{"gene":"TG_208030","gene_symbol":"MIC4","run_date":"2026-09-08T11:06:18","timeline":{"discoveries":[{"year":2000,"finding":"MIC4 is a 61-kDa protein secreted by Toxoplasma gondii tachyzoites that localizes to the micronemes of all invasive forms (tachyzoites, bradyzoites, sporozoites, merozoites). It is proteolytically processed at both N- and C-termini only upon release from the organelle. The adhesive motif maps to the most C-terminal (fifth/sixth) apple domain, and MIC4 binds efficiently to host cells.","method":"Protein purification and microsequencing, gene cloning, immunolocalization, deletion/domain mapping assays, host cell binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (purification, sequencing, localization, domain mapping, binding assays) in a single rigorous study","pmids":["11053441"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"finding":"MIC6 functions as an escorter for MIC1 and MIC4, forming a trimeric complex (MIC4-MIC1-MIC6) that is required for accurate targeting of MIC1 and MIC4 to the micronemes. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. MIC6 carries a sorting signal in its cytoplasmic tail, while its association with MIC1 involves a lumenal EGF-like domain. MIC1 is linked to a quality control system and is absolutely required for the complex to leave early secretory compartments.","method":"Gene disruption (knockouts of MIC1, MIC4, MIC6), domain mapping, Co-IP, sorting/trafficking assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockouts combined with domain mapping and interaction studies, multiple orthogonal methods","pmids":["11157983"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"finding":"MIC1 and MIC4 co-purify from tachyzoite soluble antigen by affinity chromatography on immobilized lactose, consistent with their physical association. The lectin activity (hemagglutination inhibited by beta-galactosides) was attributed to MIC1, not to native MIC4.","method":"Affinity chromatography (lactose column), N-terminal microsequencing, hemagglutination inhibition assay, blot overlay","journal":"Glycobiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — biochemical co-purification and functional assay, single lab, finding partially attributed to MIC1 rather than MIC4","pmids":["11447133"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2000,"finding":"MIC4, or a MIC4-like protein, is expressed in macrogametes and associated with oocyst wall formation. By immuno-electron microscopy, MIC4 antigen was detected in a subset of wall forming body type 1 (WFB1a) dense granules, which sequentially release their contents to form an anti-MIC4-positive outer veil on the developing oocyst wall.","method":"Immunocytochemistry, immuno-electron microscopy","journal":"International journal for parasitology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct immuno-EM localization, single lab; caveat that identity as MIC4 vs. cross-reacting protein could not be confirmed by western blot","pmids":["11027789"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2005,"finding":"The N-terminal TSR1-like domains of TgMIC1 promote association with TgMIC4. The C-terminal domain of TgMIC1 adopts a novel Galectin-like fold (without carbohydrate-binding properties) that interacts with and stabilizes MIC6, providing the structural basis for a quality-control mechanism enabling the MIC4-MIC1-MIC6 complex to exit early secretory compartments and traffic to micronemes.","method":"X-ray crystallography (3D structure of TgMIC1 C-terminal domain), NMR, interaction assays, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with functional interaction mapping and mutagenesis in a single rigorous study","pmids":["16166092"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"The microneme subtilisin protease TgSUB1, anchored via a GPI moiety, is responsible for proteolytic trimming of MIC4 (and MIC2, M2AP) after secretion onto the parasite surface. Loss of TgSUB1 abolishes this processing, decreases cell attachment and gliding efficiency, and reduces invasion rates in vitro and virulence in mice.","method":"Gene knockout (Δsub1), complementation with full-length and ΔGPI-SUB1 constructs, surface processing assays, invasion and gliding assays, mouse virulence assay","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with complementation controls, multiple orthogonal functional readouts","pmids":["20678172"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"finding":"The crystal structure of TgMIC4 reveals six apple domains; the fifth domain displays potent galactose-binding activity and is cleaved from the complex during parasite invasion. TgMIC4 binds a variety of galactose-containing carbohydrate ligands.","method":"X-ray crystallography, carbohydrate-binding assays (glycan array), structural mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with carbohydrate-binding specificity mapping and identification of cleaved domain, single rigorous study with multiple methods","pmids":["22399295"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"The cytoplasmic tail of MIC6 interacts with parasite aldolase, connecting the MIC4-MIC1-MIC6 adhesive complex to the parasite cytoskeleton during host cell invasion.","method":"Co-immunoprecipitation, pulldown assays","journal":"Parasitology