{"gene":"TG_248830","gene_symbol":"PIPLC","run_date":"2026-09-08T12:58:25","timeline":{"discoveries":[{"year":2006,"finding":"TgPI-PLC was cloned, expressed in E. coli, and shown to encode a 1097-amino acid polypeptide (~121 kDa) with conserved catalytic X and Y domains, an N-terminal PH domain, an EF hand motif, and a C-terminal C2 domain. Recombinant TgPI-PLC and the native enzyme from crude membrane extracts preferentially hydrolyze phosphatidylinositol (PI) over phosphatidylinositol 4,5-bisphosphate (PIP2). By indirect immunofluorescence, TgPI-PLC localizes to the plasma membrane of tachyzoites.","method":"Molecular cloning, recombinant expression in E. coli, in vitro enzymatic assay with PI and PIP2 substrates, indirect immunofluorescence with affinity-purified antibody","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with defined substrates plus direct localization; single lab but multiple orthogonal methods","pmids":["16288600"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"PI-PLC (together with PKG) was identified as an important factor controlling intracellular Ca2+ signals induced by estradiol in T. gondii, with cytosolic Ca2+ mobilized mainly from acidic organelles and linked to MIC2 secretion, gliding motility, and egress.","method":"Pharmacological inhibition of PI-PLC and PKG, cytosolic Ca2+ flux measurement, MIC2 secretion assay, gliding and egress assays","journal":"Frontiers in microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — multiple functional readouts but pathway placement relies on pharmacological inhibition (not genetic KO), single lab","pmids":["29946311"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1989,"finding":"PI-PLC treatment of T. gondii surface antigen P30 (SAG1) released the protein from intact parasites and removed [3H]palmitate label, demonstrating that P30 is anchored to the membrane via a glycolipid (GPI) anchor containing a 1,2-diacylglycerol moiety; PI-PLC cleavage exposed a cross-reacting determinant analogous to that of Trypanosoma VSG.","method":"Metabolic radiolabeling ([3H]palmitate, myo-[3H]inositol, [35S]methionine), PI-PLC treatment of intact parasites and purified antigen, TLC, phospholipase A2 digestion, PAGE mobility shift","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution with multiple orthogonal labeling and enzymatic cleavage methods, independently replicated (see PMID 2531282)","pmids":["2925621"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1989,"finding":"PI-PLC treatment rendered the four major surface antigens of T. gondii tachyzoites (P43, P35, P30, P22) water-soluble and removed their [3H]fatty acid labels, and exposed a cross-reacting determinant, establishing that all four major surface antigens are GPI-anchored.","method":"Biosynthetic labeling with [3H]fatty acids, [3H]ethanolamine, [3H]carbohydrates; PI-PLC treatment of parasite lysates; immunoprecipitation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical enzymatic cleavage with multiple radiolabels, independently replicated alongside PMID 2925621","pmids":["2531282"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1993,"finding":"PI-PLC treatment released gp23 from the surface of living T. gondii, and PLA2 digestion characterized the phosphatidylinositol moiety, together establishing that gp23 (a tachyzoite surface glycoprotein) is GPI-anchored with a conserved core glycan structure bearing beta-linked N-acetylgalactosamine residue(s).","method":"Metabolic radiolabeling (palmitate, myristate, ethanolamine, inositol, glucosamine, mannose, galactose), PI-PLC and PLA2 digestion, immunoprecipitation, aqueous-HF dephosphorylation, nitrous acid deamination, exo- and endoglycosidase analysis","journal":"Biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical reconstitution with multiple orthogonal enzymatic and chemical methods in a single rigorous study","pmids":["8241958"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"finding":"In hypotonically permeabilized T. gondii tachyzoites, PI-PLC hydrolysis of radiolabeled GPI biosynthetic intermediates (GlcN-PI and GlcNAc-PI) demonstrated that early GPI intermediates are predominantly in the cytoplasmic leaflet of the ER membrane, and identified a new inositol-acylated early intermediate resistant to PI-PLC, indicating inositol acylation occurs in T. gondii.","method":"Hypotonic permeabilization, metabolic labeling with UDP-[3H]GlcNAc, GDP-[3H]mannose, UDP-[3H]glucose, PI-PLC probing of transmembrane distribution, protease protection assays, electron microscopy","journal":"Eukaryotic cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct topology