["Single lab","Mechanism by which the disulfide bond activates the GH47 domain not structurally resolved","Generality across substrates beyond TCRalpha not established"]},{"year":2021,"claim":"Defined EDEM3 as the major enzyme for the second mannose-trimming step in gpERAD by reconstituting the full M8B-to-M7/M6/M5 reaction with purified protein and defined glycans.","evidence":"In vitro mannosidase assays with purified recombinant EDEM3 and pyridylamine-labeled M8B and glycoprotein substrates","pmids":["34698634"],"confidence":"High","gaps":["Kinetic determinants of product distribution not detailed","Did not address in vivo cofactor requirement"]},{"year":2021,"claim":"Mapped EDEM3 into four functional domains and assigned distinct roles, explaining how a single enzyme tunes ERAD timing for different substrates.","evidence":"EDEM3-knockout cells complemented with domain-deletion mutants, NHK and tyrosinase degradation assays, and LC/MS interactome analysis","pmids":["33671632"],"confidence":"Medium","gaps":["Mechanism of IDD-mediated negative modulation unknown","Single lab","Domain effects shown for limited substrate set"]},{"year":2021,"claim":"Connected EDEM3 catalytic function to human disease, demonstrating that loss-of-function variants cause a CDG and impair the unfolded protein response in vivo.","evidence":"Exome sequencing, patient fibroblast/plasma and mouse glycan profiling, and tunicamycin-induced UPR assays","pmids":["34143952"],"confidence":"Medium","gaps":["Link between glycan defect and UPR impairment mechanistically unresolved","No in vitro reconstitution of variant enzymes"]},{"year":2022,"claim":"Refined substrate specificity by showing EDEM3 acts on asparagine-linked but not glycine-linked oligomannose glycans and that ERp46 enhances activity, indicating glycan-protein linkage context matters.","evidence":"In vitro mannosidase assays with purified EDEM3 and synthetic N-linked versus glycine-linked M9 substrates, with and without ERp46","pmids":["35500441"],"confidence":"Medium","gaps":["Single method, no mutagenesis validation","Structural basis of linkage discrimination unknown"]},{"year":2020,"claim":"Revealed a physiological consequence of EDEM3 activity beyond protein quality control, linking it to LRP1 surface glycosylation and lipoprotein metabolism.","evidence":"EDEM3-knockout cells and mice with VLDL uptake, LRP1 expression, cell-surface glycoprotein profiling, and metabolomics","pmids":["32213464"],"confidence":"Medium","gaps":["Direct glycan changes on LRP1 not mapped","Single lab"]},{"year":2017,"claim":"Began mapping the EDEM3 interaction network, placing it among ERAD cargo-recognition and ubiquitination machinery sensitive to glycan-processing state.","evidence":"IP-mass spectrometry with Western validation and glycan-processing inhibitor treatments","pmids":["28366632"],"confidence":"Low","gaps":["Single Co-IP/MS with only partial validation","Direct versus indirect interactions not distinguished","UBA1/UBA2 functional relevance unconfirmed"]},{"year":2025,"claim":"Implicated COPB2 in controlling EDEM3 ER localization and activity, suggesting trafficking-level regulation of EDEM3 function in cancer cells.","evidence":"Co-IP, glycoproteomics, and COPB2 knockdown/overexpression in ovarian cancer cells with xenografts","pmids":["40736660"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation","No in vitro reconstitution","Direct effect on EDEM3 catalysis versus localization not separated"]},{"year":null,"claim":"How EDEM3's domain architecture and the ERp46 disulfide bond are structurally coordinated to set substrate-specific trimming kinetics in the cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental structure of EDEM3 or the EDEM3-ERp46 complex in the corpus","Mechanism of IDD-mediated turnover restraint unknown","Regulation of EDEM3 by trafficking factors not reconstituted"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,2,5]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,2,5]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,8]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[4]}],"complexes":[],"partners":["ERP46","COPB2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BZQ6","full_name":"ER degradation-enhancing alpha-mannosidase-like protein 3","aliases":["Alpha-1,2-mannosidase EDEM3"],"length_aa":932,"mass_kda":104.7,"function":"Involved in endoplasmic reticulum-associated degradation (ERAD). Accelerates the glycoprotein ERAD by proteasomes, by catalyzing mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 in the N-glycans (PubMed:25092655). May also participate in mannose trimming from all glycoproteins and not just misfolded ones targeted to ERAD (PubMed:34143952). May