{"gene":"ALG3","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":1996,"finding":"The ALG3 gene of Saccharomyces cerevisiae encodes a potential ER-transmembrane protein of 458 amino acids (53 kDa) with a C-terminal KKXX-retrieval sequence; its inactivation results in accumulation of lipid-linked Man5GlcNAc2 and transfer of Endo H-resistant (underglycosylated) carbohydrates to protein by the oligosaccharyltransferase complex, establishing ALG3 as required for dolichol-linked oligosaccharide biosynthesis beyond Man5GlcNAc2.","method":"Complementation cloning of alg3 temperature-sensitive mutant; gene disruption; biochemical analysis of lipid-linked oligosaccharides and secretory protein glycosylation","journal":"Glycobiology","confidence":"High","confidence_rationale":"Tier 1 / Strong — gene cloning with functional complementation, null mutant biochemical characterization, replicated by multiple subsequent studies","pmids":["8842708"],"is_preprint":false},{"year":2001,"finding":"ALG3 encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase itself (not an accessory protein): immunoprecipitation of an HA-tagged ALG3 fusion selectively co-precipitated mannosyltransferase activity converting Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol from detergent-solubilized yeast membranes, and the initial Man5→Man6 reaction is metal-ion independent whereas further elongation requires Mn2+.","method":"In vitro mannosyltransferase assay with [3H]Man5GlcNAc2-PP-Dol substrate; immunoprecipitation of HA-tagged ALG3 fusion protein to co-precipitate enzymatic activity","journal":"Biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic reconstitution with tagged protein immunoprecipitation, single lab but two orthogonal methods (activity assay + IP)","pmids":["11308030"],"is_preprint":false},{"year":1991,"finding":"Structural analysis of oligosaccharides from alg3,sec18 yeast by 1H NMR established that the alg3 mutant accumulates Man5GlcNAc2-PP-dolichol due to a defective alpha-1,3-mannosyltransferase required for the next elongation step, and that the transferred Man5GlcNAc2 structure is Man-alpha1,2Man-alpha1,2Man-alpha1,3(Man-alpha1,6)Man-beta1,4GlcNAc-beta1,4GlcNAc.","method":"1H NMR spectroscopy of oligosaccharides released from ER-restricted invertase in alg3,sec18 yeast; peptide-N-glycosidase F and Endo H digestion","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structural determination with biochemical validation, foundational structural proof replicated by subsequent work","pmids":["2005096"],"is_preprint":false},{"year":1993,"finding":"In the alg3,sec18,gls1 triple mutant, the majority (>75%) of ER N-linked glycosylation occurs by transfer of Man5GlcNAc2 without prior addition of the 3 glucoses normally present on the lipid-linked precursor; 2D DQF-COSY 1H NMR revealed Glc3Man5GlcNAc2 and Man8GlcNAc2 as the glucosylated species, demonstrating that the alg3 yeast still glucosylates both Man5 and Man9 precursors.","method":"500-MHz 2D DQF-COSY 1H NMR spectroscopy; Endo H and PNGase F digestion; Bio-Gel P-4 oligosaccharide sizing of invertase glycans from alg3,sec18,gls1 yeast","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structural analysis with multiple orthogonal biochemical methods, directly characterizes ALG3-dependent glycan structure in the ER","pmids":["8505333"],"is_preprint":false},{"year":1993,"finding":"In the delta-och1 mnn1 alg3 triple mutant of S. cerevisiae, N-linked oligosaccharides accumulated as Man5GlcNAc2 and Man8GlcNAc2 in total cell mannoprotein, confirming the lack of outer chain addition to the incomplete core-like oligosaccharide and the leaky phenotype of the alg3 mutation; this established that the ALG3-dependent Man8GlcNAc2 core is the substrate for OCH1-initiated alpha-1,6-polymannose outer chain addition.","method":"Structural analysis of pyridylaminated neutral oligosaccharides from purified periplasmic invertase by hydrazinolysis, N-acetylation, and chromatography","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct structural characterization of glycans in defined triple mutant, multiple orthogonal methods, replicated by subsequent studies","pmids":["8253757"],"is_preprint":false},{"year":2004,"finding":"The Pichia pastoris ALG3 homolog encodes the enzyme converting Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP; deletion in an och1 background leads to secretion of Man5GlcNAc2-containing glycoproteins trimmable by alpha-1,2-mannosidase, plus additional larger glycans (Hex6-Hex12GlcNAc2) resistant to mannosidase digestion, revealing divergent Golgi processing relative to S. cerevisiae alg3.","method":"Gene deletion (alg3Δ) in P. pastoris; glycan profiling by mass spectrometry; in vitro alpha-1,2-mannosidase digestion of secreted glycoproteins","journal":"Glycobiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — functional gene deletion with direct biochemical characterization of glycan products, two orthogonal methods (MS + enzymatic digestion)","pmids":["15033937"],"is_preprint":false},{"year":2004,"finding":"A silent mutation (c.165C>T) in exon 1 of the human ALG3 gene activates a cryptic donor splice site, deleting c.160_196 and generating a premature termination codon after the first N-terminal transmembrane domain (p.Val54fsX66); the resulting truncated ALG3 mRNA escapes nonsense-mediated mRNA decay (NMD) despite fulfilling NMD criteria, as demonstrated by cycloheximide treatment having no effect on transcript levels.","method":"RT-PCR analysis of patient transcripts; cycloheximide treatment to suppress NMD; expression studies in patient cells","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct patient transcript analysis with NMD suppression experiment, two methods but single lab and disease-context study","pmids":["15108280"],"is_preprint":false},{"year":2021,"finding":"ALG3 promotes radioresistance and cancer stemness in breast cancer cells by inducing glycosylation of TGF-β receptor II (TGFBR2); both pharmacological inhibition of glycosylation (tunicamycin) and TGFBR2 inhibition (LY2109761) abrogated the stimulatory effects of ALG3 overexpression on stemness and radioresistance, establishing TGFBR2 glycosylation as the mechanistic link.","method":"Immunoprecipitation; Western blot; shRNA knockdown; overexpression in breast cancer cell lines; orthotopic xenograft models; pharmacological inhibitor experiments","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and functional rescue/inhibition experiments in vitro and in vivo, single lab, multiple orthogonal approaches","pmids":["33931075"],"is_preprint":false},{"year":2022,"finding":"ALG3 promotes ovarian cancer peritoneal metastasis by catalyzing alpha-1,3-mannosylation of urokinase plasminogen activator receptor (uPAR), which enhances uPA/uPAR activation and the interaction between uPAR and ADAM8, thereby activating the ADAM8/Ras/ERK signaling pathway.","method":"Lectin chip; Western blot; Lectin blot; Co-immunoprecipitation of uPAR and ADAM8; ALG3 knockdown in ovarian cancer cells; mouse peritoneal metastasis model","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (lectin profiling, Co-IP, in vivo model), single lab","pmids":["36231102"],"is_preprint":false},{"year":2022,"finding":"ALG3 inhibition induces deficiency of post-translational N-linked glycosylation, leading to excessive lipid accumulation through SREBP1-dependent lipogenesis in cancer cells; N-linked glycosylation deficiency-mediated lipid hyperperoxidation triggers immunogenic ferroptosis and promotes a pro-inflammatory tumor microenvironment that boosts anti-tumor