assay using PI-PLC as a probe in permeabilized cells with orthogonal controls; single lab","pmids":["16896225"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"Treatment with edelfosine, a phospholipid analogue that inhibits PI-PLC, significantly reduced GRA1 and MIC3 mRNA and protein expression in T. gondii both in vitro (infected Vero cells) and in vivo (mouse model), supporting the role of PI-PLC in regulating dense granule and microneme secretory antigen expression.","method":"In vitro infection of Vero cells with edelfosine treatment, in vivo mouse model, qRT-PCR, Western blot","journal":"Parasitology research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological inhibition only (no genetic manipulation of PI-PLC), single lab, indirect evidence for PI-PLC mechanism","pmids":["31970471"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgPI-PLC is a plasma membrane-localized phospholipase C (PH-EF hand-X/Y-C2 domain architecture) that preferentially hydrolyzes phosphatidylinositol over PIP2, participates in intracellular Ca2+ signaling downstream of PKG to regulate microneme secretion, gliding, and egress, and has been experimentally used as a diagnostic tool to demonstrate that the major tachyzoite surface antigens (P30/SAG1, P43, P35, P22, gp23) are GPI-anchored to the parasite plasma membrane."},"narrative":{"mechanistic_narrative":"TgPI-PLC is a plasma membrane-localized phosphatidylinositol-specific phospholipase C of Toxoplasma gondii that couples lipid hydrolysis to intracellular Ca2+ signaling governing tachyzoite motility and egress [PMID:16288600, PMID:29946311]. The cloned enzyme is a ~121 kDa polypeptide organized as an N-terminal PH domain, an EF-hand motif, the catalytic X and Y domains, and a C-terminal C2 domain, and both recombinant and native enzyme preferentially hydrolyze phosphatidylinositol over PIP2 [PMID:16288600]. Functionally, PI-PLC acts together with PKG to control cytosolic Ca2+ signals mobilized from acidic organelles, linking the enzyme to MIC2 secretion, gliding motility, and egress [PMID:29946311]. Independently, PI-PLC has served as a definitive biochemical probe establishing that the major tachyzoite surface antigens—P30/SAG1, P43, P35, P22, and gp23—are attached to the plasma membrane through GPI anchors, since PI-PLC cleavage renders them water-soluble, removes their fatty-acyl labels, and exposes a trypanosome-VSG-like cross-reacting determinant [PMID:2925621, PMID:2531282, PMID:8241958]. Used as a topological tool in permeabilized parasites, PI-PLC also mapped early GPI biosynthetic intermediates to the cytoplasmic leaflet of the ER and revealed an inositol-acylated, PI-PLC-resistant intermediate [PMID:16896225].","teleology":[{"year":1989,"claim":"Establishing how major surface antigens attach to the parasite membrane was open; PI-PLC cleavage demonstrated that P30/SAG1 and the other major tachyzoite surface antigens are GPI-anchored via a diacylglycerol-containing glycolipid.","evidence":"Metabolic radiolabeling, PI-PLC treatment of intact parasites and lysates, PLA2 digestion, and PAGE mobility shift/immunoprecipitation in T. gondii tachyzoites","pmids":["2925621","2531282"],"confidence":"High","gaps":["Did not identify the enzyme's endogenous parasite gene","GPI core glycan fine structure not fully resolved in these reports"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1993,"claim":"Extending the GPI-anchor model to additional surface glycoproteins, PI-PLC release of gp23 combined with glycan analysis defined a conserved GPI core bearing beta-linked N-acetylgalactosamine.","evidence":"Multi-radiolabel metabolic labeling, PI-PLC and PLA2 digestion, chemical dephosphorylation/deamination, and exo/endoglycosidase analysis in living T. gondii","pmids":["8241958"],"confidence":"High","gaps":["Biological function of the GalNAc modification not addressed","Did not link cleavage to the endogenous PI-PLC enzyme"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"claim":"The identity and biochemical properties of the parasite enzyme itself were unknown; cloning and recombinant expression defined a PH-EF-X/Y-C2 phospholipase C that preferentially hydrolyzes PI over PIP2 and localizes to the plasma membrane.","evidence":"Molecular cloning, recombinant expression in E. coli, in vitro PI/PIP2 hydrolysis assays, and indirect immunofluorescence in T. gondii","pmids":["16288600"],"confidence":"High","gaps":["No genetic knockout to establish essentiality or in vivo function","Regulation of catalytic activity not characterized"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"claim":"The membrane topology of