{"gene":"TG_200010","gene_symbol":"GRA20","run_date":"2026-09-07T10:25:42","timeline":{"discoveries":[{"year":2015,"finding":"GRA20 contains a PEXEL-like (HT/PEXEL) motif that is cleaved by the Golgi-resident aspartyl protease ASP5; in the absence of ASP5, GRA20 is not cleaved at this motif.","method":"Genetic deletion of ASP5 combined with analysis of GRA20 processing (loss-of-function, biochemical characterization of cleavage)","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined molecular phenotype (loss of cleavage), single lab, two orthogonal readouts (protease deletion + motif analysis)","pmids":["26473595"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2013,"finding":"GRA20 contains an HT/PEXEL-like motif (RxLxD/E) and localizes to the parasitophorous vacuole membrane (PVM) but is NOT exported into the host cell; cleavage of the HT/PEXEL motif promotes tighter PVM association, while mutations preventing cleavage reduce PVM association without abolishing PV targeting.","method":"HA-tagging with co-localization microscopy, site-directed mutagenesis of the HT/PEXEL motif, Myc-tag placement before and after cleavage site","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with mutagenesis, single lab, two orthogonal approaches (localization + mutagenesis)","pmids":["23356236"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"Targeted deletion of GRA20 in the RH strain via CRISPR-Cas9 has no significant effect on in vitro parasite growth (plaque assay), egress from host cells, or virulence in mice, indicating GRA20 is dispensable for these processes in this genetic background.","method":"CRISPR-Cas9 gene disruption, in vitro plaque and egress assays, in vivo mouse virulence model","journal":"Frontiers in cellular and infection microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with multiple defined phenotypic readouts (negative result mechanistically informative), single lab","pmids":["30211128"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"current_model":"GRA20 is a dense granule protein that localizes to the parasitophorous vacuole membrane (PVM) via its HT/PEXEL-like motif, which is cleaved by the Golgi-resident aspartyl protease ASP5 to promote tighter PVM association; unlike its Plasmodium counterparts, GRA20 is not exported beyond the PVM into the host cell, and its deletion in the RH strain causes no detectable defect in parasite growth, egress, or virulence in mice."},"narrative":{"mechanistic_narrative":"GRA20 is a Toxoplasma gondii dense granule protein that localizes to the parasitophorous vacuole membrane (PVM) but, unlike its Plasmodium PEXEL-motif counterparts, is not exported beyond the PVM into the host cell [PMID:23356236]. GRA20 carries an HT/PEXEL-like motif (RxLxD/E) that is cleaved by the Golgi-resident aspartyl protease ASP5; this processing promotes tighter PVM association, whereas cleavage-blocking mutations reduce PVM association without abolishing PV targeting [PMID:26473595, PMID:23356236]. CRISPR-Cas9 deletion of GRA20 in the RH strain produces no detectable defect in in vitro growth, egress, or mouse virulence, indicating it is dispensable for these processes in this background [PMID:30211128]. Beyond its ASP5-dependent processing and PVM targeting, no molecular effector function for GRA20 has been characterized in the available corpus.","teleology":[{"year":2013,"claim":"It was unknown how GRA20 associates with the PVM and whether, like Plasmodium PEXEL proteins, it is exported into the host cell; localization and mutagenesis established that GRA20 targets the PVM without host export and that HT/PEXEL cleavage strengthens membrane association.","evidence":"HA/Myc tagging with co-localization microscopy and site-directed mutagenesis of the HT/PEXEL motif in T. gondii","pmids":["23356236"],"confidence":"Medium","gaps":["The identity of the protease responsible for cleavage was not yet defined","No molecular partners at the PVM were identified","The functional consequence of PVM association for the parasite was not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"It was unclear which protease processes the GRA20 HT/PEXEL motif; deletion of ASP5 showed that this Golgi-resident aspartyl protease is responsible for the cleavage, placing GRA20 processing within the ASP5-dependent maturation pathway.","evidence":"Genetic deletion of ASP5 and biochemical analysis of GRA20 cleavage in T. gondii","pmids":["26473595"],"confidence":"Medium","gaps":["Downstream functional role of ASP5-mediated GRA20 cleavage beyond membrane association not resolved","Single lab, no reciprocal biochemical reconstitution of the cleavage reaction"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"The requirement of GRA20 for parasite fitness was untested; CRISPR-Cas9 deletion in RH established that GRA20 is dispensable for growth, egress, and virulence in this strain, indicating any function is non-essential or redundant in this background.","evidence":"CRISPR-Cas9 gene disruption with plaque, egress, and mouse virulence assays in the RH strain","pmids":["30211128"],"confidence":"Medium","gaps":["Negative result does not exclude functional redundancy with other GRA proteins","Not tested in other strains or under stress/immune-competent conditions that might reveal a phenotype","No positive molecular function assigned"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":null,"claim":"The molecular effector function of GRA20 at the PVM and the biological purpose of its ASP5-dependent membrane association remain unknown.