research","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP/pulldown, single lab; finding is for MIC6 tail rather than MIC4 directly","pmids":["19308454"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"MIC4's galactose-recognition activity (via its carbohydrate recognition domain, CRD) enables it to interact physically with N-glycans on TLR2 and TLR4 on host innate immune cells, triggering NF-κB activation and pro-inflammatory cytokine (IL-12, TNF-α, IL-6) production. Point mutations in the CRD of MIC4 abolish innate immune cell activation. Parasites expressing CRD-mutant MIC4 fail to induce wild-type IL-12 levels in vitro.","method":"Recombinant protein stimulation of dendritic cells/macrophages, CRD point mutants, HEK cell TLR glycomutant transfections, Co-IP, in vitro infection with MIC4-knockout or CRD-mutant parasites, cytokine measurement","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (mutagenesis, transfection systems, genetic knockouts, cytokine readouts), single lab but rigorous multi-method approach","pmids":["31226171"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"TLR2 heterodimerization (with TLR1 or TLR6) augments but is not critical for MIC4-induced cell activation; CD14 and CD36 enhance the response. MIC4 (and MIC1) activate macrophages through a TAK1-, p38 MAPK-, and NF-κB-dependent signaling pathway. The interaction of MIC4 with TLR2 N-glycans alone is sufficient to induce cell activation.","method":"Macrophage stimulation with recombinant MICs, HEK293T transfection with TLR2/TLR1/TLR6/CD14/CD36 constructs, NF-κB reporter assay, cytokine ELISA, pathway inhibitor assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transfection system with pathway inhibitors, single lab","pmids":["31658592"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"finding":"MIC4 (and MIC1) trigger IL-10 production from macrophages through TLR4 endocytosis/internalization. Blocking endocytosis with Dynasore prevents IL-10 but not TNF-α production in response to MIC4. MIC4-stimulated macrophages acquire transient tolerance to LPS.","method":"Macrophage stimulation with recombinant MIC4, Dynasore-mediated endocytosis blockade, cytokine ELISA, in vitro T. gondii infection","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological blockade with functional cytokine readout, single lab","pmids":["33912181"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"Recombinant MIC4 binds Jurkat T lymphocytes in a carbohydrate-dependent manner and induces apoptosis through activation of both extrinsic and intrinsic caspase pathways, driven by reactive oxygen species (ROS) production via the NADPH oxidase complex and activation of p38 and JNK MAPK signaling.","method":"Flow cytometry (cell binding, apoptosis/caspase assays), ROS detection, MAPK inhibitor assays, viability assays","journal":"Pathogens (Basel, Switzerland)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple functional assays in a single study but single lab, in vitro cell line model","pmids":["40333130"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"TgMIC4 binds desialylated (galactose-exposing) glycoproteins (asialofetuin) preferentially over sialylated glycoproteins (fetuin), with a conformational change accompanying galactose binding, as detected by a slower kinetic step in QCM-D measurements. This galactose-induced conformational change may contribute to host cell internalization.","method":"Quartz crystal microbalance with dissipation monitoring (QCM-D) using recombinant TgMIC4 immobilized on sensor surface, binding kinetics with fetuin vs. asialofetuin","journal":"Langmuir : the ACS journal of surfaces and colloids","confidence":"Low","confidence_rationale":"Tier 3 / Weak — biophysical assay with recombinant protein, single lab, mechanistic interpretation (conformational change) inferred from kinetic data","pmids":["26488670"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgMIC4 is a secreted, apple-domain lectin that forms the MIC4-MIC1-MIC6 trimeric adhesin complex in micronemes; MIC4 binds directly to MIC1 (acting as passive cargo), MIC6 escorts the complex to micronemes via its cytoplasmic sorting signal and aldolase interaction, the fifth apple domain of MIC4 mediates galactose-specific host cell attachment, and upon surface secretion the complex is proteolytically processed by the GPI-anchored subtilisin TgSUB1; additionally, MIC4's galactose-binding carbohydrate recognition domain engages N-glycans on host TLR2 and TLR4, activating innate immune signaling through TAK1/p38/NF-κB (driving IL-12/TNF-α) and TLR4 endocytosis (driving IL-10), and can induce T-cell apoptosis via ROS-dependent caspase activation."