GPI biosynthesis was unresolved; using PI-PLC as a leaflet-specific probe placed early GPI intermediates in the cytoplasmic ER leaflet and revealed a PI-PLC-resistant inositol-acylated intermediate.","evidence":"Hypotonic permeabilization, metabolic labeling of GPI intermediates, PI-PLC probing of transmembrane distribution, protease protection, and electron microscopy in T. gondii","pmids":["16896225"],"confidence":"Medium","gaps":["Uses PI-PLC as a reagent rather than testing its own cellular role","Enzyme(s) performing inositol acylation not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Whether PI-PLC contributes to Ca2+-driven invasion/egress behavior was untested; pharmacological inhibition placed PI-PLC with PKG upstream of cytosolic Ca2+ mobilization from acidic stores, MIC2 secretion, gliding, and egress.","evidence":"Pharmacological inhibition of PI-PLC and PKG with cytosolic Ca2+ flux measurement, MIC2 secretion, gliding and egress assays in T. gondii","pmids":["29946311"],"confidence":"Medium","gaps":["Pathway placement rests on inhibitors, not genetic knockout","Direct enzymatic product coupling Ca2+ release not demonstrated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Whether PI-PLC influences secretory-antigen expression was addressed by inhibitor treatment, which reduced GRA1 and MIC3 mRNA and protein in vitro and in vivo.","evidence":"Edelfosine (PI-PLC inhibitor) treatment of infected Vero cells and a mouse model, qRT-PCR and Western blot in T. gondii","pmids":["31970471"],"confidence":"Low","gaps":["Pharmacological inhibition only, no genetic manipulation of PI-PLC","Edelfosine effects may be indirect on the enzyme"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The endogenous, genetically defined function of TgPI-PLC in Ca2+ signaling and its physiological lipid substrate/product in vivo remain to be established.","evidence":"","pmids":[],"confidence":"Low","gaps":["No conditional knockout or complementation of TgPI-PLC reported","In vivo substrate preference (PI vs PIP2) during signaling not resolved","Mechanism linking PI-PLC activity to acidic-store Ca2+ release undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["PKG (FUNCTIONAL CO-REQUIREMENT IN CA2+ SIGNALING, PHARMACOLOGICAL EVIDENCE) [#1]"],"other_free_text":["Domain architecture: N-terminal PH domain, EF-hand motif, catalytic X and Y domains, C-terminal C2 domain; ~1097 aa / ~121 kDa [#0].","Preferentially hydrolyzes phosphatidylinositol over PIP2 in vitro (recombinant and native enzyme) [#0].","Used experimentally as a diagnostic/topological probe to demonstrate GPI anchoring of surface antigens P30/SAG1, P43, P35, P22, and gp23 [#2, #3, #4] and to map GPI biosynthetic intermediates to the cytoplasmic ER leaflet [#5].","Functionally linked (via pharmacological inhibition) to cytosolic Ca2+ mobilization from acidic organelles, MIC2 secretion, gliding, egress, and to GRA1/MIC3 secretory-antigen expression [#1, #6]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"2925621","id":"PMC_2925621","title":"The major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid.","date":"1989","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2925621","citation_count":98,"is_preprint":false},{"pmid":"2531282","id":"PMC_2531282","title":"Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens.","date":"1989","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/2531282","citation_count":93,"is_preprint":false},{"pmid":"16288600","id":"PMC_16288600","title":"A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol.","date":"2006","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/16288600","citation_count":43,"is_preprint":false},{"pmid":"29946311","id":"PMC_29946311","title":"Effects of Estradiol and Progesterone-Induced Intracellular Calcium Fluxes on Toxoplasma gondii Gliding, Microneme Secretion, and Egress.","date":"2018","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/29946311","citation_count":17,"is_preprint":false},{"pmid":"8241958","id":"PMC_8241958","title":"Structural analysis of glycosyl-phosphatidylinositol membrane anchor of the Toxoplasma gondii tachyzoite surface glycoprotein gp23.","date":"1993","source":"Biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/8241958","citation_count":14,"is_preprint":false},{"pmid":"16896225","id":"PMC_16896225","title":"Membrane topology and transient acylation of Toxoplasma gondii glycosylphosphatidylinositols.","date":"2006","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/16896225","citation_count":6,"is_preprint":false},{"pmid":"31970471","id":"PMC_31970471","title":"The effect of edelfosine on GRA1 