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No binding partners or biochemical activity identified","No phenotype linking PVM association to parasite biology","Function in non-RH strains uncharacterized"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["ASP5 (ASPARTYL PROTEASE THAT CLEAVES THE GRA20 HT/PEXEL MOTIF)"],"other_free_text":["GRA20 is a dense granule protein bearing an HT/PEXEL-like motif (RxLxD/E) cleaved by Golgi-resident ASP5, which promotes tighter PVM association [#0, #1].","Unlike Plasmodium PEXEL proteins, GRA20 is retained at the PVM and not exported into the host cell [#1].","GRA20 is dispensable for growth, egress, and virulence in the RH strain [#2]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"26473595","id":"PMC_26473595","title":"Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface.","date":"2015","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/26473595","citation_count":95,"is_preprint":false},{"pmid":"23356236","id":"PMC_23356236","title":"A HT/PEXEL motif in Toxoplasma dense granule proteins is a signal for protein cleavage but not export into the host cell.","date":"2013","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/23356236","citation_count":53,"is_preprint":false},{"pmid":"30211128","id":"PMC_30211128","title":"Functional Characterization of Dense Granule Proteins in Toxoplasma gondii RH Strain Using CRISPR-Cas9 System.","date":"2018","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/30211128","citation_count":30,"is_preprint":false},{"pmid":"26174830","id":"PMC_26174830","title":"Genetic Diversity of Toxoplasma gondii Strains from Different Hosts and Geographical Regions by Sequence Analysis of GRA20 Gene.","date":"2015","source":"The Korean journal of parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/26174830","citation_count":3,"is_preprint":false},{"pmid":"42058244","id":"PMC_42058244","title":"Phylogenetic study of the protozoan Toxoplasma gondii using cytochrome b and GRA20 genes.","date":"2025","source":"Archives of Razi Institute","url":"https://pubmed.ncbi.nlm.nih.gov/42058244","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4823,"output_tokens":906,"usd":0.01403,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5725,"output_tokens":1822,"usd":0.037088,"stage2_stop_reason":"end_turn"},"total_usd":0.051118,"stage1_batch_id":"msgbatch_018xTxyBPcQFK9jdgubtxgsj","stage2_batch_id":"msgbatch_01UiG5BTaJP7BvKKL6RLeyv2","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"GRA20 contains a PEXEL-like (HT/PEXEL) motif that is cleaved by the Golgi-resident aspartyl protease ASP5; in the absence of ASP5, GRA20 is not cleaved at this motif.\",\n      \"method\": \"Genetic deletion of ASP5 combined with analysis of GRA20 processing (loss-of-function, biochemical characterization of cleavage)\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined molecular phenotype (loss of cleavage), single lab, two orthogonal readouts (protease deletion + motif analysis)\",\n      \"pmids\": [\"26473595\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"GRA20 contains an HT/PEXEL-like motif (RxLxD/E) and localizes to the parasitophorous vacuole membrane (PVM) but is NOT exported into the host cell; cleavage of the HT/PEXEL motif promotes tighter PVM association, while mutations preventing cleavage reduce PVM association without abolishing PV targeting.\",\n      \"method\": \"HA-tagging with co-localization microscopy, site-directed mutagenesis of the HT/PEXEL motif, Myc-tag placement before and after cleavage site\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with mutagenesis, single lab, two orthogonal approaches (localization + mutagenesis)\",\n      \"pmids\": [\"23356236\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Targeted deletion of GRA20 in the RH strain via CRISPR-Cas9 has no significant effect on in vitro parasite growth (plaque assay), egress from host cells, or virulence in mice, indicating GRA20 is dispensable for these processes in this genetic background.\",\n      \"method\": \"CRISPR-Cas9 gene disruption, in vitro plaque and egress assays, in vivo mouse virulence model\",\n      \"journal\": \"Frontiers in cellular and infection microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with multiple defined phenotypic readouts (negative result mechanistically informative), single lab\",\n      \"pmids\": [\"30211128\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"current_model\": \"GRA20 is a dense granule protein that localizes to the parasitophorous vacuole membrane (PVM) via its HT/PEXEL-like motif, which is cleaved by the Golgi-resident aspartyl protease ASP5 to promote tighter PVM association; unlike its Plasmodium counterparts, GRA20 is not exported beyond the PVM into the host cell, and its deletion in the RH strain causes no detectable defect in parasite growth, egress, or virulence in mice.