},"narrative":{"mechanistic_narrative":"TgMIC4 is a secreted, multi-apple-domain adhesin of Toxoplasma gondii that localizes to the micronemes of all invasive forms and functions both as a host-cell attachment factor during invasion and as a modulator of host innate immunity [PMID:11053441, PMID:22399295]. It is delivered to micronemes as a passive cargo of the trimeric MIC4-MIC1-MIC6 complex: MIC4 binds directly to MIC1, while MIC6 provides the cytoplasmic sorting signal and MIC1 serves as a quality-control checkpoint that permits the complex to exit early secretory compartments and reach the organelle [PMID:11157983, PMID:16166092]. Host recognition is mediated by a galactose-specific carbohydrate recognition domain in the fifth apple domain, which binds galactose-terminal glycans and is proteolytically cleaved from the complex during invasion [PMID:22399295]. Upon secretion onto the parasite surface the complex is trimmed at its N- and C-termini by the GPI-anchored subtilisin TgSUB1, a processing step that supports efficient attachment, gliding, invasion, and virulence [PMID:11053441, PMID:20678172]. Beyond invasion, MIC4's carbohydrate recognition domain engages N-glycans on host TLR2 and TLR4 to drive innate immune signaling: TLR2 engagement activates a TAK1/p38/NF-κB axis producing IL-12, TNF-α and IL-6, TLR4 endocytosis drives IL-10 and LPS tolerance, and CRD point mutations abolish this activation [PMID:31226171, PMID:31658592, PMID:33912181]. MIC4 also binds T lymphocytes in a carbohydrate-dependent manner and induces ROS- and MAPK-driven caspase-dependent apoptosis [PMID:40333130].","teleology":[{"year":2000,"claim":"Establishing that MIC4 is a micronemal secreted adhesin defined its role in host attachment and localized the adhesive activity to a C-terminal apple domain.","evidence":"Protein purification, microsequencing, immunolocalization, domain-mapping and host cell binding assays in T. gondii","pmids":["11053441"],"confidence":"High","gaps":["Did not define binding partners within the microneme complex","Ligand identity of the adhesive apple domain not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"Detection of MIC4 antigen in macrogamete wall-forming bodies raised the possibility of a role beyond invasion in oocyst wall formation.","evidence":"Immuno-EM and immunocytochemistry in T. gondii macrogametes","pmids":["11027789"],"confidence":"Medium","gaps":["Identity as MIC4 versus a cross-reacting protein not confirmed by western blot","Functional contribution to oocyst wall not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"claim":"Genetic dissection resolved how MIC4 reaches micronemes, showing it is passive cargo escorted by MIC6 within a trimeric MIC4-MIC1-MIC6 complex.","evidence":"Knockouts of MIC1/MIC4/MIC6 with domain mapping and Co-IP trafficking assays in T. gondii","pmids":["11157983"],"confidence":"High","gaps":["Structural basis of MIC4-MIC1 contact not defined","MIC4's own contribution to complex stability unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"claim":"Lactose-affinity co-purification confirmed the MIC1-MIC4 physical association but attributed the native lectin activity to MIC1 rather than MIC4.","evidence":"Lactose affinity chromatography, hemagglutination inhibition and blot overlay in T. gondii RH","pmids":["11447133"],"confidence":"Medium","gaps":["Left MIC4's intrinsic carbohydrate-binding activity unresolved","Single-lab biochemical inference"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2005,"claim":"Structural analysis of MIC1 explained the quality-control basis of complex assembly, showing TSR1 domains engage MIC4 and a Galectin-like fold stabilizes MIC6 to enable secretory exit.","evidence":"X-ray crystallography, NMR, interaction mapping and mutagenesis of TgMIC1 in T. gondii","pmids":["16166092"],"confidence":"High","gaps":["Atomic details of the MIC4-MIC1 interface not solved","Dynamics of complex disassembly during secretion unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Identifying an aldolase interaction on the MIC6 cytoplasmic tail connected the MIC4-containing complex to the parasite cytoskeleton during invasion.","evidence":"Co-IP and pulldown assays in T. gondii","pmids":["19308454"],"confidence":"Medium","gaps":["Interaction is with MIC6 tail, not MIC4 directly","Single Co-IP/pulldown without reciprocal validation"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Knockout of TgSUB1 established that surface proteolytic trimming of MIC4 is functionally required for efficient attachment, gliding, invasion and virulence.","evidence":"Δsub1 knockout with complementation, surface processing, invasion, gliding and mouse virulence assays in T. gondii","pmids":["20678172"],"confidence":"High","gaps":["Precise cleavage sites on MIC4 not mapped","Functional consequence of MIC4-specific trimming not isolated from other substrates"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"claim":"The MIC4 crystal structure defined six apple domains and localized potent galactose-binding to the fifth domain, which is cleaved during invasion, establishing the molecular basis of its host lectin activity.","evidence":"X-ray crystallography, glycan-array binding assays and structural mutagenesis of TgMIC4 in T. gondii","pmids":["22399295"],"confidence":"High","gaps":["In vivo host glycan targets during invasion not identified","Functional consequence of the fifth-domain cleavage not directly tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Biophysical binding kinetics indicated MIC4 prefers galactose-exposed (desialylated) glycoproteins and undergoes a galactose-induced conformational change potentially linked to internalization.","evidence":"QCM-D binding kinetics of recombinant TgMIC4 with fetuin versus asialofetuin","pmids":["26488670"],"confidence":"Low","gaps":["Conformational change inferred from kinetics, not directly observed","Single-lab biophysical assay with recombinant protein","Link to host