and MIC3 expressions in acute toxoplasmosis.","date":"2020","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/31970471","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":5330,"output_tokens":2205,"usd":0.024532,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7615,"output_tokens":3338,"usd":0.060762,"stage2_stop_reason":"end_turn"},"total_usd":0.085294,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"TgPI-PLC was cloned, expressed in E. coli, and shown to encode a 1097-amino acid polypeptide (~121 kDa) with conserved catalytic X and Y domains, an N-terminal PH domain, an EF hand motif, and a C-terminal C2 domain. Recombinant TgPI-PLC and the native enzyme from crude membrane extracts preferentially hydrolyze phosphatidylinositol (PI) over phosphatidylinositol 4,5-bisphosphate (PIP2). By indirect immunofluorescence, TgPI-PLC localizes to the plasma membrane of tachyzoites.\",\n      \"method\": \"Molecular cloning, recombinant expression in E. coli, in vitro enzymatic assay with PI and PIP2 substrates, indirect immunofluorescence with affinity-purified antibody\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with defined substrates plus direct localization; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16288600\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PI-PLC (together with PKG) was identified as an important factor controlling intracellular Ca2+ signals induced by estradiol in T. gondii, with cytosolic Ca2+ mobilized mainly from acidic organelles and linked to MIC2 secretion, gliding motility, and egress.\",\n      \"method\": \"Pharmacological inhibition of PI-PLC and PKG, cytosolic Ca2+ flux measurement, MIC2 secretion assay, gliding and egress assays\",\n      \"journal\": \"Frontiers in microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — multiple functional readouts but pathway placement relies on pharmacological inhibition (not genetic KO), single lab\",\n      \"pmids\": [\"29946311\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"PI-PLC treatment of T. gondii surface antigen P30 (SAG1) released the protein from intact parasites and removed [3H]palmitate label, demonstrating that P30 is anchored to the membrane via a glycolipid (GPI) anchor containing a 1,2-diacylglycerol moiety; PI-PLC cleavage exposed a cross-reacting determinant analogous to that of Trypanosoma VSG.\",\n      \"method\": \"Metabolic radiolabeling ([3H]palmitate, myo-[3H]inositol, [35S]methionine), PI-PLC treatment of intact parasites and purified antigen, TLC, phospholipase A2 digestion, PAGE mobility shift\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution with multiple orthogonal labeling and enzymatic cleavage methods, independently replicated (see PMID 2531282)\",\n      \"pmids\": [\"2925621\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"PI-PLC treatment rendered the four major surface antigens of T. gondii tachyzoites (P43, P35, P30, P22) water-soluble and removed their [3H]fatty acid labels, and exposed a cross-reacting determinant, establishing that all four major surface antigens are GPI-anchored.\",\n      \"method\": \"Biosynthetic labeling with [3H]fatty acids, [3H]ethanolamine, [3H]carbohydrates; PI-PLC treatment of parasite lysates; immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical enzymatic cleavage with multiple radiolabels, independently replicated alongside PMID 2925621\",\n      \"pmids\": [\"2531282\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"PI-PLC treatment released gp23 from the surface of living T. gondii, and PLA2 digestion characterized the phosphatidylinositol moiety, together establishing that gp23 (a tachyzoite surface glycoprotein) is GPI-anchored with a conserved core glycan structure bearing beta-linked N-acetylgalactosamine residue(s).\",\n      \"method\": \"Metabolic radiolabeling (palmitate, myristate, ethanolamine, inositol, glucosamine, mannose, galactose), PI-PLC and PLA2 digestion, immunoprecipitation, aqueous-HF dephosphorylation, nitrous acid deamination, exo- and endoglycosidase analysis\",\n      \"journal\": \"Biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical reconstitution with multiple orthogonal enzymatic and chemical methods in a single rigorous study\",\n      \"pmids\": [\"8241958\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In hypotonically permeabilized T. gondii tachyzoites, PI-PLC hydrolysis of radiolabeled GPI biosynthetic intermediates (GlcN-PI and GlcNAc-PI) demonstrated that early GPI intermediates are predominantly in the cytoplasmic leaflet of the ER membrane, and identified a new inositol-acylated early intermediate resistant to PI-PLC, indicating inositol acylation occurs in T. gondii.