{"gene":"TG_273550","gene_symbol":"POFUT2","run_date":"2026-09-08T14:08:30","timeline":{"discoveries":[{"year":2018,"finding":"T. gondii POFUT2 (encoded by TGGT1_273550) is the enzyme responsible for O-fucosylation of MIC2 TSR domains; disruption of POFUT2 abolished O-fucosylation of MIC2 (detected by anti-GlcFuc disaccharide antibody) and markedly reduced cellular MIC2 levels. In 10-15% of Δpofut2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes, indicating a role for O-fucosylation in MIC2 processing and trafficking.","method":"Gene knockout (Δpofut2), MS analysis of glycosylation, antibody detection of GlcFuc disaccharide, immunolocalization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and biochemical evidence (KO + MS glycan detection + localization), replicated across two independent studies","pmids":["30538131"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"Loss of POFUT2 (Δpofut2) in T. gondii resulted in reduced host cell attachment and invasion comparable to the Δmic2 phenotype, and decreased dissemination in human foreskin fibroblasts, establishing that O-fucosylation of TSRs by POFUT2 is required for efficient host cell invasion.","method":"Gene knockout, host cell attachment and invasion assays, plaque/dissemination assay in human foreskin fibroblasts","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined cellular phenotype (invasion/attachment), single lab; contradicted by a second study","pmids":["30538131"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"TgPOFUT2 (TGGT1_273550) mediates O-glycosylation of at least three O-linked sites in MIC2 TSR domains with >95% occupancy, as confirmed by affinity-purified MIC2 MS/MS analysis and enzymatic digestion; loss of TgPOFUT2 had only a modest impact on MIC2 protein levels and the wider proteome, and did not significantly affect plaque formation or tachyzoite invasion.","method":"Gene knockout, affinity purification of MIC2, MS/MS glycan site mapping, enzymatic digestion assays, plaque assay, invasion assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — MS/MS glycan mapping + KO phenotyping with multiple readouts, single lab; conflicts with Bandini et al. 2018 on invasion phenotype severity","pmids":["30514763"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"MS analysis of T. gondii MIC2 revealed that the four TSRs containing POFUT2 acceptor sites are modified by a dHexHex (GlcFuc) disaccharide, and three TSR Trp residues are additionally modified with C-mannose, demonstrating co-occurrence of O-fucosylation and C-mannosylation on MIC2.","method":"Mass spectrometry analysis of affinity-purified MIC2","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct MS characterization of glycan structures, replicated by independent lab (Khurana et al. 2018)","pmids":["30538131","30514763"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"Disruption of the putative GDP-fucose transporter gene NST2 also abolished MIC2 O-fucosylation and phenocopied the Δpofut2 invasion defect, placing POFUT2 in a pathway dependent on NST2-mediated GDP-fucose transport into the secretory pathway.","method":"Gene knockout (Δnst2), antibody-based detection of O-fucosylation, host cell invasion assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via two independent KOs with consistent phenotype, single lab","pmids":["30538131"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"T. gondii POFUT2 (TGGT1_273550) is the O-fucosyltransferase that modifies thrombospondin type I repeats of the microneme adhesin MIC2 with a GlcFuc disaccharide in the secretory pathway, a modification dependent on the GDP-fucose transporter NST2; O-fucosylation by POFUT2 is required for proper MIC2 trafficking to micronemes and for its stability, though the functional impact on host cell invasion remains contested between two independent studies."},"narrative":{"mechanistic_narrative":"POFUT2 is the protein O-fucosyltransferase of Toxoplasma gondii (encoded by TGGT1_273550) that installs O-linked fucose on the thrombospondin type I repeat (TSR) domains of the microneme adhesin MIC2 within the secretory pathway [PMID:30538131, PMID:30514763]. Genetic disruption abolishes MIC2 O-fucosylation, and mass spectrometry shows that at least three TSR acceptor sites are modified with high (>95%) occupancy by a GlcFuc (dHexHex) disaccharide, a modification that co-occurs with C-mannosylation of TSR tryptophans on the same protein [PMID:30514763, PMID:30538131]. This O-fucosylation depends on GDP-fucose delivery into the secretory pathway by the transporter NST2, whose disruption phenocopies loss of POFUT2 [PMID:30538131]. Functionally, POFUT2-mediated O-fucosylation