immune responses and synergizes with anti-PD1 therapy.","method":"CRISPR/Cas9 ALG3 deletion in mouse cancer cells; lipid accumulation assays; SREBP1 pathway analysis; in vivo syngeneic tumor models with anti-PD1 combination; tunicamycin treatment as pharmacological comparator","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with multiple functional readouts and in vivo validation, single lab, mechanism partially inferred from pathway inhibitors","pmids":["35676564"],"is_preprint":false},{"year":2025,"finding":"AKT directly phosphorylates ALG3 at Ser11/Ser13 in the amino-terminal region downstream of the PI3K/AKT pathway in growth factor-stimulated and PI3K/AKT-hyperactive cancer cells; CRISPR/Cas9 depletion of ALG3 causes improper glycan formation, ER stress, unfolded protein response induction, and impaired cell proliferation; phosphorylation at Ser11/Ser13 is required for proper glycosylation of cell surface receptors EGFR, HER3, and E-cadherin.","method":"In vitro AKT kinase assay with ALG3 substrate; phosphorylation site identification (Ser11/Ser13); CRISPR/Cas9 ALG3 depletion; phospho-site mutagenesis; glycosylation analysis of EGFR, HER3, E-cadherin by Western blot; UPR marker analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase assay plus CRISPR KO plus mutagenesis plus substrate glycosylation readouts, multiple orthogonal methods in single rigorous study","pmids":["40789468","40236010"],"is_preprint":false},{"year":2025,"finding":"ALG3 directly interacts with transcription factor FOXD1 and induces N-glycosylation at Asn176 of FOXD1, which increases FOXD1 protein stability and nuclear localization; this in turn allows FOXD1 to transcriptionally activate BNIP3, promoting mitophagy and gemcitabine resistance in nasopharyngeal carcinoma cells.","method":"Co-immunoprecipitation of ALG3 and FOXD1; glycosylation site mutagenesis (Asn176); nuclear fractionation; Western blot; BNIP3 transcriptional reporter/ChIP; cell proliferation and drug sensitivity assays","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus mutagenesis plus functional downstream pathway analysis, single lab","pmids":["40083705"],"is_preprint":false},{"year":2026,"finding":"Cryo-EM structures of pseudo-Michaelis complexes of ALG3, ALG9, and ALG12 at high resolution revealed how the branched glycan is accurately synthesized in four consecutive mannosylation reactions converting GlcNAc2Man5 to GlcNAc2Man9; molecular dynamics simulations and mutagenesis uncovered the mechanism by which ALG3 selects the dolichylphosphomannose donor over dolichylphosphoglucose; the structures also provide mechanistic explanations for enzyme dysfunction in CDGs.","method":"Cryo-electron microscopy of pseudo-Michaelis complexes; chemoenzymatic synthesis of lipid-linked glycan analogs for in vitro reconstitution; molecular dynamics simulations; site-directed mutagenesis","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure combined with in vitro reconstitution, MD simulations, and mutagenesis in a single rigorous study","pmids":["41807832"],"is_preprint":false},{"year":2018,"finding":"The human ALG3 protein (hNOT-1/ALG3-1) forms homodimers and interacts in vivo with OSBP, OSBPL9, LRP1, SYPL1, and the transcription factor CREB3 precursor (but not with CREB3 proteolytic products); binding to CREB3 precursor is a prerequisite for CREB3's proteolytic activation; different post-translationally processed ALG3 products localize to distinct cellular compartments and interact with different partners.","method":"Yeast two-hybrid screening; co-immunoprecipitation validation of selected interactions (OSBP, OSBPL9, LRP1, CREB3) in vivo","journal":"Human molecular genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid plus single Co-IP validation, single lab, limited mechanistic follow-up","pmids":["29547901"],"is_preprint":false},{"year":2018,"finding":"The human ALG3 (hNOT-1/ALG3-1) protein undergoes N-glycosylation and sequential proteolytic cleavage generating derivatives destined to distinct cellular compartments; truncated transcripts are not translated; two full-length precursor proteins (hNOT-1 and hNOT-4) are encoded by alternatively spliced transcripts differing at exon 1.","method":"Polyclonal antibodies against diverse protein regions; subcellular fractionation; Western blot analysis of N-glycosylation; RT-PCR and translation analysis of truncated transcripts","journal":"Human molecular genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, antibody-based localization and biochemical characterization without functional consequence established","pmids":["30192950"],"is_preprint":false},{"year":2024,"finding":"ALG3 deficiency in patient-derived fibroblasts results in constitutive activation of the unfolded protein response via the IRE1-α pathway, increased ER-associated degradation activity, and accumulation of N-linked Man3-4 glycans in cellular and secreted glycoproteins; in transferrin, the Man5 intermediate is further processed to a mono-antennary glycan NeuAc1Gal1GlcNAc1Man3GlcNAc2.","method":"Western blot of UPR markers in patient-derived fibroblasts; glycoprotein glycan profiling by mass spectrometry; IRE1-α pathway activation analysis","journal":"Journal of inherited metabolic disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with multiple biochemical readouts (UPR markers, glycan MS profiling), single lab, two orthogonal methods","pmids":["38597022"],"is_preprint":false}],"current_model":"Human ALG3 (alpha-1,3-mannosyltransferase) is an ER-resident transmembrane enzyme that catalyzes the first luminal mannosylation step in N-glycan biosynthesis—adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 to form Dol-PP-Man6GlcNAc2—a rate-limiting reaction whose structural basis has been resolved by cryo-EM; ALG3 activity is regulated by direct AKT-mediated phosphorylation at Ser11/Ser13, which is required for proper glycosylation of surface receptors including EGFR, HER3, and E-cadherin; ALG3 also N-glycosylates specific substrates such as TGF-β receptor II and FOXD1, thereby modulating TGF-β signaling, FOXD1 stability/nuclear localization, and downstream transcriptional programs; loss of ALG3 function causes ER stress, constitutive UPR activation via IRE1-α, impaired cell proliferation, and, in cancer contexts, immunogenic ferroptosis through SREBP1-dependent lipid accumulation."},"narrative":{"mechanistic_narrative":"ALG3 is an ER-resident transmembrane alpha-1,3-mannosyltransferase that performs the first luminal mannosylation in dolichol-linked N-glycan biosynthesis, transferring mannose from Dol-P-Man onto Dol-PP-Man5GlcNAc2 to extend the precursor beyond Man5GlcNAc2 [PMID:8842708, PMID:11308030, PMID:2005096]. Loss of this activity causes accumulation of lipid-linked Man5GlcNAc2 and transfer of underglycosylated, truncated glycans to protein [PMID:8842708, PMID:2005096, PMID:8505333], and the ALG3-dependent core serves as the substrate for downstream elongation and outer-chain addition [PMID:8253757]. Cryo-EM of pseudo-Michaelis complexes resolved how ALG3 builds the branched glycan in concert with ALG9 and ALG12 and how it selects the dolichylphosphomannose donor over dolichylphosphoglucose, and provided a structural basis for ALG3 dysfunction in congenital disorders of glycosylation [PMID:41807832]. ALG3 activity is controlled by the PI3K/AKT pathway through direct AKT phosphorylation at Ser11/Ser13, which is required for proper N-glycosylation of cell-surface receptors including EGFR, HER3, and E-cadherin; ALG3 depletion produces improper glycan formation, ER stress, UPR