\",\n      \"method\": \"Hypotonic permeabilization, metabolic labeling with UDP-[3H]GlcNAc, GDP-[3H]mannose, UDP-[3H]glucose, PI-PLC probing of transmembrane distribution, protease protection assays, electron microscopy\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct topology assay using PI-PLC as a probe in permeabilized cells with orthogonal controls; single lab\",\n      \"pmids\": [\"16896225\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Treatment with edelfosine, a phospholipid analogue that inhibits PI-PLC, significantly reduced GRA1 and MIC3 mRNA and protein expression in T. gondii both in vitro (infected Vero cells) and in vivo (mouse model), supporting the role of PI-PLC in regulating dense granule and microneme secretory antigen expression.\",\n      \"method\": \"In vitro infection of Vero cells with edelfosine treatment, in vivo mouse model, qRT-PCR, Western blot\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological inhibition only (no genetic manipulation of PI-PLC), single lab, indirect evidence for PI-PLC mechanism\",\n      \"pmids\": [\"31970471\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgPI-PLC is a plasma membrane-localized phospholipase C (PH-EF hand-X/Y-C2 domain architecture) that preferentially hydrolyzes phosphatidylinositol over PIP2, participates in intracellular Ca2+ signaling downstream of PKG to regulate microneme secretion, gliding, and egress, and has been experimentally used as a diagnostic tool to demonstrate that the major tachyzoite surface antigens (P30/SAG1, P43, P35, P22, gp23) are GPI-anchored to the parasite plasma membrane.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgPI-PLC is a plasma membrane-localized phosphatidylinositol-specific phospholipase C of Toxoplasma gondii that couples lipid hydrolysis to intracellular Ca2+ signaling governing tachyzoite motility and egress [#0, #1]. The cloned enzyme is a ~121 kDa polypeptide organized as an N-terminal PH domain, an EF-hand motif, the catalytic X and Y domains, and a C-terminal C2 domain, and both recombinant and native enzyme preferentially hydrolyze phosphatidylinositol over PIP2 [#0]. Functionally, PI-PLC acts together with PKG to control cytosolic Ca2+ signals mobilized from acidic organelles, linking the enzyme to MIC2 secretion, gliding motility, and egress [#1]. Independently, PI-PLC has served as a definitive biochemical probe establishing that the major tachyzoite surface antigens\\u2014P30/SAG1, P43, P35, P22, and gp23\\u2014are attached to the plasma membrane through GPI anchors, since PI-PLC cleavage renders them water-soluble, removes their fatty-acyl labels, and exposes a trypanosome-VSG-like cross-reacting determinant [#2, #3, #4]. Used as a topological tool in permeabilized parasites, PI-PLC also mapped early GPI biosynthetic intermediates to the cytoplasmic leaflet of the ER and revealed an inositol-acylated, PI-PLC-resistant intermediate [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 1989,\n      \"claim\": \"Establishing how major surface antigens attach to the parasite membrane was open; PI-PLC cleavage demonstrated that P30/SAG1 and the other major tachyzoite surface antigens are GPI-anchored via a diacylglycerol-containing glycolipid.\",\n      \"evidence\": \"Metabolic radiolabeling, PI-PLC treatment of intact parasites and lysates, PLA2 digestion, and PAGE mobility shift/immunoprecipitation in T. gondii tachyzoites\",\n      \"pmids\": [\"2925621\", \"2531282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the enzyme's endogenous parasite gene\", \"GPI core glycan fine structure not fully resolved in these reports\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Extending the GPI-anchor model to additional surface glycoproteins, PI-PLC release of gp23 combined with glycan analysis defined a conserved GPI core bearing beta-linked N-acetylgalactosamine.\",\n      \"evidence\": \"Multi-radiolabel metabolic labeling, PI-PLC and PLA2 digestion, chemical dephosphorylation/deamination, and exo/endoglycosidase analysis in living T. gondii\",\n      \"pmids\": [\"8241958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biological function of the GalNAc modification not addressed\", \"Did not link cleavage to the endogenous PI-PLC enzyme\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The identity and biochemical properties of the parasite enzyme itself were unknown; cloning and recombinant expression defined a PH-EF-X/Y-C2 phospholipase C that preferentially hydrolyzes PI over PIP2 and localizes to the plasma membrane.