supports MIC2 stability and trafficking to the micronemes: loss of the enzyme reduces cellular MIC2 and causes a fraction of MIC2 to accumulate earlier in the secretory pathway [PMID:30538131]. The consequence for host cell invasion is divergent across studies, with one reporting an invasion and attachment defect comparable to Δmic2 [PMID:30538131] and another finding little impact on invasion or plaque formation [PMID:30514763].","teleology":[{"year":2018,"claim":"Establishing that POFUT2 is the enzyme responsible for MIC2 TSR O-fucosylation answered what glycosyltransferase generates this modification and linked it to MIC2 processing and trafficking.","evidence":"Δpofut2 knockout with MS glycan analysis, anti-GlcFuc antibody detection, and immunolocalization in T. gondii","pmids":["30538131"],"confidence":"High","gaps":["Structural basis of substrate recognition not resolved","Mechanism by which O-fucose promotes MIC2 folding/trafficking not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Mapping the glycan structures defined the chemistry of the modification, showing GlcFuc disaccharide addition at TSR acceptor sites co-occurring with TSR Trp C-mannosylation.","evidence":"MS/MS glycan site mapping and enzymatic digestion of affinity-purified MIC2 across two independent labs","pmids":["30538131","30514763"],"confidence":"High","gaps":["Whether C-mannosylation and O-fucosylation are functionally interdependent not tested","Full inventory of POFUT2 substrates beyond MIC2 not determined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Disruption of NST2 placed POFUT2 in a GDP-fucose-dependent pathway, identifying the upstream nucleotide-sugar supply required for the modification.","evidence":"Δnst2 knockout with antibody-based O-fucosylation detection and invasion assay in T. gondii","pmids":["30538131"],"confidence":"Medium","gaps":["Direct demonstration of NST2 GDP-fucose transport activity not shown","Whether NST2 serves other fucosyltransferases not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Phenotyping Δpofut2 addressed whether O-fucosylation is required for host cell invasion, but the two studies reached divergent conclusions on the severity of the invasion defect.","evidence":"Independent Δpofut2 knockouts with attachment, invasion, plaque and dissemination assays in human foreskin fibroblasts","pmids":["30538131","30514763"],"confidence":"Medium","gaps":["Source of the invasion-phenotype discrepancy between labs unresolved","Contribution of residual MIC2 versus trafficking defect to invasion not separated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The molecular mechanism by which O-fucosylation stabilizes and traffics MIC2, and the reason for the discordant invasion phenotypes, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of POFUT2-TSR recognition","Full substrate repertoire beyond MIC2 unknown","Basis of cross-study invasion discrepancy undetermined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["MIC2 (SUBSTRATE ADHESIN BEARING TSR DOMAINS)","NST2 (GDP-FUCOSE TRANSPORTER, UPSTREAM IN THE SAME PATHWAY)"],"other_free_text":["Modifies TSR domains with a GlcFuc (dHexHex) disaccharide at >95% occupancy at three or more O-linked sites","O-fucosylation co-occurs with C-mannosylation of TSR tryptophan residues on MIC2","Loss of POFUT2 reduces cellular MIC2 and causes partial accumulation of MIC2 earlier in the secretory pathway rather than at micronemes"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"30538131","id":"PMC_30538131","title":"O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30538131","citation_count":23,"is_preprint":false},{"pmid":"30514763","id":"PMC_30514763","title":"Protein O-fucosyltransferase 2-mediated O-glycosylation of the adhesin MIC2 is dispensable for Toxoplasma gondii tachyzoite infection.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30514763","citation_count":17,"is_preprint":false},{"pmid":"33158988","id":"PMC_33158988","title":"The nucleocytosolic O-fucosyltransferase SPINDLY affects protein expression and virulence in Toxoplasma gondii.