cell internalization not demonstrated in cells"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Demonstrating that MIC4's CRD binds TLR2/TLR4 N-glycans to drive NF-κB and pro-inflammatory cytokines extended MIC4's function from invasion to direct activation of host innate immunity.","evidence":"Recombinant protein and knockout/CRD-mutant parasite stimulation of macrophages/DCs, TLR glycomutant transfection, Co-IP and cytokine assays in T. gondii","pmids":["31226171","31658592"],"confidence":"High","gaps":["Contribution relative to MIC1 not fully separable","Signaling pathway data partly from transfection/inhibitor systems"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"claim":"Showing that MIC4 drives IL-10 via TLR4 endocytosis and confers LPS tolerance revealed a distinct immunomodulatory arm separable from its pro-inflammatory signaling.","evidence":"Recombinant MIC4 macrophage stimulation with Dynasore endocytosis blockade and cytokine ELISA in T. gondii","pmids":["33912181"],"confidence":"Medium","gaps":["Pharmacological blockade only, no genetic confirmation of endocytic route","In vivo relevance of tolerance not established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Identifying carbohydrate-dependent T-cell binding and ROS/MAPK-driven apoptosis positioned MIC4 as a potential immune-evasion effector acting on adaptive lymphocytes.","evidence":"Flow cytometry binding/apoptosis assays, ROS detection and MAPK inhibitor assays with recombinant MIC4 on Jurkat cells","pmids":["40333130"],"confidence":"Medium","gaps":["In vitro cell line model only","Physiological relevance during infection not tested","Host T-cell glycan target not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The in vivo host glycan targets of the MIC4 fifth-domain CRD during invasion, and the mechanism coupling galactose binding to host cell entry, remain undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of the MIC4-MIC1 interface","In vivo relevance of immunomodulatory functions untested","Relative in vivo contributions of MIC4 vs MIC1 signaling unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MIC4-MIC1-MIC6 trimeric micronemal adhesin complex"],"partners":["TGMIC1 (DIRECT BINDING PARTNER WITHIN THE TRIMERIC COMPLEX)","TGMIC6 (ESCORTER WITHIN THE COMPLEX)","TGSUB1 (GPI-ANCHORED SUBTILISIN THAT PROCESSES MIC4 AFTER SECRETION)","HOST TLR2 (VIA N-GLYCANS)","HOST TLR4 (VIA N-GLYCANS)"],"other_free_text":["MIC4 is passive cargo delivered to micronemes; MIC6 provides the cytoplasmic sorting signal and MIC1 acts as a quality-control checkpoint for secretory exit [#1, #4].","The galactose-binding fifth apple domain is proteolytically cleaved from the complex during invasion [#6].","CRD point mutations abolish MIC4-driven innate immune cell activation [#8].","MIC4-induced IL-10 depends on TLR4 endocytosis and is separable from TNF-α production [#10].","A MIC4-like antigen was detected in macrogamete wall-forming bodies, though identity could not be confirmed by western blot [#3]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"11157983","id":"PMC_11157983","title":"Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii.","date":"2001","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11157983","citation_count":178,"is_preprint":false},{"pmid":"11566297","id":"PMC_11566297","title":"Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii.","date":"2001","source":"International journal for 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colloids","url":"https://pubmed.ncbi.nlm.nih.gov/26488670","citation_count":5,"is_preprint":false},{"pmid":"34039408","id":"PMC_34039408","title":"Proteomics analysis reveals that the proto-oncogene eIF-5A indirectly influences the growth, invasion and replication of Toxoplasma gondii tachyzoite.","date":"2021","source":"Parasites & vectors","url":"https://pubmed.ncbi.nlm.nih.gov/34039408","citation_count":3,"is_preprint":false},{"pmid":"39350555","id":"PMC_39350555","title":"Structural Prediction and Antigenic Analysis of ROP18, MIC4, and SAG1 Proteins to Improve Vaccine Design against Toxoplasma gondii: An In silico Approach.","date":"2025","source":"Infectious disorders drug targets","url":"https://pubmed.ncbi.nlm.nih.gov/39350555","citation_count":2,"is_preprint":false},{"pmid":"36162598","id":"PMC_36162598","title":"Protective immunity induced with a DNA vaccine encoding B- and T-cells multi-epitope SAG1, ROP16, MIC4, GRA12, M2AP, and multi-epitope ROP8 against acute and chronic toxoplasmosis in BALB/c mice.","date":"2022","source":"Experimental parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/36162598","citation_count":2,"is_preprint":false},{"pmid":"40333130","id":"PMC_40333130","title":"Recombinant Microneme Proteins MIC1 and MIC4 from Toxoplasma gondii Cause Cytotoxic Effects in the Human Jurkat T-Lymphocyte Cell Line.","date":"2025","source":"Pathogens (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/40333130","citation_count":1,"is_preprint":false},{"pmid":"32306345","id":"PMC_32306345","title":"MIC4 from Toxoplasma gondii: A Lectin Acting as a Toll-Like Receptor Agonist.","date":"2020","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/32306345","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18729,"output_tokens":3763,"usd":0.056316,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9826,"output_tokens":4932,"usd":0.086215,"stage2_stop_reason":"end_turn"},"total_usd":0.142531,"stage1_batch_id":"msgbatch_01WokvBYTB6JLnYrpD4rXnyV","stage2_batch_id":"msgbatch_01M7hsQDvz6AqVC9DLVjQzzc","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"MIC4 is a 61-kDa protein secreted by Toxoplasma gondii tachyzoites that localizes to the micronemes of all invasive forms (tachyzoites, bradyzoites, sporozoites, merozoites). It is proteolytically processed at both N- and C-termini only upon release from the organelle. The adhesive motif maps to the most C-terminal (fifth/sixth) apple domain, and MIC4 binds efficiently to host cells.