have alpha 1,2-mannosidase activity (By similarity)","subcellular_location":"Endoplasmic reticulum lumen","url":"https://www.uniprot.org/uniprotkb/Q9BZQ6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EDEM3","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EDEM3","total_profiled":1310},"omim":[{"mim_id":"619493","title":"CONGENITAL DISORDER OF GLYCOSYLATION, TYPE 2v; CDG2V","url":"https://www.omim.org/entry/619493"},{"mim_id":"610302","title":"ENDOPLASMIC RETICULUM DEGRADATION-ENHANCING ALPHA-MANNOSIDASE-LIKE PROTEIN 2; EDEM2","url":"https://www.omim.org/entry/610302"},{"mim_id":"610214","title":"ENDOPLASMIC RETICULUM DEGRADATION-ENHANCING ALPHA-MANNOSIDASE-LIKE PROTEIN 3; EDEM3","url":"https://www.omim.org/entry/610214"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Endoplasmic reticulum","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EDEM3"},"hgnc":{"alias_symbol":[],"prev_symbol":["C1orf22"]},"alphafold":{"accession":"Q9BZQ6","domains":[{"cath_id":"-","chopping":"92-221","consensus_level":"medium","plddt":96.4591,"start":92,"end":221},{"cath_id":"-","chopping":"583-644","consensus_level":"high","plddt":79.8352,"start":583,"end":644},{"cath_id":"3.50.30.30","chopping":"649-797","consensus_level":"high","plddt":90.3974,"start":649,"end":797}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BZQ6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BZQ6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BZQ6-F1-predicted_aligned_error_v6.png","plddt_mean":78.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EDEM3","jax_strain_url":"https://www.jax.org/strain/search?query=EDEM3"},"sequence":{"accession":"Q9BZQ6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BZQ6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BZQ6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BZQ6"}},"corpus_meta":[{"pmid":"16431915","id":"PMC_16431915","title":"EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16431915","citation_count":199,"is_preprint":false},{"pmid":"17499246","id":"PMC_17499246","title":"Glycoprotein folding and the role of EDEM1, EDEM2 and EDEM3 in degradation of folding-defective glycoproteins.","date":"2007","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/17499246","citation_count":112,"is_preprint":false},{"pmid":"29784879","id":"PMC_29784879","title":"ER-resident protein 46 (ERp46) triggers the mannose-trimming activity of ER degradation-enhancing α-mannosidase-like protein 3 (EDEM3).","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29784879","citation_count":33,"is_preprint":false},{"pmid":"34143952","id":"PMC_34143952","title":"Bi-allelic variants in the ER quality-control mannosidase gene EDEM3 cause a congenital disorder of glycosylation.","date":"2021","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34143952","citation_count":19,"is_preprint":false},{"pmid":"34698634","id":"PMC_34698634","title":"Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/34698634","citation_count":15,"is_preprint":false},{"pmid":"33671632","id":"PMC_33671632","title":"EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33671632","citation_count":11,"is_preprint":false},{"pmid":"35897761","id":"PMC_35897761","title":"Pro-Survival Factor EDEM3 Confers Therapy Resistance in Prostate Cancer.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35897761","citation_count":11,"is_preprint":false},{"pmid":"32213464","id":"PMC_32213464","title":"EDEM3 Modulates Plasma Triglyceride Level through Its Regulation of LRP1 Expression.","date":"2020","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/32213464","citation_count":9,"is_preprint":false},{"pmid":"35500441","id":"PMC_35500441","title":"In vitro mannosidase activity of EDEM3 against asparagine-linked oligomannose-type glycans.","date":"2022","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/35500441","citation_count":7,"is_preprint":false},{"pmid":"39754316","id":"PMC_39754316","title":"Disrupting EDEM3-induced M2-like macrophage trafficking by glucose restriction overcomes resistance to PD-1/PD-L1 blockade.","date":"2025","source":"Clinical and translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39754316","citation_count":6,"is_preprint":false},{"pmid":"28366632","id":"PMC_28366632","title":"Inhibition of N-glycan processing modulates the network of EDEM3 interactors.","date":"2017","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/28366632","citation_count":6,"is_preprint":false},{"pmid":"40736660","id":"PMC_40736660","title":"COPB2 facilitates EDEM3-mediated mannose trimming to sustain ER homeostasis in ovarian cancer.","date":"2025","source":"Cellular oncology (Dordrecht, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/40736660","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8005,"output_tokens":2430,"usd":0.030232,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9480,"output_tokens":2977,"usd":0.060913,"stage2_stop_reason":"end_turn"},"total_usd":0.091145,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"EDEM3 has alpha1,2-mannosidase activity in vivo, accelerating mannose trimming from misfolded glycoproteins and total glycoproteins. Mutation of the conserved active-site residue E147Q abolishes mannose trimming and greatly reduces ERAD stimulation, demonstrating that mannosidase activity is required for EDEM3's ERAD function.