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33158988","citation_count":13,"is_preprint":false},{"pmid":"36250576","id":"PMC_36250576","title":"Spindly is a nucleocytosolic O-fucosyltransferase in Dictyostelium and related proteins are widespread in protists and bacteria.","date":"2023","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/36250576","citation_count":6,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4788,"output_tokens":1502,"usd":0.018447,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6605,"output_tokens":2250,"usd":0.044637,"stage2_stop_reason":"end_turn"},"total_usd":0.063084,"stage1_batch_id":"msgbatch_015xmQKDRqnTh9anE4m7LiD6","stage2_batch_id":"msgbatch_01JLpkFTVhoQBNgqHCKFrVVU","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"T. gondii POFUT2 (encoded by TGGT1_273550) is the enzyme responsible for O-fucosylation of MIC2 TSR domains; disruption of POFUT2 abolished O-fucosylation of MIC2 (detected by anti-GlcFuc disaccharide antibody) and markedly reduced cellular MIC2 levels. In 10-15% of Δpofut2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes, indicating a role for O-fucosylation in MIC2 processing and trafficking.\",\n      \"method\": \"Gene knockout (Δpofut2), MS analysis of glycosylation, antibody detection of GlcFuc disaccharide, immunolocalization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and biochemical evidence (KO + MS glycan detection + localization), replicated across two independent studies\",\n      \"pmids\": [\"30538131\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Loss of POFUT2 (Δpofut2) in T. gondii resulted in reduced host cell attachment and invasion comparable to the Δmic2 phenotype, and decreased dissemination in human foreskin fibroblasts, establishing that O-fucosylation of TSRs by POFUT2 is required for efficient host cell invasion.\",\n      \"method\": \"Gene knockout, host cell attachment and invasion assays, plaque/dissemination assay in human foreskin fibroblasts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined cellular phenotype (invasion/attachment), single lab; contradicted by a second study\",\n      \"pmids\": [\"30538131\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TgPOFUT2 (TGGT1_273550) mediates O-glycosylation of at least three O-linked sites in MIC2 TSR domains with >95% occupancy, as confirmed by affinity-purified MIC2 MS/MS analysis and enzymatic digestion; loss of TgPOFUT2 had only a modest impact on MIC2 protein levels and the wider proteome, and did not significantly affect plaque formation or tachyzoite invasion.\",\n      \"method\": \"Gene knockout, affinity purification of MIC2, MS/MS glycan site mapping, enzymatic digestion assays, plaque assay, invasion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — MS/MS glycan mapping + KO phenotyping with multiple readouts, single lab; conflicts with Bandini et al. 2018 on invasion phenotype severity\",\n      \"pmids\": [\"30514763\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MS analysis of T. gondii MIC2 revealed that the four TSRs containing POFUT2 acceptor sites are modified by a dHexHex (GlcFuc) disaccharide, and three TSR Trp residues are additionally modified with C-mannose, demonstrating co-occurrence of O-fucosylation and C-mannosylation on MIC2.\",\n      \"method\": \"Mass spectrometry analysis of affinity-purified MIC2\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct MS characterization of glycan structures, replicated by independent lab (Khurana et al. 2018)\",\n      \"pmids\": [\"30538131\", \"30514763\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Disruption of the putative GDP-fucose transporter gene NST2 also abolished MIC2 O-fucosylation and phenocopied the Δpofut2 invasion defect, placing POFUT2 in a pathway dependent on NST2-mediated GDP-fucose transport into the secretory pathway.\",\n      \"method\": \"Gene knockout (Δnst2), antibody-based detection of O-fucosylation, host cell invasion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via two independent KOs with consistent phenotype, single lab\",\n      \"pmids\": [\"30538131\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"T. gondii POFUT2 (TGGT1_273550) is the O-fucosyltransferase that modifies thrombospondin type I repeats of the microneme adhesin MIC2 with a GlcFuc disaccharide in the secretory pathway, a modification dependent on the GDP-fucose transporter NST2; O-fucosylation by POFUT2 is required for proper MIC2 trafficking to micronemes and for its stability, though the functional impact on host cell invasion remains contested between two independent studies.