\",\n      \"method\": \"Protein purification and microsequencing, gene cloning, immunolocalization, deletion/domain mapping assays, host cell binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (purification, sequencing, localization, domain mapping, binding assays) in a single rigorous study\",\n      \"pmids\": [\"11053441\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"MIC6 functions as an escorter for MIC1 and MIC4, forming a trimeric complex (MIC4-MIC1-MIC6) that is required for accurate targeting of MIC1 and MIC4 to the micronemes. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. MIC6 carries a sorting signal in its cytoplasmic tail, while its association with MIC1 involves a lumenal EGF-like domain. MIC1 is linked to a quality control system and is absolutely required for the complex to leave early secretory compartments.\",\n      \"method\": \"Gene disruption (knockouts of MIC1, MIC4, MIC6), domain mapping, Co-IP, sorting/trafficking assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockouts combined with domain mapping and interaction studies, multiple orthogonal methods\",\n      \"pmids\": [\"11157983\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"MIC1 and MIC4 co-purify from tachyzoite soluble antigen by affinity chromatography on immobilized lactose, consistent with their physical association. The lectin activity (hemagglutination inhibited by beta-galactosides) was attributed to MIC1, not to native MIC4.\",\n      \"method\": \"Affinity chromatography (lactose column), N-terminal microsequencing, hemagglutination inhibition assay, blot overlay\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — biochemical co-purification and functional assay, single lab, finding partially attributed to MIC1 rather than MIC4\",\n      \"pmids\": [\"11447133\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MIC4, or a MIC4-like protein, is expressed in macrogametes and associated with oocyst wall formation. By immuno-electron microscopy, MIC4 antigen was detected in a subset of wall forming body type 1 (WFB1a) dense granules, which sequentially release their contents to form an anti-MIC4-positive outer veil on the developing oocyst wall.\",\n      \"method\": \"Immunocytochemistry, immuno-electron microscopy\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct immuno-EM localization, single lab; caveat that identity as MIC4 vs. cross-reacting protein could not be confirmed by western blot\",\n      \"pmids\": [\"11027789\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The N-terminal TSR1-like domains of TgMIC1 promote association with TgMIC4. The C-terminal domain of TgMIC1 adopts a novel Galectin-like fold (without carbohydrate-binding properties) that interacts with and stabilizes MIC6, providing the structural basis for a quality-control mechanism enabling the MIC4-MIC1-MIC6 complex to exit early secretory compartments and traffic to micronemes.\",\n      \"method\": \"X-ray crystallography (3D structure of TgMIC1 C-terminal domain), NMR, interaction assays, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with functional interaction mapping and mutagenesis in a single rigorous study\",\n      \"pmids\": [\"16166092\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The microneme subtilisin protease TgSUB1, anchored via a GPI moiety, is responsible for proteolytic trimming of MIC4 (and MIC2, M2AP) after secretion onto the parasite surface. Loss of TgSUB1 abolishes this processing, decreases cell attachment and gliding efficiency, and reduces invasion rates in vitro and virulence in mice.\",\n      \"method\": \"Gene knockout (Δsub1), complementation with full-length and ΔGPI-SUB1 constructs, surface processing assays, invasion and gliding assays, mouse virulence assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with complementation controls, multiple orthogonal functional readouts\",\n      \"pmids\": [\"20678172\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The crystal structure of TgMIC4 reveals six apple domains; the fifth domain displays potent galactose-binding activity and is cleaved from the complex during parasite invasion. TgMIC4 binds a variety of galactose-containing carbohydrate ligands.\",\n      \"method\": \"X-ray crystallography, carbohydrate-binding assays (glycan array), structural mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with carbohydrate-binding specificity mapping and identification of cleaved domain, single rigorous study with multiple methods\",\n      \"pmids\": [\"22399295\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The cytoplasmic tail of MIC6 interacts with parasite aldolase, connecting the MIC4-MIC1-MIC6 adhesive complex to the parasite cytoskeleton during host cell invasion.