induction, and impaired proliferation [PMID:40789468, PMID:40236010]. In cancer contexts, ALG3 glycosylates specific substrates—TGF-β receptor II to drive stemness and radioresistance [PMID:33931075], uPAR to activate ADAM8/Ras/ERK-driven metastasis [PMID:36231102], and the transcription factor FOXD1 at Asn176 to stabilize it and promote its nuclear localization and BNIP3-dependent mitophagy [PMID:40083705]—while ALG3 inhibition triggers SREBP1-dependent lipid accumulation and immunogenic ferroptosis that synergizes with anti-PD1 therapy [PMID:35676564]. Human ALG3 deficiency causes a congenital disorder of glycosylation, with patient cells showing constitutive IRE1-α UPR activation and accumulation of truncated N-glycans [PMID:15108280, PMID:38597022].","teleology":[{"year":1991,"claim":"Before ALG3 was cloned, the structural defect of the alg3 mutant was unknown; NMR established the precise glycan that accumulates, defining the exact biosynthetic step that fails.","evidence":"1H NMR of oligosaccharides from alg3,sec18 yeast invertase with endoglycosidase digestion","pmids":["2005096"],"confidence":"High","gaps":["Did not identify the gene or enzyme responsible","No demonstration of which protein catalyzes the missing transfer"]},{"year":1993,"claim":"It was unclear whether the truncated Man5GlcNAc2 could be glucosylated or serve as a substrate for further processing; structural analyses showed both glucosylation of the Man5 precursor and outer-chain extension, placing ALG3 within the larger assembly pathway.","evidence":"2D DQF-COSY NMR and oligosaccharide sizing of glycans from alg3,sec18,gls1 and och1 mnn1 alg3 yeast mutants","pmids":["8505333","8253757"],"confidence":"High","gaps":["Did not identify the ALG3 gene product","Mechanism of donor/acceptor recognition unaddressed"]},{"year":1996,"claim":"The gene encoding the activity was unknown; cloning of yeast ALG3 identified an ER transmembrane protein with a KKXX retrieval motif required for dolichol-linked oligosaccharide biosynthesis beyond Man5GlcNAc2.","evidence":"Complementation cloning of an alg3 ts mutant, gene disruption, and biochemical analysis of lipid-linked oligosaccharides","pmids":["8842708"],"confidence":"High","gaps":["Did not establish whether ALG3 is the catalytic enzyme or an accessory factor","No mammalian characterization"]},{"year":2001,"claim":"Whether ALG3 was itself the mannosyltransferase or merely required for the reaction was open; immunoprecipitation of tagged ALG3 co-purified the Man5→Man6 transfer activity, identifying ALG3 as the catalytic enzyme.","evidence":"In vitro mannosyltransferase assay with [3H]Man5GlcNAc2-PP-Dol and IP of HA-tagged ALG3 from yeast membranes","pmids":["11308030"],"confidence":"High","gaps":["No structural basis for catalysis or donor selection","Metal-ion dependence of subsequent steps only partially defined"]},{"year":2004,"claim":"The conservation and glycan-processing consequences of ALG3 loss across species and the molecular basis of a human disease allele were unknown; work in Pichia and in a CDG patient extended ALG3 function to other organisms and to human pathology.","evidence":"P. pastoris alg3Δ deletion with MS glycan profiling and mannosidase digestion; RT-PCR and NMD-suppression analysis of a human ALG3 splice mutation","pmids":["15033937","15108280"],"confidence":"Medium","gaps":["Human disease mechanism inferred from transcript analysis without functional rescue","Divergent Golgi processing in Pichia not mechanistically explained"]},{"year":2018,"claim":"Human ALG3 protein processing and potential non-glycosyltransferase interactions were uncharacterized; antibody and yeast two-hybrid studies reported proteolytic processing, homodimerization, and interactions with OSBP, LRP1, and CREB3 precursor.","evidence":"Yeast two-hybrid screening with single Co-IP validation; polyclonal antibodies and subcellular fractionation","pmids":["29547901","30192950"],"confidence":"Low","gaps":["Yeast two-hybrid interactions lack reciprocal validation and functional follow-up","Reported processing not reconciled with canonical ER mannosyltransferase role","Functional consequences of compartment-specific products not established"]},{"year":2022,"claim":"Whether ALG3 has substrate-specific roles in cancer beyond bulk glycosylation was unclear; studies identified TGFBR2 and uPAR as glycosylation substrates driving stemness, radioresistance, and metastasis, and showed ALG3 loss triggers SREBP1-dependent lipid accumulation and immunogenic ferroptosis.","evidence":"Reciprocal Co-IP, lectin profiling, shRNA/CRISPR knockdown, inhibitor rescue, and in vivo xenograft/syngeneic tumor models","pmids":["33931075","36231102","35676564"],"confidence":"Medium","gaps":["Substrate-specificity mechanism for individual glycoproteins not resolved","Ferroptosis mechanism partly inferred from pathway inhibitors","Single-lab findings per substrate"]},{"year":2024,"claim":"The cellular stress consequences of human ALG3 deficiency were undefined; patient fibroblasts revealed constitutive IRE1-α UPR activation, elevated ERAD, and accumulation of truncated Man3-4 glycans.","evidence":"UPR marker Western blot and MS glycan profiling in patient-derived fibroblasts","pmids":["38597022"],"confidence":"Medium","gaps":["Causal link between glycan truncation and IRE1-α activation not dissected","Single patient-cell context"]},{"year":2025,"claim":"How ALG3 activity is regulated by signaling and whether it modulates transcription factors was unknown; AKT phosphorylation at Ser11/Ser13 was shown to control surface-receptor glycosylation, and ALG3 was shown to glycosylate FOXD1 at Asn176 to stabilize it and drive BNIP3-dependent mitophagy.","evidence":"In vitro AKT kinase assay, phospho-site mutagenesis, CRISPR depletion, Co-IP, and downstream transcriptional/drug-sensitivity assays","pmids":["40789468","40236010","40083705"],"confidence":"Medium","gaps":["FOXD1 interaction from single-lab Co-IP","How phosphorylation alters ALG3 catalytic behavior mechanistically unresolved"]},{"year":2026,"claim":"The structural basis for ALG3's accurate branched-glycan synthesis and donor selectivity was unknown; cryo-EM of pseudo-Michaelis complexes with ALG9 and ALG12 revealed the four-step mannosylation mechanism and how ALG3 discriminates Dol-P-Man from Dol-P-Glc.","evidence":"Cryo-EM of pseudo-Michaelis complexes, chemoenzymatic substrate reconstitution, MD simulations, and mutagenesis","pmids":["41807832"],"confidence":"High","gaps":["Structural impact of AKT phosphorylation not captured","Structural basis of substrate-specific glycosylation of client proteins not addressed"]},{"year":null,"claim":"How signaling-driven ALG3 phosphorylation, substrate-selective client glycosylation, and the structural catalytic mechanism integrate to produce its diverse cancer and disease phenotypes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model linking Ser11/Ser13 phosphorylation to catalysis","Determinants of selectivity for specific client glycoproteins unknown","Causal chain from glycan truncation to IRE1-α UPR and ferroptosis not fully mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,5,12]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[7,8,11]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,10,15]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,12]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[10,15]}],"complexes":[],"partners":["TGFBR2","UPAR","FOXD1","ALG9","ALG12","AKT","ADAM8"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q92685","full_name":"Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase","aliases":["Asparagine-linked