\",\n      \"evidence\": \"Molecular cloning, recombinant expression in E. coli, in vitro PI/PIP2 hydrolysis assays, and indirect immunofluorescence in T. gondii\",\n      \"pmids\": [\"16288600\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No genetic knockout to establish essentiality or in vivo function\", \"Regulation of catalytic activity not characterized\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The membrane topology of GPI biosynthesis was unresolved; using PI-PLC as a leaflet-specific probe placed early GPI intermediates in the cytoplasmic ER leaflet and revealed a PI-PLC-resistant inositol-acylated intermediate.\",\n      \"evidence\": \"Hypotonic permeabilization, metabolic labeling of GPI intermediates, PI-PLC probing of transmembrane distribution, protease protection, and electron microscopy in T. gondii\",\n      \"pmids\": [\"16896225\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Uses PI-PLC as a reagent rather than testing its own cellular role\", \"Enzyme(s) performing inositol acylation not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Whether PI-PLC contributes to Ca2+-driven invasion/egress behavior was untested; pharmacological inhibition placed PI-PLC with PKG upstream of cytosolic Ca2+ mobilization from acidic stores, MIC2 secretion, gliding, and egress.\",\n      \"evidence\": \"Pharmacological inhibition of PI-PLC and PKG with cytosolic Ca2+ flux measurement, MIC2 secretion, gliding and egress assays in T. gondii\",\n      \"pmids\": [\"29946311\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pathway placement rests on inhibitors, not genetic knockout\", \"Direct enzymatic product coupling Ca2+ release not demonstrated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether PI-PLC influences secretory-antigen expression was addressed by inhibitor treatment, which reduced GRA1 and MIC3 mRNA and protein in vitro and in vivo.\",\n      \"evidence\": \"Edelfosine (PI-PLC inhibitor) treatment of infected Vero cells and a mouse model, qRT-PCR and Western blot in T. gondii\",\n      \"pmids\": [\"31970471\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Pharmacological inhibition only, no genetic manipulation of PI-PLC\", \"Edelfosine effects may be indirect on the enzyme\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The endogenous, genetically defined function of TgPI-PLC in Ca2+ signaling and its physiological lipid substrate/product in vivo remain to be established.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No conditional knockout or complementation of TgPI-PLC reported\", \"In vivo substrate preference (PI vs PIP2) during signaling not resolved\", \"Mechanism linking PI-PLC activity to acidic-store Ca2+ release undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004435\", \"supporting_discovery_ids\": [0, 2, 3, 4, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0004436\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0019722\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006506\", \"supporting_discovery_ids\": [2, 3, 4, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PKG (functional co-requirement in Ca2+ signaling, pharmacological evidence) [#1]\"],\n    \"other_free_text\": [\n      \"Domain architecture: N-terminal PH domain, EF-hand motif, catalytic X and Y domains, C-terminal C2 domain; ~1097 aa / ~121 kDa [#0].\",\n      \"Preferentially hydrolyzes phosphatidylinositol over PIP2 in vitro (recombinant and native enzyme) [#0].\",\n      \"Used experimentally as a diagnostic/topological probe to demonstrate GPI anchoring of surface antigens P30/SAG1, P43, P35, P22, and gp23 [#2, #3, #4] and to map GPI biosynthetic intermediates to the cytoplasmic ER leaflet [#5].\",\n      \"Functionally linked (via pharmacological inhibition) to cytosolic Ca2+ mobilization from acidic organelles, MIC2 secretion, gliding, egress, and to GRA1/MIC3 secretory-antigen expression [#1, #6].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_248830","name":"PIPLC","product":"phosphoinositide phospholipase PIPLC","uniprot_acc":"A0A125YZD6","chromosome":"TGME49_chrXII","start_pos":4196822},{"strain":"GT1","gene_id":"TGGT1_248830","name":null,"product":"phosphoinositide phospholipase PIPLC","uniprot_acc":null,"chromosome":"TGGT1_chrXII","start_pos":4131545},{"strain":"RH88","gene_id":"TGRH88_062700","name":null,"product":"phosphoinositide phospholipase PIPLC","uniprot_acc":null,"chromosome":"CM023093","start_pos":4252131},{"strain":"VEG","gene_id":"TGVEG_248830","name":null,"product":"phosphoinositide phospholipase PIPLC","uniprot_acc":"B6KHA6","chromosome":"TGVEG_chrXII","start_pos":4169678}]}