\",\n      \"method\": \"Transfection of HEK293 cells with wild-type and E147Q catalytic mutant EDEM3; degradation assays of misfolded alpha1-antitrypsin NHK and TCRα; mannose trimming assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — active-site mutagenesis combined with functional ERAD and mannose-trimming assays in a single focused study, clear mechanistic dissection\",\n      \"pmids\": [\"16431915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ERp46, an ER-resident oxidoreductase, stably associates with EDEM3 via a disulfide bond between ERp46 redox-active cysteine residues and the EDEM3 alpha-mannosidase domain. This covalent interaction is required for EDEM3 mannose-trimming activity toward misfolded TCRα substrate, as reconstituted in a defined in vitro system with purified recombinant proteins.\",\n      \"method\": \"Co-immunoprecipitation, in vitro reconstitution with purified recombinant proteins from HEK293 cells, disulfide bond analysis, mannose-trimming activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins plus disulfide bond mapping, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"29784879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Purified EDEM3 alone is the major alpha1,2-mannosidase responsible for the second step of N-glycan trimming in gpERAD, converting M8B to M7 (M7A and M7C), M6, and M5 oligosaccharides. EDEM3 also efficiently trims M8B from intact glycoproteins.\",\n      \"method\": \"In vitro mannosidase assay with purified recombinant EDEM3 and pyridylamine-labeled M8B glycan substrates; glycoprotein substrate trimming assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro enzymatic activity with purified protein and defined substrates, functional glycoprotein assay, peer-reviewed\",\n      \"pmids\": [\"34698634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EDEM3 consists of four functional domains: GH47 (mannosidase), intermediate (IMD), protease-associated (PA), and intrinsically disordered (IDD). The GH47 domain mediates substrate binding even without mannose trimming; IMD is required for GH47 folding; PA domain positively modulates ERAD of specific substrates; IDD domain negatively modulates substrate turnover, providing unique ERAD timing.\",\n      \"method\": \"EDEM3 knockout cell line complemented with domain-deletion mutants; ERAD substrate degradation assays for NHK and soluble tyrosinase mutant; LC/MS interactome analysis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO rescue with domain mutants and defined phenotypic readouts, single lab, multiple mutants tested\",\n      \"pmids\": [\"33671632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss-of-function EDEM3 variants in humans cause a congenital disorder of glycosylation (EDEM3-CDG) characterized by decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, and impaired Man5GlcNAc2-to-Man4GlcNAc2 conversion with accumulation of Glc1Man5GlcNAc2, confirming EDEM3's enzymatic role in vivo. Loss of EDEM3 also impairs the unfolded protein response (reduced PERK/EIF2AK3 induction upon ER stress).\",\n      \"method\": \"Exome sequencing; glycan profiling in patient fibroblasts, human plasma, and mouse plasma/brain tissue; tunicamycin-induced UPR assay in human fibroblasts\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cell and plasma glycan profiling with multiple independent families, functional UPR assay, but no in vitro reconstitution\",\n      \"pmids\": [\"34143952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EDEM3 exhibits in vitro alpha1,2-mannosidase activity toward asparagine-linked (N-linked) oligomannose glycans, converting M9 to M8 and M7, but not toward glycine-linked M9 glycan. ERp46 co-incubation enhances this activity.