glycosylation protein 3 homolog","Dol-P-Man-dependent alpha(1-3)-mannosyltransferase","Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase","Dolichyl-phosphate-mannose--glycolipid alpha-mannosyltransferase","Not56-like protein"],"length_aa":438,"mass_kda":50.1,"function":"Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase that operates in the biosynthetic pathway of dolichol-linked oligosaccharides, the glycan precursors employed in protein asparagine (N)-glycosylation. The assembly of dolichol-linked oligosaccharides begins on the cytosolic side of the endoplasmic reticulum membrane and finishes in its lumen. The sequential addition of sugars to dolichol pyrophosphate produces dolichol-linked oligosaccharides containing fourteen sugars, including two GlcNAcs, nine mannoses and three glucoses. Once assembled, the oligosaccharide is transferred from the lipid to nascent proteins by oligosaccharyltransferases. In the lumen of the endoplasmic reticulum, adds the first dolichyl beta-D-mannosyl phosphate derived mannose in an alpha-1,3 linkage to Man(5)GlcNAc(2)-PP-dolichol to produce Man(6)GlcNAc(2)-PP-dolichol (PubMed:10581255). Man(6)GlcNAc(2)-PP-dolichol is a substrate for ALG9, the following enzyme in the biosynthetic pathway (PubMed:10581255)","subcellular_location":"Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q92685/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ALG3","classification":"Not Classified","n_dependent_lines":35,"n_total_lines":1208,"dependency_fraction":0.028973509933774833},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ALG3","total_profiled":1310},"omim":[{"mim_id":"608750","title":"ALG3 ALPHA-1,3-MANNOSYLTRANSFERASE; ALG3","url":"https://www.omim.org/entry/608750"},{"mim_id":"601110","title":"CONGENITAL DISORDER OF GLYCOSYLATION, TYPE Id; CDG1D","url":"https://www.omim.org/entry/601110"},{"mim_id":"600759","title":"PRESENILIN 2; PSEN2","url":"https://www.omim.org/entry/600759"},{"mim_id":"212065","title":"CONGENITAL DISORDER OF GLYCOSYLATION, TYPE Ia; CDG1A","url":"https://www.omim.org/entry/212065"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ALG3"},"hgnc":{"alias_symbol":["NOT56L","Not56","CDGS4","D16Ertd36e"],"prev_symbol":[]},"alphafold":{"accession":"Q92685","domains":[{"cath_id":"-","chopping":"41-423","consensus_level":"medium","plddt":93.3935,"start":41,"end":423}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92685","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q92685-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q92685-F1-predicted_aligned_error_v6.png","plddt_mean":89.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ALG3","jax_strain_url":"https://www.jax.org/strain/search?query=ALG3"},"sequence":{"accession":"Q92685","fasta_url":"https://rest.uniprot.org/uniprotkb/Q92685.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q92685/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q92685"}},"corpus_meta":[{"pmid":"8560270","id":"PMC_8560270","title":"Interfering with apoptosis: Ca(2+)-binding protein ALG-2 and Alzheimer's disease gene ALG-3.","date":"1996","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/8560270","citation_count":451,"is_preprint":false},{"pmid":"20133686","id":"PMC_20133686","title":"Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans.","date":"2010","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/20133686","citation_count":186,"is_preprint":false},{"pmid":"8253757","id":"PMC_8253757","title":"Structure of the N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae.","date":"1993","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8253757","citation_count":135,"is_preprint":false},{"pmid":"8940094","id":"PMC_8940094","title":"Requirement of the familial Alzheimer's disease gene PS2 for apoptosis. Opposing effect of ALG-3.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8940094","citation_count":121,"is_preprint":false},{"pmid":"8842708","id":"PMC_8842708","title":"Cloning and characterization of the ALG3 gene of Saccharomyces cerevisiae.","date":"1996","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/8842708","citation_count":106,"is_preprint":false},{"pmid":"33931075","id":"PMC_33931075","title":"ALG3 contributes to stemness and radioresistance through regulating glycosylation of TGF-β receptor II in breast cancer.","date":"2021","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/33931075","citation_count":65,"is_preprint":false},{"pmid":"15033937","id":"PMC_15033937","title":"Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man5GlcNAc2-PP-Dol mannosyltransferase enzyme of P. pastoris.","date":"2004","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/15033937","citation_count":53,"is_preprint":false},{"pmid":"15108280","id":"PMC_15108280","title":"An activated 5' cryptic splice site in the human ALG3 gene generates a premature termination codon insensitive to nonsense-mediated mRNA decay in a new case of congenital disorder of glycosylation type Id (CDG-Id).","date":"2004","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/15108280","citation_count":51,"is_preprint":false},{"pmid":"16053906","id":"PMC_16053906","title":"CDG-Id caused by homozygosity for an ALG3 mutation due to segmental maternal isodisomy UPD3(q21.3-qter).","date":"2005","source":"European journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/16053906","citation_count":46,"is_preprint":false},{"pmid":"9164928","id":"PMC_9164928","title":"Dissociation of apoptosis and activation of IL-1beta-converting enzyme/Ced-3 proteases by ALG-2 and the truncated Alzheimer's gene ALG-3.","date":"1997","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/9164928","citation_count":46,"is_preprint":false},{"pmid":"8505333","id":"PMC_8505333","title":"Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum.","date":"1993","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8505333","citation_count":45,"is_preprint":false},{"pmid":"11308030","id":"PMC_11308030","title":"Biosynthesis of lipid-linked oligosaccharides in yeast: the ALG3 gene encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase.","date":"2001","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11308030","citation_count":41,"is_preprint":false},{"pmid":"31899049","id":"PMC_31899049","title":"ALG3 contributes to the malignancy of non-small cell lung cancer and is negatively regulated by MiR-98-5p.","date":"2019","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/31899049","citation_count":38,"is_preprint":false},{"pmid":"29880818","id":"PMC_29880818","title":"Aberrant mannosylation profile and FTX/miR-342/ALG3-axis contribute to development of drug resistance in acute myeloid leukemia.","date":"2018","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/29880818","citation_count":38,"is_preprint":false},{"pmid":"35676564","id":"PMC_35676564","title":"Inhibition of ALG3 stimulates cancer cell immunogenic ferroptosis to potentiate immunotherapy.","date":"2022","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/35676564","citation_count":33,"is_preprint":false},{"pmid":"2005096","id":"PMC_2005096","title":"Structure of Saccharomyces cerevisiae alg3, sec18 mutant oligosaccharides.","date":"1991","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2005096","citation_count":32,"is_preprint":false},{"pmid":"26126960","id":"PMC_26126960","title":"ALG3-CDG: Report of two siblings with antenatal features carrying homozygous p.Gly96Arg