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA20 is a Toxoplasma gondii dense granule protein that localizes to the parasitophorous vacuole membrane (PVM) but, unlike its Plasmodium PEXEL-motif counterparts, is not exported beyond the PVM into the host cell [#1]. GRA20 carries an HT/PEXEL-like motif (RxLxD/E) that is cleaved by the Golgi-resident aspartyl protease ASP5; this processing promotes tighter PVM association, whereas cleavage-blocking mutations reduce PVM association without abolishing PV targeting [#0, #1]. CRISPR-Cas9 deletion of GRA20 in the RH strain produces no detectable defect in in vitro growth, egress, or mouse virulence, indicating it is dispensable for these processes in this background [#2]. Beyond its ASP5-dependent processing and PVM targeting, no molecular effector function for GRA20 has been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"It was unknown how GRA20 associates with the PVM and whether, like Plasmodium PEXEL proteins, it is exported into the host cell; localization and mutagenesis established that GRA20 targets the PVM without host export and that HT/PEXEL cleavage strengthens membrane association.\",\n      \"evidence\": \"HA/Myc tagging with co-localization microscopy and site-directed mutagenesis of the HT/PEXEL motif in T. gondii\",\n      \"pmids\": [\"23356236\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The identity of the protease responsible for cleavage was not yet defined\",\n        \"No molecular partners at the PVM were identified\",\n        \"The functional consequence of PVM association for the parasite was not tested\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"It was unclear which protease processes the GRA20 HT/PEXEL motif; deletion of ASP5 showed that this Golgi-resident aspartyl protease is responsible for the cleavage, placing GRA20 processing within the ASP5-dependent maturation pathway.\",\n      \"evidence\": \"Genetic deletion of ASP5 and biochemical analysis of GRA20 cleavage in T. gondii\",\n      \"pmids\": [\"26473595\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Downstream functional role of ASP5-mediated GRA20 cleavage beyond membrane association not resolved\",\n        \"Single lab, no reciprocal biochemical reconstitution of the cleavage reaction\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The requirement of GRA20 for parasite fitness was untested; CRISPR-Cas9 deletion in RH established that GRA20 is dispensable for growth, egress, and virulence in this strain, indicating any function is non-essential or redundant in this background.\",\n      \"evidence\": \"CRISPR-Cas9 gene disruption with plaque, egress, and mouse virulence assays in the RH strain\",\n      \"pmids\": [\"30211128\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Negative result does not exclude functional redundancy with other GRA proteins\",\n        \"Not tested in other strains or under stress/immune-competent conditions that might reveal a phenotype\",\n        \"No positive molecular function assigned\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular effector function of GRA20 at the PVM and the biological purpose of its ASP5-dependent membrane association remain unknown.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No binding partners or biochemical activity identified\",\n        \"No phenotype linking PVM association to parasite biology\",\n        \"Function in non-RH strains uncharacterized\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [],\n    \"localization\": [\n      {\"term_id\": \"GO:0020005\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [],\n    \"complexes\": [],\n    \"partners\": [\"ASP5 (aspartyl protease that cleaves the GRA20 HT/PEXEL motif)\"],\n    \"other_free_text\": [\n      \"GRA20 is a dense granule protein bearing an HT/PEXEL-like motif (RxLxD/E) cleaved by Golgi-resident ASP5, which promotes tighter PVM association [#0, #1].\",\n      \"Unlike Plasmodium PEXEL proteins, GRA20 is retained at the PVM and not exported into the host cell [#1].\",\n      \"GRA20 is dispensable for growth, egress, and virulence in the RH strain [#2].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_200010","name":"GRA20","product":"dense granule protein GRA20","uniprot_acc":"S8ER97","chromosome":"TGME49_chrXII","start_pos":2245476},{"strain":"GT1","gene_id":"TGGT1_200010","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrXII","start_pos":2184571},{"strain":"RH88","gene_id":"TGRH88_060010","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023093","start_pos":2275317},{"strain":"VEG","gene_id":"TGVEG_245428","name":null,"product":"hypothetical protein","uniprot_acc":"B9QCB5","chromosome":"TGVEG_chrXII","start_pos":2215872}]}