\",\n      \"method\": \"In vitro mannosidase assay with purified EDEM3 and synthetic asparagine-linked vs. glycine-linked M9 glycan substrates; activity measured with and without ERp46\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical assay with defined substrates, single lab, single method, no mutagenesis validation\",\n      \"pmids\": [\"35500441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EDEM3 interacts with ER-resident proteins including components of the ERAD cargo recognition and targeting machinery, as well as UBA1 and UBA2 ubiquitinating enzymes. This interaction network is sensitive to perturbation of early ER N-glycan processing by kifunensine and NB-DNJ.\",\n      \"method\": \"Immunoprecipitation coupled with mass spectrometry; Western blot validation; glycan processing inhibitor treatments\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/MS experiment, single lab, partial Western blot validation only\",\n      \"pmids\": [\"28366632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EDEM3 deficiency increases LRP1 cell-surface expression and VLDL uptake by upregulating surface mannose-containing glycoproteins, thereby reducing plasma triglyceride levels. EDEM3 deletion upregulates RNA and ER protein processing/transport pathways.\",\n      \"method\": \"EDEM3 knockout cell and mouse models; VLDL uptake assays; LRP1 expression analysis; cell-surface glycoprotein profiling; metabolomics\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell and in vivo KO models with multiple functional readouts (uptake, receptor expression, metabolomics), single lab\",\n      \"pmids\": [\"32213464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"COPB2 physically interacts with EDEM3 and enhances its ER localization and mannose-trimming activity. COPB2 depletion impairs EDEM3 function, causing glycan processing defects and ER stress accumulation.\",\n      \"method\": \"Co-immunoprecipitation; protein interaction analysis; glycoproteomic analysis; COPB2 knockdown and overexpression in ovarian cancer cells; xenograft models\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP interaction plus functional KD assay, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"40736660\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EDEM3 is an ER-resident soluble alpha1,2-mannosidase (GH47 family) that acts as the major enzyme for the second step of N-glycan mannose trimming in glycoprotein ERAD, converting M8B to M7/M6/M5 oligosaccharides; its catalytic activity requires formation of a disulfide-bond-dependent complex with the ER oxidoreductase ERp46, is modulated by distinct functional domains (GH47, IMD, PA, IDD), and is further regulated by COPB2-dependent ER localization, with loss-of-function causing a congenital disorder of glycosylation and impaired unfolded protein response in humans.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EDEM3 is an ER-resident alpha1,2-mannosidase that catalyzes the second step of N-glycan mannose trimming during glycoprotein ER-associated degradation (ERAD), and its activity directly drives clearance of misfolded glycoproteins [#0, #2]. Purified EDEM3 is the major enzyme converting Man8GlcNAc2 isomer B (M8B) to M7 (M7A/M7C), M6, and M5 oligosaccharides, and it also trims intact glycoproteins, with catalysis depending on the conserved active-site glutamate (E147) [#0, #2]. Full activity requires a covalent partnership with the ER oxidoreductase ERp46, which forms a disulfide bond with the EDEM3 alpha-mannosidase domain and is needed for mannose trimming of substrates such as misfolded TCRalpha [#1, #5]. The protein is organized into discrete functional domains — a catalytic GH47 domain that binds substrate even without trimming, an intermediate (IMD) domain required for GH47 folding, and protease-associated (PA) and intrinsically disordered (IDD) domains that respectively promote and restrain degradation of specific substrates to set ERAD timing [#3]. Human loss-of-function variants cause a congenital disorder of glycosylation (EDEM3-CDG) marked by defective M8B trimming, accumulation of Glc1Man5GlcNAc2, and a blunted unfolded protein response [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established that EDEM3 is an active mannosidase whose enzymatic activity, not merely lectin binding, is required for its ERAD-promoting function — resolving whether EDEM proteins act catalytically.\",\n      \"evidence\": \"Wild-type versus E147Q active-site mutant transfection in HEK293 cells with NHK and TCRalpha degradation and mannose-trimming assays\",\n      \"pmids\": [\"16431915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the precise glycan products generated\", \"In-cell assay could not exclude contributions from associated factors\", \"No purified-protein reconstitution\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified ERp46 as a disulfide-bonded partner essential for EDEM3 catalysis, showing the enzyme requires a redox-active cofactor rather than acting alone.