mutation.","date":"2015","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/26126960","citation_count":22,"is_preprint":false},{"pmid":"31067009","id":"PMC_31067009","title":"Novel variants and clinical symptoms in four new ALG3-CDG patients, review of the literature, and identification of AAGRP-ALG3 as a novel ALG3 variant with alanine and glycine-rich N-terminus.","date":"2019","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/31067009","citation_count":21,"is_preprint":false},{"pmid":"24076077","id":"PMC_24076077","title":"Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger.","date":"2013","source":"Fungal genetics and biology : FG & B","url":"https://pubmed.ncbi.nlm.nih.gov/24076077","citation_count":21,"is_preprint":false},{"pmid":"29799832","id":"PMC_29799832","title":"ALG3 Is Activated by Heat Shock Factor 2 and Promotes Breast Cancer Growth.","date":"2018","source":"Medical science monitor : international medical journal of experimental and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/29799832","citation_count":19,"is_preprint":false},{"pmid":"33084111","id":"PMC_33084111","title":"ALG3 contributes to the malignant properties of OSCC cells by regulating CDK-Cyclin pathway.","date":"2020","source":"Oral diseases","url":"https://pubmed.ncbi.nlm.nih.gov/33084111","citation_count":15,"is_preprint":false},{"pmid":"36231102","id":"PMC_36231102","title":"ALG3 Promotes Peritoneal Metastasis of Ovarian Cancer through Increasing Interaction of α1,3-mannosylated uPAR and ADAM8.","date":"2022","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/36231102","citation_count":15,"is_preprint":false},{"pmid":"35782861","id":"PMC_35782861","title":"Expression of ALG3 in Hepatocellular Carcinoma and Its Clinical Implication.","date":"2022","source":"Frontiers in molecular biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/35782861","citation_count":12,"is_preprint":false},{"pmid":"37757993","id":"PMC_37757993","title":"Yi-shen-hua-shi granules modulate immune and inflammatory damage via the ALG3/PPARγ/NF-κB pathway in the treatment of immunoglobulin a nephropathy.","date":"2023","source":"Journal of ethnopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37757993","citation_count":12,"is_preprint":false},{"pmid":"33187827","id":"PMC_33187827","title":"Fetal glycosylation defect due to ALG3 and COG5 variants detected via amniocentesis: Complex glycosylation defect with embryonic lethal phenotype.","date":"2020","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/33187827","citation_count":12,"is_preprint":false},{"pmid":"35980117","id":"PMC_35980117","title":"CircPTK2 promotes cell viability, cell cycle process, and glycolysis and inhibits cell apoptosis in acute myeloid leukemia by regulating miR-582-3p/ALG3 axis.","date":"2022","source":"Expert review of hematology","url":"https://pubmed.ncbi.nlm.nih.gov/35980117","citation_count":10,"is_preprint":false},{"pmid":"29547901","id":"PMC_29547901","title":"Molecular partners of hNOT/ALG3, the human counterpart of the Drosophila NOT and yeast ALG3 gene, suggest its involvement in distinct cellular processes relevant to congenital disorders of glycosylation, cancer, neurodegeneration and a variety of further pathologies.","date":"2018","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29547901","citation_count":9,"is_preprint":false},{"pmid":"34090370","id":"PMC_34090370","title":"ALG3-CDG: a patient with novel variants and review of the genetic and ophthalmic findings.","date":"2021","source":"BMC ophthalmology","url":"https://pubmed.ncbi.nlm.nih.gov/34090370","citation_count":7,"is_preprint":false},{"pmid":"38820773","id":"PMC_38820773","title":"Deep pan-cancer analysis and multi-omics evidence reveal that ALG3 inhibits CD8+ T cell infiltration by suppressing chemokine secretion and is associated with 5-fluorouracil sensitivity.","date":"2024","source":"Computers in biology and medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38820773","citation_count":7,"is_preprint":false},{"pmid":"38597022","id":"PMC_38597022","title":"Deficient glycan extension and endoplasmic reticulum stresses in ALG3-CDG.","date":"2024","source":"Journal of inherited metabolic disease","url":"https://pubmed.ncbi.nlm.nih.gov/38597022","citation_count":6,"is_preprint":false},{"pmid":"34549171","id":"PMC_34549171","title":"Meiotic H3K9me2 distribution is influenced by the ALG-3 and ALG-4 pathway and by poly(U) polymerase activity.","date":"2021","source":"microPublication biology","url":"https://pubmed.ncbi.nlm.nih.gov/34549171","citation_count":6,"is_preprint":false},{"pmid":"22997240","id":"PMC_22997240","title":"Identification of alg3 in the mushroom-forming fungus Schizophyllum commune and analysis of the Δalg3 knockout mutant.","date":"2012","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/22997240","citation_count":5,"is_preprint":false},{"pmid":"40083705","id":"PMC_40083705","title":"Targeting ALG3/FOXD1/BNIP3 Axis Prevents Mitophagy and Gemcitabine Resistance of Nasopharyngeal Carcinoma.","date":"2025","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40083705","citation_count":2,"is_preprint":false},{"pmid":"38329424","id":"PMC_38329424","title":"Comprehensive analysis of ALG3 in pan-cancer and validation of ALG3 as an onco-immunological biomarker in breast cancer.","date":"2024","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/38329424","citation_count":2,"is_preprint":false},{"pmid":"40789468","id":"PMC_40789468","title":"The glycosyltransferase ALG3 is an AKT substrate that regulates protein N-glycosylation.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40789468","citation_count":1,"is_preprint":false},{"pmid":"40236010","id":"PMC_40236010","title":"The glycosyltransferase ALG3 is an AKT substrate that regulates protein N-glycosylation.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40236010","citation_count":1,"is_preprint":false},{"pmid":"30192950","id":"PMC_30192950","title":"Sequential cleavage of the proteins encoded by HNOT/ALG3, the human counterpart of the Drosophila NOT and yeast ALG3 gene, results in products acting in distinct cellular compartments.","date":"2018","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30192950","citation_count":1,"is_preprint":false},{"pmid":"40905205","id":"PMC_40905205","title":"CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production.","date":"2025","source":"Plant biotechnology 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Neurophysiology","url":"https://pubmed.ncbi.nlm.nih.gov/39831946","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":23642,"output_tokens":4915,"usd":0.072326,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13026,"output_tokens":3984,"usd":0.082365,"stage2_stop_reason":"end_turn"},"total_usd":0.154691,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"The ALG3 gene of Saccharomyces cerevisiae encodes a potential ER-transmembrane protein of 458 amino acids (53 kDa) with a C-terminal KKXX-retrieval sequence; its inactivation results in accumulation of lipid-linked Man5GlcNAc2 and transfer of Endo H-resistant (underglycosylated) carbohydrates to protein by the oligosaccharyltransferase complex, establishing ALG3 as required for dolichol-linked oligosaccharide biosynthesis beyond Man5GlcNAc2.