\",\n      \"method\": \"Co-immunoprecipitation, pulldown assays\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/pulldown, single lab; finding is for MIC6 tail rather than MIC4 directly\",\n      \"pmids\": [\"19308454\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MIC4's galactose-recognition activity (via its carbohydrate recognition domain, CRD) enables it to interact physically with N-glycans on TLR2 and TLR4 on host innate immune cells, triggering NF-κB activation and pro-inflammatory cytokine (IL-12, TNF-α, IL-6) production. Point mutations in the CRD of MIC4 abolish innate immune cell activation. Parasites expressing CRD-mutant MIC4 fail to induce wild-type IL-12 levels in vitro.\",\n      \"method\": \"Recombinant protein stimulation of dendritic cells/macrophages, CRD point mutants, HEK cell TLR glycomutant transfections, Co-IP, in vitro infection with MIC4-knockout or CRD-mutant parasites, cytokine measurement\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (mutagenesis, transfection systems, genetic knockouts, cytokine readouts), single lab but rigorous multi-method approach\",\n      \"pmids\": [\"31226171\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TLR2 heterodimerization (with TLR1 or TLR6) augments but is not critical for MIC4-induced cell activation; CD14 and CD36 enhance the response. MIC4 (and MIC1) activate macrophages through a TAK1-, p38 MAPK-, and NF-κB-dependent signaling pathway. The interaction of MIC4 with TLR2 N-glycans alone is sufficient to induce cell activation.\",\n      \"method\": \"Macrophage stimulation with recombinant MICs, HEK293T transfection with TLR2/TLR1/TLR6/CD14/CD36 constructs, NF-κB reporter assay, cytokine ELISA, pathway inhibitor assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transfection system with pathway inhibitors, single lab\",\n      \"pmids\": [\"31658592\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MIC4 (and MIC1) trigger IL-10 production from macrophages through TLR4 endocytosis/internalization. Blocking endocytosis with Dynasore prevents IL-10 but not TNF-α production in response to MIC4. MIC4-stimulated macrophages acquire transient tolerance to LPS.\",\n      \"method\": \"Macrophage stimulation with recombinant MIC4, Dynasore-mediated endocytosis blockade, cytokine ELISA, in vitro T. gondii infection\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological blockade with functional cytokine readout, single lab\",\n      \"pmids\": [\"33912181\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Recombinant MIC4 binds Jurkat T lymphocytes in a carbohydrate-dependent manner and induces apoptosis through activation of both extrinsic and intrinsic caspase pathways, driven by reactive oxygen species (ROS) production via the NADPH oxidase complex and activation of p38 and JNK MAPK signaling.\",\n      \"method\": \"Flow cytometry (cell binding, apoptosis/caspase assays), ROS detection, MAPK inhibitor assays, viability assays\",\n      \"journal\": \"Pathogens (Basel, Switzerland)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple functional assays in a single study but single lab, in vitro cell line model\",\n      \"pmids\": [\"40333130\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TgMIC4 binds desialylated (galactose-exposing) glycoproteins (asialofetuin) preferentially over sialylated glycoproteins (fetuin), with a conformational change accompanying galactose binding, as detected by a slower kinetic step in QCM-D measurements. This galactose-induced conformational change may contribute to host cell internalization.\",\n      \"method\": \"Quartz crystal microbalance with dissipation monitoring (QCM-D) using recombinant TgMIC4 immobilized on sensor surface, binding kinetics with fetuin vs. asialofetuin\",\n      \"journal\": \"Langmuir : the ACS journal of surfaces and colloids\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — biophysical assay with recombinant protein, single lab, mechanistic interpretation (conformational change) inferred from kinetic data\",\n      \"pmids\": [\"26488670\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgMIC4 is a secreted, apple-domain lectin that forms the MIC4-MIC1-MIC6 trimeric adhesin complex in micronemes; MIC4 binds directly to MIC1 (acting as passive cargo), MIC6 escorts the complex to micronemes via its cytoplasmic sorting signal and aldolase interaction, the fifth apple domain of MIC4 mediates galactose-specific host cell attachment, and upon surface secretion the complex is proteolytically processed by the GPI-anchored subtilisin TgSUB1; additionally, MIC4's galactose-binding carbohydrate recognition domain engages N-glycans on host TLR2 and TLR4, activating innate immune signaling through TAK1/p38/NF-κB (driving IL-12/TNF-α) and TLR4 endocytosis (driving IL-10), and can induce T-cell apoptosis via ROS-dependent caspase activation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgMIC4 is a secreted, multi-apple-domain adhesin of Toxoplasma gondii that localizes to the micronemes of all invasive forms and functions both as a host-cell attachment factor during invasion and as a modulator of host innate immunity [#0, #6]. It is delivered to micronemes as a