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"POFUT2 is the protein O-fucosyltransferase of Toxoplasma gondii (encoded by TGGT1_273550) that installs O-linked fucose on the thrombospondin type I repeat (TSR) domains of the microneme adhesin MIC2 within the secretory pathway [#0, #2]. Genetic disruption abolishes MIC2 O-fucosylation, and mass spectrometry shows that at least three TSR acceptor sites are modified with high (>95%) occupancy by a GlcFuc (dHexHex) disaccharide, a modification that co-occurs with C-mannosylation of TSR tryptophans on the same protein [#2, #3]. This O-fucosylation depends on GDP-fucose delivery into the secretory pathway by the transporter NST2, whose disruption phenocopies loss of POFUT2 [#4]. Functionally, POFUT2-mediated O-fucosylation supports MIC2 stability and trafficking to the micronemes: loss of the enzyme reduces cellular MIC2 and causes a fraction of MIC2 to accumulate earlier in the secretory pathway [#0]. The consequence for host cell invasion is divergent across studies, with one reporting an invasion and attachment defect comparable to \\u0394mic2 [#1] and another finding little impact on invasion or plaque formation [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"Establishing that POFUT2 is the enzyme responsible for MIC2 TSR O-fucosylation answered what glycosyltransferase generates this modification and linked it to MIC2 processing and trafficking.\",\n      \"evidence\": \"\\u0394pofut2 knockout with MS glycan analysis, anti-GlcFuc antibody detection, and immunolocalization in T. gondii\",\n      \"pmids\": [\"30538131\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of substrate recognition not resolved\", \"Mechanism by which O-fucose promotes MIC2 folding/trafficking not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Mapping the glycan structures defined the chemistry of the modification, showing GlcFuc disaccharide addition at TSR acceptor sites co-occurring with TSR Trp C-mannosylation.\",\n      \"evidence\": \"MS/MS glycan site mapping and enzymatic digestion of affinity-purified MIC2 across two independent labs\",\n      \"pmids\": [\"30538131\", \"30514763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether C-mannosylation and O-fucosylation are functionally interdependent not tested\", \"Full inventory of POFUT2 substrates beyond MIC2 not determined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Disruption of NST2 placed POFUT2 in a GDP-fucose-dependent pathway, identifying the upstream nucleotide-sugar supply required for the modification.\",\n      \"evidence\": \"\\u0394nst2 knockout with antibody-based O-fucosylation detection and invasion assay in T. gondii\",\n      \"pmids\": [\"30538131\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demonstration of NST2 GDP-fucose transport activity not shown\", \"Whether NST2 serves other fucosyltransferases not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Phenotyping \\u0394pofut2 addressed whether O-fucosylation is required for host cell invasion, but the two studies reached divergent conclusions on the severity of the invasion defect.\",\n      \"evidence\": \"Independent \\u0394pofut2 knockouts with attachment, invasion, plaque and dissemination assays in human foreskin fibroblasts\",\n      \"pmids\": [\"30538131\", \"30514763\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Source of the invasion-phenotype discrepancy between labs unresolved\", \"Contribution of residual MIC2 versus trafficking defect to invasion not separated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular mechanism by which O-fucosylation stabilizes and traffics MIC2, and the reason for the discordant invasion phenotypes, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of POFUT2-TSR recognition\", \"Full substrate repertoire beyond MIC2 unknown\", \"Basis of cross-study invasion discrepancy undetermined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0046922\", \"supporting_discovery_ids\": [0, 2, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0036065\", \"supporting_discovery_ids\": [0, 3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MIC2 (substrate adhesin bearing TSR domains)\",\n      \"NST2 (GDP-fucose transporter, upstream in the same pathway)\"\n    ],\n    \"other_free_text\": [\n      \"Modifies TSR domains with a GlcFuc (dHexHex) disaccharide at >95% occupancy at three or more O-linked sites\",\n      \"O-fucosylation co-occurs with C-mannosylation of TSR tryptophan residues on MIC2\",\n      \"Loss of POFUT2 reduces cellular MIC2 and causes partial accumulation of MIC2 earlier in the secretory pathway rather than at micronemes\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_273550","name":"POFUT2","product":"GDP-fucose protein O-fucosyltransferase 2","uniprot_acc":"S8EYY4","chromosome":"TGME49_chrVIII","start_pos":3405913},{"strain":"GT1","gene_id":"TGGT1_273550","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrVIII","start_pos":3402961},{"strain":"RH88","gene_id":"TGRH88_078470","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023089","start_pos":8514346},{"strain":"VEG","gene_id":"TGVEG_273550","name":null,"product":"GDP-fucose protein O-fucosyltransferase","uniprot_acc":"A0A125YVF4","chromosome":"TGVEG_chrVIII","start_pos":3415428}]}