\",\n      \"method\": \"Complementation cloning of alg3 temperature-sensitive mutant; gene disruption; biochemical analysis of lipid-linked oligosaccharides and secretory protein glycosylation\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — gene cloning with functional complementation, null mutant biochemical characterization, replicated by multiple subsequent studies\",\n      \"pmids\": [\"8842708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ALG3 encodes the Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase itself (not an accessory protein): immunoprecipitation of an HA-tagged ALG3 fusion selectively co-precipitated mannosyltransferase activity converting Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol from detergent-solubilized yeast membranes, and the initial Man5→Man6 reaction is metal-ion independent whereas further elongation requires Mn2+.\",\n      \"method\": \"In vitro mannosyltransferase assay with [3H]Man5GlcNAc2-PP-Dol substrate; immunoprecipitation of HA-tagged ALG3 fusion protein to co-precipitate enzymatic activity\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic reconstitution with tagged protein immunoprecipitation, single lab but two orthogonal methods (activity assay + IP)\",\n      \"pmids\": [\"11308030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"Structural analysis of oligosaccharides from alg3,sec18 yeast by 1H NMR established that the alg3 mutant accumulates Man5GlcNAc2-PP-dolichol due to a defective alpha-1,3-mannosyltransferase required for the next elongation step, and that the transferred Man5GlcNAc2 structure is Man-alpha1,2Man-alpha1,2Man-alpha1,3(Man-alpha1,6)Man-beta1,4GlcNAc-beta1,4GlcNAc.\",\n      \"method\": \"1H NMR spectroscopy of oligosaccharides released from ER-restricted invertase in alg3,sec18 yeast; peptide-N-glycosidase F and Endo H digestion\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structural determination with biochemical validation, foundational structural proof replicated by subsequent work\",\n      \"pmids\": [\"2005096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"In the alg3,sec18,gls1 triple mutant, the majority (>75%) of ER N-linked glycosylation occurs by transfer of Man5GlcNAc2 without prior addition of the 3 glucoses normally present on the lipid-linked precursor; 2D DQF-COSY 1H NMR revealed Glc3Man5GlcNAc2 and Man8GlcNAc2 as the glucosylated species, demonstrating that the alg3 yeast still glucosylates both Man5 and Man9 precursors.\",\n      \"method\": \"500-MHz 2D DQF-COSY 1H NMR spectroscopy; Endo H and PNGase F digestion; Bio-Gel P-4 oligosaccharide sizing of invertase glycans from alg3,sec18,gls1 yeast\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structural analysis with multiple orthogonal biochemical methods, directly characterizes ALG3-dependent glycan structure in the ER\",\n      \"pmids\": [\"8505333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"In the delta-och1 mnn1 alg3 triple mutant of S. cerevisiae, N-linked oligosaccharides accumulated as Man5GlcNAc2 and Man8GlcNAc2 in total cell mannoprotein, confirming the lack of outer chain addition to the incomplete core-like oligosaccharide and the leaky phenotype of the alg3 mutation; this established that the ALG3-dependent Man8GlcNAc2 core is the substrate for OCH1-initiated alpha-1,6-polymannose outer chain addition.\",\n      \"method\": \"Structural analysis of pyridylaminated neutral oligosaccharides from purified periplasmic invertase by hydrazinolysis, N-acetylation, and chromatography\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct structural characterization of glycans in defined triple mutant, multiple orthogonal methods, replicated by subsequent studies\",\n      \"pmids\": [\"8253757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The Pichia pastoris ALG3 homolog encodes the enzyme converting Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP; deletion in an och1 background leads to secretion of Man5GlcNAc2-containing glycoproteins trimmable by alpha-1,2-mannosidase, plus additional larger glycans (Hex6-Hex12GlcNAc2) resistant to mannosidase digestion, revealing divergent Golgi processing relative to S. cerevisiae alg3.\",\n      \"method\": \"Gene deletion (alg3Δ) in P. pastoris; glycan profiling by mass spectrometry; in vitro alpha-1,2-mannosidase digestion of secreted glycoproteins\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — functional gene deletion with direct biochemical characterization of glycan products, two orthogonal methods (MS + enzymatic digestion)\",\n      \"pmids\": [\"15033937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"A silent mutation (c.165C>T) in exon 1 of the human ALG3 gene activates a cryptic donor splice site, deleting c.160_196 and generating a premature termination codon after the first N-terminal transmembrane domain (p.Val54fsX66); the resulting truncated ALG3 mRNA escapes nonsense-mediated mRNA decay (NMD) despite fulfilling NMD criteria, as demonstrated by cycloheximide treatment having no effect on transcript levels.\",\n      \"method\": \"RT-PCR analysis of patient transcripts; cycloheximide treatment to suppress NMD; expression studies in patient cells\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct patient transcript analysis with NMD suppression experiment, two methods but single lab and disease-context study\",\n      \"pmids\": [\"15108280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALG3 promotes radioresistance and cancer stemness in breast cancer cells by inducing glycosylation of TGF-β receptor II (TGFBR2); both pharmacological inhibition of glycosylation (tunicamycin) and TGFBR2 inhibition (LY2109761) abrogated the stimulatory effects of ALG3 overexpression on stemness and radioresistance, establishing TGFBR2 glycosylation as the mechanistic link.\",\n      \"method\": \"Immunoprecipitation; Western blot; shRNA knockdown; overexpression in breast cancer cell lines; orthotopic xenograft models; pharmacological inhibitor experiments\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and functional rescue/inhibition experiments in vitro and in vivo, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"33931075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALG3 promotes ovarian cancer peritoneal metastasis by catalyzing alpha-1,3-mannosylation of urokinase plasminogen activator receptor (uPAR), which enhances uPA/uPAR activation and the interaction between uPAR and ADAM8, thereby activating the ADAM8/Ras/ERK signaling pathway.\",\n      \"method\": \"Lectin chip; Western blot; Lectin blot; Co-immunoprecipitation of uPAR and ADAM8; ALG3 knockdown in ovarian cancer cells; mouse peritoneal metastasis model\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (lectin profiling, Co-IP, in vivo model), single lab\",\n      \"pmids\": [\"36231102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALG3 inhibition induces deficiency of post-translational N-linked glycosylation, leading to excessive lipid accumulation through SREBP1-dependent lipogenesis in cancer cells; N-linked glycosylation deficiency-mediated lipid hyperperoxidation triggers immunogenic ferroptosis and promotes a pro-inflammatory tumor microenvironment that boosts anti-tumor immune responses and synergizes with anti-PD1 therapy.\",\n      \"method\": \"CRISPR/Cas9 ALG3 deletion in mouse cancer cells; lipid accumulation assays; SREBP1 pathway analysis; in vivo syngeneic tumor models with anti-PD1 combination; tunicamycin treatment as pharmacological comparator\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with multiple functional readouts and in vivo validation, single lab, mechanism partially inferred from pathway inhibitors\",\n      \"pmids\": [\"35676564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AKT directly phosphorylates ALG3 at Ser11/Ser13 in the amino-terminal region downstream of the PI3K/AKT pathway in growth factor-stimulated and PI3K/AKT-hyperactive cancer cells; CRISPR/Cas9 depletion of ALG3 causes improper glycan formation, ER stress, unfolded protein response induction, and impaired cell proliferation; phosphorylation at Ser11/Ser13 is required for proper glycosylation of cell surface receptors EGFR, HER3, and E-cadherin.