\",\n      \"evidence\": \"Co-IP, disulfide bond mapping, and in vitro reconstitution with purified recombinant proteins from HEK293 cells\",\n      \"pmids\": [\"29784879\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single lab\", \"Mechanism by which the disulfide bond activates the GH47 domain not structurally resolved\", \"Generality across substrates beyond TCRalpha not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined EDEM3 as the major enzyme for the second mannose-trimming step in gpERAD by reconstituting the full M8B-to-M7/M6/M5 reaction with purified protein and defined glycans.\",\n      \"evidence\": \"In vitro mannosidase assays with purified recombinant EDEM3 and pyridylamine-labeled M8B and glycoprotein substrates\",\n      \"pmids\": [\"34698634\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetic determinants of product distribution not detailed\", \"Did not address in vivo cofactor requirement\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapped EDEM3 into four functional domains and assigned distinct roles, explaining how a single enzyme tunes ERAD timing for different substrates.\",\n      \"evidence\": \"EDEM3-knockout cells complemented with domain-deletion mutants, NHK and tyrosinase degradation assays, and LC/MS interactome analysis\",\n      \"pmids\": [\"33671632\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of IDD-mediated negative modulation unknown\", \"Single lab\", \"Domain effects shown for limited substrate set\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected EDEM3 catalytic function to human disease, demonstrating that loss-of-function variants cause a CDG and impair the unfolded protein response in vivo.\",\n      \"evidence\": \"Exome sequencing, patient fibroblast/plasma and mouse glycan profiling, and tunicamycin-induced UPR assays\",\n      \"pmids\": [\"34143952\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Link between glycan defect and UPR impairment mechanistically unresolved\", \"No in vitro reconstitution of variant enzymes\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Refined substrate specificity by showing EDEM3 acts on asparagine-linked but not glycine-linked oligomannose glycans and that ERp46 enhances activity, indicating glycan-protein linkage context matters.\",\n      \"evidence\": \"In vitro mannosidase assays with purified EDEM3 and synthetic N-linked versus glycine-linked M9 substrates, with and without ERp46\",\n      \"pmids\": [\"35500441\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method, no mutagenesis validation\", \"Structural basis of linkage discrimination unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a physiological consequence of EDEM3 activity beyond protein quality control, linking it to LRP1 surface glycosylation and lipoprotein metabolism.\",\n      \"evidence\": \"EDEM3-knockout cells and mice with VLDL uptake, LRP1 expression, cell-surface glycoprotein profiling, and metabolomics\",\n      \"pmids\": [\"32213464\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct glycan changes on LRP1 not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Began mapping the EDEM3 interaction network, placing it among ERAD cargo-recognition and ubiquitination machinery sensitive to glycan-processing state.\",\n      \"evidence\": \"IP-mass spectrometry with Western validation and glycan-processing inhibitor treatments\",\n      \"pmids\": [\"28366632\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP/MS with only partial validation\", \"Direct versus indirect interactions not distinguished\", \"UBA1/UBA2 functional relevance unconfirmed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated COPB2 in controlling EDEM3 ER localization and activity, suggesting trafficking-level regulation of EDEM3 function in cancer cells.\",\n      \"evidence\": \"Co-IP, glycoproteomics, and COPB2 knockdown/overexpression in ovarian cancer cells with xenografts\",\n      \"pmids\": [\"40736660\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"No in vitro reconstitution\", \"Direct effect on EDEM3 catalysis versus localization not separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EDEM3's domain architecture and the ERp46 disulfide bond are structurally coordinated to set substrate-specific trimming kinetics in the cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental structure of EDEM3 or the EDEM3-ERp46 complex in the corpus\", \"Mechanism of IDD-mediated turnover restraint unknown\", \"Regulation of EDEM3 by trafficking factors not reconstituted\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 2, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ERP46\", \"COPB2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}