passive cargo of the trimeric MIC4-MIC1-MIC6 complex: MIC4 binds directly to MIC1, while MIC6 provides the cytoplasmic sorting signal and MIC1 serves as a quality-control checkpoint that permits the complex to exit early secretory compartments and reach the organelle [#1, #4]. Host recognition is mediated by a galactose-specific carbohydrate recognition domain in the fifth apple domain, which binds galactose-terminal glycans and is proteolytically cleaved from the complex during invasion [#6]. Upon secretion onto the parasite surface the complex is trimmed at its N- and C-termini by the GPI-anchored subtilisin TgSUB1, a processing step that supports efficient attachment, gliding, invasion, and virulence [#0, #5]. Beyond invasion, MIC4's carbohydrate recognition domain engages N-glycans on host TLR2 and TLR4 to drive innate immune signaling: TLR2 engagement activates a TAK1/p38/NF-\\u03baB axis producing IL-12, TNF-\\u03b1 and IL-6, TLR4 endocytosis drives IL-10 and LPS tolerance, and CRD point mutations abolish this activation [#8, #9, #10]. MIC4 also binds T lymphocytes in a carbohydrate-dependent manner and induces ROS- and MAPK-driven caspase-dependent apoptosis [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing that MIC4 is a micronemal secreted adhesin defined its role in host attachment and localized the adhesive activity to a C-terminal apple domain.\",\n      \"evidence\": \"Protein purification, microsequencing, immunolocalization, domain-mapping and host cell binding assays in T. gondii\",\n      \"pmids\": [\"11053441\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define binding partners within the microneme complex\", \"Ligand identity of the adhesive apple domain not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Detection of MIC4 antigen in macrogamete wall-forming bodies raised the possibility of a role beyond invasion in oocyst wall formation.\",\n      \"evidence\": \"Immuno-EM and immunocytochemistry in T. gondii macrogametes\",\n      \"pmids\": [\"11027789\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity as MIC4 versus a cross-reacting protein not confirmed by western blot\", \"Functional contribution to oocyst wall not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Genetic dissection resolved how MIC4 reaches micronemes, showing it is passive cargo escorted by MIC6 within a trimeric MIC4-MIC1-MIC6 complex.\",\n      \"evidence\": \"Knockouts of MIC1/MIC4/MIC6 with domain mapping and Co-IP trafficking assays in T. gondii\",\n      \"pmids\": [\"11157983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of MIC4-MIC1 contact not defined\", \"MIC4's own contribution to complex stability unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Lactose-affinity co-purification confirmed the MIC1-MIC4 physical association but attributed the native lectin activity to MIC1 rather than MIC4.\",\n      \"evidence\": \"Lactose affinity chromatography, hemagglutination inhibition and blot overlay in T. gondii RH\",\n      \"pmids\": [\"11447133\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Left MIC4's intrinsic carbohydrate-binding activity unresolved\", \"Single-lab biochemical inference\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Structural analysis of MIC1 explained the quality-control basis of complex assembly, showing TSR1 domains engage MIC4 and a Galectin-like fold stabilizes MIC6 to enable secretory exit.\",\n      \"evidence\": \"X-ray crystallography, NMR, interaction mapping and mutagenesis of TgMIC1 in T. gondii\",\n      \"pmids\": [\"16166092\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic details of the MIC4-MIC1 interface not solved\", \"Dynamics of complex disassembly during secretion unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying an aldolase interaction on the MIC6 cytoplasmic tail connected the MIC4-containing complex to the parasite cytoskeleton during invasion.\",\n      \"evidence\": \"Co-IP and pulldown assays in T. gondii\",\n      \"pmids\": [\"19308454\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction is with MIC6 tail, not MIC4 directly\", \"Single Co-IP/pulldown without reciprocal validation\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Knockout of TgSUB1 established that surface proteolytic trimming of MIC4 is functionally required for efficient attachment, gliding, invasion and virulence.\",\n      \"evidence\": \"\\u0394sub1 knockout with complementation, surface processing, invasion, gliding and mouse virulence assays in T. gondii\",\n      \"pmids\": [\"20678172\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise cleavage sites on MIC4 not mapped\", \"Functional consequence of MIC4-specific trimming not isolated from other substrates\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The MIC4 crystal structure defined six apple domains and localized potent galactose-binding to the fifth domain, which is cleaved during invasion, establishing the molecular basis of its host lectin activity.\",\n      \"evidence\": \"X-ray crystallography, glycan-array binding assays and structural mutagenesis of TgMIC4 in T. gondii\",\n      \"pmids\": [\"22399295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo host glycan targets during invasion not identified\", \"Functional consequence of the fifth-domain cleavage not directly tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Biophysical binding kinetics indicated MIC4 prefers galactose-exposed (desialylated) glycoproteins and undergoes a galactose-induced conformational change potentially linked to internalization.