\",\n      \"method\": \"In vitro AKT kinase assay with ALG3 substrate; phosphorylation site identification (Ser11/Ser13); CRISPR/Cas9 ALG3 depletion; phospho-site mutagenesis; glycosylation analysis of EGFR, HER3, E-cadherin by Western blot; UPR marker analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase assay plus CRISPR KO plus mutagenesis plus substrate glycosylation readouts, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"40789468\", \"40236010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALG3 directly interacts with transcription factor FOXD1 and induces N-glycosylation at Asn176 of FOXD1, which increases FOXD1 protein stability and nuclear localization; this in turn allows FOXD1 to transcriptionally activate BNIP3, promoting mitophagy and gemcitabine resistance in nasopharyngeal carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation of ALG3 and FOXD1; glycosylation site mutagenesis (Asn176); nuclear fractionation; Western blot; BNIP3 transcriptional reporter/ChIP; cell proliferation and drug sensitivity assays\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus mutagenesis plus functional downstream pathway analysis, single lab\",\n      \"pmids\": [\"40083705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Cryo-EM structures of pseudo-Michaelis complexes of ALG3, ALG9, and ALG12 at high resolution revealed how the branched glycan is accurately synthesized in four consecutive mannosylation reactions converting GlcNAc2Man5 to GlcNAc2Man9; molecular dynamics simulations and mutagenesis uncovered the mechanism by which ALG3 selects the dolichylphosphomannose donor over dolichylphosphoglucose; the structures also provide mechanistic explanations for enzyme dysfunction in CDGs.\",\n      \"method\": \"Cryo-electron microscopy of pseudo-Michaelis complexes; chemoenzymatic synthesis of lipid-linked glycan analogs for in vitro reconstitution; molecular dynamics simulations; site-directed mutagenesis\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure combined with in vitro reconstitution, MD simulations, and mutagenesis in a single rigorous study\",\n      \"pmids\": [\"41807832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The human ALG3 protein (hNOT-1/ALG3-1) forms homodimers and interacts in vivo with OSBP, OSBPL9, LRP1, SYPL1, and the transcription factor CREB3 precursor (but not with CREB3 proteolytic products); binding to CREB3 precursor is a prerequisite for CREB3's proteolytic activation; different post-translationally processed ALG3 products localize to distinct cellular compartments and interact with different partners.\",\n      \"method\": \"Yeast two-hybrid screening; co-immunoprecipitation validation of selected interactions (OSBP, OSBPL9, LRP1, CREB3) in vivo\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid plus single Co-IP validation, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"29547901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The human ALG3 (hNOT-1/ALG3-1) protein undergoes N-glycosylation and sequential proteolytic cleavage generating derivatives destined to distinct cellular compartments; truncated transcripts are not translated; two full-length precursor proteins (hNOT-1 and hNOT-4) are encoded by alternatively spliced transcripts differing at exon 1.\",\n      \"method\": \"Polyclonal antibodies against diverse protein regions; subcellular fractionation; Western blot analysis of N-glycosylation; RT-PCR and translation analysis of truncated transcripts\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, antibody-based localization and biochemical characterization without functional consequence established\",\n      \"pmids\": [\"30192950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALG3 deficiency in patient-derived fibroblasts results in constitutive activation of the unfolded protein response via the IRE1-α pathway, increased ER-associated degradation activity, and accumulation of N-linked Man3-4 glycans in cellular and secreted glycoproteins; in transferrin, the Man5 intermediate is further processed to a mono-antennary glycan NeuAc1Gal1GlcNAc1Man3GlcNAc2.\",\n      \"method\": \"Western blot of UPR markers in patient-derived fibroblasts; glycoprotein glycan profiling by mass spectrometry; IRE1-α pathway activation analysis\",\n      \"journal\": \"Journal of inherited metabolic disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with multiple biochemical readouts (UPR markers, glycan MS profiling), single lab, two orthogonal methods\",\n      \"pmids\": [\"38597022\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Human ALG3 (alpha-1,3-mannosyltransferase) is an ER-resident transmembrane enzyme that catalyzes the first luminal mannosylation step in N-glycan biosynthesis—adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 to form Dol-PP-Man6GlcNAc2—a rate-limiting reaction whose structural basis has been resolved by cryo-EM; ALG3 activity is regulated by direct AKT-mediated phosphorylation at Ser11/Ser13, which is required for proper glycosylation of surface receptors including EGFR, HER3, and E-cadherin; ALG3 also N-glycosylates specific substrates such as TGF-β receptor II and FOXD1, thereby modulating TGF-β signaling, FOXD1 stability/nuclear localization, and downstream transcriptional programs; loss of ALG3 function causes ER stress, constitutive UPR activation via IRE1-α, impaired cell proliferation, and, in cancer contexts, immunogenic ferroptosis through SREBP1-dependent lipid accumulation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALG3 is an ER-resident transmembrane alpha-1,3-mannosyltransferase that performs the first luminal mannosylation in dolichol-linked N-glycan biosynthesis, transferring mannose from Dol-P-Man onto Dol-PP-Man5GlcNAc2 to extend the precursor beyond Man5GlcNAc2 [#0, #1, #2]. Loss of this activity causes accumulation of lipid-linked Man5GlcNAc2 and transfer of underglycosylated, truncated glycans to protein [#0, #2, #3], and the ALG3-dependent core serves as the substrate for downstream elongation and outer-chain addition [#4]. Cryo-EM of pseudo-Michaelis complexes resolved how ALG3 builds the branched glycan in concert with ALG9 and ALG12 and how it selects the dolichylphosphomannose donor over dolichylphosphoglucose, and provided a structural basis for ALG3 dysfunction in congenital disorders of glycosylation [#12]. ALG3 activity is controlled by the PI3K/AKT pathway through direct AKT phosphorylation at Ser11/Ser13, which is required for proper N-glycosylation of cell-surface receptors including EGFR, HER3, and E-cadherin; ALG3 depletion produces improper glycan formation, ER stress, UPR induction, and impaired proliferation [#10]. In cancer contexts, ALG3 glycosylates specific substrates—TGF-\\u03b2 receptor II to drive stemness and radioresistance [#7], uPAR to activate ADAM8/Ras/ERK-driven metastasis [#8], and the transcription factor FOXD1 at Asn176 to stabilize it and promote its nuclear localization and BNIP3-dependent mitophagy [#11]—while ALG3 inhibition triggers SREBP1-dependent lipid accumulation and immunogenic ferroptosis that synergizes with anti-PD1 therapy [#9]. Human ALG3 deficiency causes a congenital disorder of glycosylation, with patient cells showing constitutive IRE1-\\u03b1 UPR activation and accumulation of truncated N-glycans [#6, #15].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Before ALG3 was cloned, the structural defect of the alg3 mutant was unknown; NMR established the precise glycan that accumulates, defining the exact biosynthetic step that fails.\",\n      \"evidence\": \"1H NMR of oligosaccharides from alg3,sec18 yeast invertase with endoglycosidase digestion\",\n      \"pmids\": [\"2005096\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the gene or enzyme responsible\", \"No demonstration of which protein catalyzes the missing transfer\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"It was unclear whether the truncated Man5GlcNAc2 could be glucosylated or serve as a substrate for further processing; structural analyses showed both glucosylation of the Man5 precursor and outer-chain extension, placing ALG3 within the larger assembly pathway.\",\n      \"evidence\": \"2D DQF-COSY NMR and oligosaccharide sizing of glycans from alg3,sec18,gls1 and och1 mnn1 alg3 yeast mutants\",\n      \"pmids\": [\"8505333\", \"8253757\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the ALG3 gene product\", \"Mechanism of donor/acceptor recognition unaddressed\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"The gene encoding the activity was unknown; cloning of yeast ALG3 identified an ER transmembrane protein with a KKXX retrieval motif required for dolichol-linked oligosaccharide biosynthesis beyond Man5GlcNAc2.\",\n      \"evidence\": \"Complementation cloning of an alg3 ts mutant, gene disruption, and biochemical analysis of lipid-linked oligosaccharides\",\n      \"pmids\": [\"8842708\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish whether ALG3 is the catalytic enzyme or an accessory factor\", \"No mammalian characterization\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Whether ALG3 was itself the mannosyltransferase or merely required for the reaction was open; immunoprecipitation of tagged ALG3 co-purified the Man5\\u2192Man6 transfer activity, identifying ALG3 as the catalytic enzyme.\",\n      \"evidence\": \"In vitro mannosyltransferase assay with [3H]Man5GlcNAc2-PP-Dol and IP of HA-tagged ALG3 from yeast membranes\",\n      \"pmids\": [\"11308030\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural basis for catalysis or donor selection\", \"Metal-ion dependence of subsequent steps only partially defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The conservation and glycan-processing consequences of ALG3 loss across species and the molecular basis of a human disease allele were unknown; work in Pichia and in a CDG patient extended ALG3 function to other organisms and to human pathology.\",\n      \"evidence\": \"P. pastoris alg3\\u0394 deletion with MS glycan profiling and mannosidase digestion; RT-PCR and NMD-suppression analysis of a human ALG3 splice mutation\",\n      \"pmids\": [\"15033937\", \"15108280\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Human disease mechanism inferred from transcript analysis without functional rescue\", \"Divergent Golgi processing in Pichia not mechanistically explained\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Human ALG3 protein processing and potential non-glycosyltransferase interactions were uncharacterized; antibody and yeast two-hybrid studies reported proteolytic processing, homodimerization, and interactions with OSBP, LRP1, and CREB3 precursor.\",\n      \"evidence\": \"Yeast two-hybrid screening with single Co-IP validation; polyclonal antibodies and subcellular fractionation\",\n      \"pmids\": [\"29547901\", \"30192950\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Yeast two-hybrid interactions lack reciprocal validation and functional follow-up\", \"Reported processing not reconciled with canonical ER mannosyltransferase role\", \"Functional consequences of compartment-specific products not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Whether ALG3 has substrate-specific roles in cancer beyond bulk glycosylation was unclear; studies identified TGFBR2 and uPAR as glycosylation substrates driving stemness, radioresistance, and metastasis, and showed ALG3 loss triggers SREBP1-dependent lipid accumulation and immunogenic ferroptosis.\",\n      \"evidence\": \"Reciprocal Co-IP, lectin profiling, shRNA/CRISPR knockdown, inhibitor rescue, and in vivo xenograft/syngeneic tumor models\",\n      \"pmids\": [\"33931075\", \"36231102\", \"35676564\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate-specificity mechanism for individual glycoproteins not resolved\", \"Ferroptosis mechanism partly inferred from pathway inhibitors\", \"Single-lab findings per substrate\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The cellular stress consequences of human ALG3 deficiency were undefined; patient fibroblasts revealed constitutive IRE1-\\u03b1 UPR activation, elevated ERAD, and accumulation of truncated Man3-4 glycans.\",\n      \"evidence\": \"UPR marker Western blot and MS glycan profiling in patient-derived fibroblasts\",\n      \"pmids\": [\"38597022\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal link between glycan truncation and IRE1-\\u03b1 activation not dissected\", \"Single patient-cell context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"How ALG3 activity is regulated by signaling and whether it modulates transcription factors was unknown; AKT phosphorylation at Ser11/Ser13 was shown to control surface-receptor glycosylation, and ALG3 was shown to glycosylate FOXD1 at Asn176 to stabilize it and drive BNIP3-dependent mitophagy.\",\n      \"evidence\": \"In vitro AKT kinase assay, phospho-site mutagenesis, CRISPR depletion, Co-IP, and downstream transcriptional/drug-sensitivity assays\",\n      \"pmids\": [\"40789468\", \"40236010\", \"40083705\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"FOXD1 interaction from single-lab Co-IP\", \"How phosphorylation alters ALG3 catalytic behavior mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"The structural basis for ALG3's accurate branched-glycan synthesis and donor selectivity was unknown; cryo-EM of pseudo-Michaelis complexes with ALG9 and ALG12 revealed the four-step mannosylation mechanism and how ALG3 discriminates Dol-P-Man from Dol-P-Glc.\",\n      \"evidence\": \"Cryo-EM of pseudo-Michaelis complexes, chemoenzymatic substrate reconstitution, MD simulations, and mutagenesis\",\n      \"pmids\": [\"41807832\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural impact of AKT phosphorylation not captured\", \"Structural basis of substrate-specific glycosylation of client proteins not addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How signaling-driven ALG3 phosphorylation, substrate-selective client glycosylation, and the structural catalytic mechanism integrate to produce its diverse cancer and disease phenotypes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model linking Ser11/Ser13 phosphorylation to catalysis\", \"Determinants of selectivity for specific client glycoproteins unknown\", \"Causal chain from glycan truncation to IRE1-\\u03b1 UPR and ferroptosis not fully mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 5, 12]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [7, 8, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 10, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 12]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [10, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"TGFBR2\", \"uPAR\", \"FOXD1\", \"ALG9\", \"ALG12\", \"AKT\", \"ADAM8\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}