\",\n      \"evidence\": \"QCM-D binding kinetics of recombinant TgMIC4 with fetuin versus asialofetuin\",\n      \"pmids\": [\"26488670\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Conformational change inferred from kinetics, not directly observed\", \"Single-lab biophysical assay with recombinant protein\", \"Link to host cell internalization not demonstrated in cells\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating that MIC4's CRD binds TLR2/TLR4 N-glycans to drive NF-\\u03baB and pro-inflammatory cytokines extended MIC4's function from invasion to direct activation of host innate immunity.\",\n      \"evidence\": \"Recombinant protein and knockout/CRD-mutant parasite stimulation of macrophages/DCs, TLR glycomutant transfection, Co-IP and cytokine assays in T. gondii\",\n      \"pmids\": [\"31226171\", \"31658592\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Contribution relative to MIC1 not fully separable\", \"Signaling pathway data partly from transfection/inhibitor systems\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showing that MIC4 drives IL-10 via TLR4 endocytosis and confers LPS tolerance revealed a distinct immunomodulatory arm separable from its pro-inflammatory signaling.\",\n      \"evidence\": \"Recombinant MIC4 macrophage stimulation with Dynasore endocytosis blockade and cytokine ELISA in T. gondii\",\n      \"pmids\": [\"33912181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pharmacological blockade only, no genetic confirmation of endocytic route\", \"In vivo relevance of tolerance not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying carbohydrate-dependent T-cell binding and ROS/MAPK-driven apoptosis positioned MIC4 as a potential immune-evasion effector acting on adaptive lymphocytes.\",\n      \"evidence\": \"Flow cytometry binding/apoptosis assays, ROS detection and MAPK inhibitor assays with recombinant MIC4 on Jurkat cells\",\n      \"pmids\": [\"40333130\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro cell line model only\", \"Physiological relevance during infection not tested\", \"Host T-cell glycan target not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The in vivo host glycan targets of the MIC4 fifth-domain CRD during invasion, and the mechanism coupling galactose binding to host cell entry, remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of the MIC4-MIC1 interface\", \"In vivo relevance of immunomodulatory functions untested\", \"Relative in vivo contributions of MIC4 vs MIC1 signaling unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005534\", \"supporting_discovery_ids\": [6, 8, 11, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005102\", \"supporting_discovery_ids\": [1, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0035325\", \"supporting_discovery_ids\": [8, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020009\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0009986\", \"supporting_discovery_ids\": [0, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [0, 5, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0007249\", \"supporting_discovery_ids\": [8, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0038123\", \"supporting_discovery_ids\": [8, 9, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006915\", \"supporting_discovery_ids\": [11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"MIC4-MIC1-MIC6 trimeric micronemal adhesin complex\"\n    ],\n    \"partners\": [\n      \"TgMIC1 (direct binding partner within the trimeric complex)\",\n      \"TgMIC6 (escorter within the complex)\",\n      \"TgSUB1 (GPI-anchored subtilisin that processes MIC4 after secretion)\",\n      \"host TLR2 (via N-glycans)\",\n      \"host TLR4 (via N-glycans)\"\n    ],\n    \"other_free_text\": [\n      \"MIC4 is passive cargo delivered to micronemes; MIC6 provides the cytoplasmic sorting signal and MIC1 acts as a quality-control checkpoint for secretory exit [#1, #4].\",\n      \"The galactose-binding fifth apple domain is proteolytically cleaved from the complex during invasion [#6].\",\n      \"CRD point mutations abolish MIC4-driven innate immune cell activation [#8].\",\n      \"MIC4-induced IL-10 depends on TLR4 endocytosis and is separable from TNF-\\u03b1 production [#10].\",\n      \"A MIC4-like antigen was detected in macrogamete wall-forming bodies, though identity could not be confirmed by western blot [#3].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_208030","name":"MIC4","product":"microneme protein MIC4","uniprot_acc":"A0A125YP11","chromosome":"TGME49_chrIb","start_pos":531351},{"strain":"GT1","gene_id":"TGGT1_208030","name":null,"product":"microneme protein MIC4","uniprot_acc":null,"chromosome":"TGGT1_chrIb","start_pos":547335},{"strain":"RH88","gene_id":"TGRH88_022630","name":null,"product":"microneme protein MIC4","uniprot_acc":null,"chromosome":"CM023082","start_pos":1479049},{"strain":"VEG","gene_id":"TGVEG_208030","name":null,"product":"microneme protein MIC4","uniprot_acc":"B9PXJ3","chromosome":"TGVEG_chrIb","start_pos":529858}]}