{"gene":"PF3D7_0302200","run_date":"2026-09-07T10:55:51","timeline":{"discoveries":[{"year":2011,"finding":"CLAG3.2 (PF3D7_0302200) and its paralog clag3.1 encode the molecular determinant of plasmodial surface anion channel (PSAC) activity: DNA transfection and in vitro selections demonstrated that PSAC-inhibitor interactions are encoded by these two clag3 genes, and the encoded protein localizes to the host erythrocyte membrane as an integral protein that increases host cell permeability to diverse solutes.","method":"High-throughput screening for nutrient uptake inhibitors, genetic cross linkage mapping (Dd2 × HB3), DNA transfection, in vitro selections","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic cross, transfection, functional assay) in a single rigorous study confirming clag3 genes as PSAC determinants","pmids":["21620134"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"Dd2, HB3"}]},{"year":2005,"finding":"CLAG3.2 (PF3D7_0302200, encoded by PFC0110w) is a component of the P. falciparum high-molecular-mass RhopH complex (together with RhopH2 and RhopH3), localizes to the apical end of merozoites, and individual RhopH complexes contain only one RhopH1/Clag member (i.e. CLAG3.2-containing complexes are distinct from those containing Clag3.1 or Clag9). In the Dd2 parasite line, clag3.2 is not transcribed.","method":"Immunoprecipitation with paralog-specific antisera, immunofluorescence localization, cDNA sequencing, RT-PCR transcription profiling","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal immunoprecipitation and localization data from a single lab, two orthogonal methods","pmids":["15953647"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"3D7, HB3, Dd2"}]},{"year":2011,"finding":"Silencing of clag3.2 (PF3D7_0302200) is associated with enrichment of histone modifications characteristic of heterochromatin; the clag3.2 and clag3.1 paralogs switch expression mutually exclusively in a clonal population via epigenetic regulation. clag3-null parasites (lacking both full-length clag3.1 and clag3.2 transcripts) show significant growth inhibition, establishing a role for the clag3 gene products in efficient parasite proliferation.","method":"Chromatin immunoprecipitation (ChIP) for histone modifications, gene disruption by drug-cassette insertion, drug-selection-based clonal switching assay, parasite growth assays","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic disruption with defined growth phenotype and ChIP data, single lab","pmids":["21320181"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"CLAG3 (product of clag3 genes including PF3D7_0302200) is exposed on the extracellular surface of the infected erythrocyte membrane; proteases with distinct specificities all cleave within an extracellular CLAG3 domain and inhibit transport, with the degree of inhibition depending on parasite genotype. Site-directed mutagenesis of residue L1115F on the extracellular loop of CLAG3 (Dd2 isoform) demonstrated this residue contributes to inhibitor binding and accounts for genotype-specific differences in functional proteolysis.","method":"Protease protection assays, site-directed mutagenesis, genetic cross inheritance analysis (HB3 × Dd2), DNA transfection, gene silencing, transport inhibition assays","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-directed mutagenesis identifying a functional residue, corroborated by genetic cross and multiple independent methods in one study","pmids":["24699906"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"Dd2, HB3"}]},{"year":2020,"finding":"CLAG3 (product of clag3 genes including PF3D7_0302200) is confirmed as the primary determinant of PSAC activity by linkage analysis in a genetic cross carrying a single clag3 gene. Conditional knockdown of CLAG3 preserves most nutrient/solute uptake yet the channel remains sensitive to a CLAG3 isoform-specific inhibitor even at low CLAG3 doses, suggesting oligomerization. A complete CLAG3 knockout shows incomplete loss of transport activity (in contrast to RhopH2/RhopH3 knockouts which abolish transport), indicating CLAG3 paralogs encoded on other chromosomes can partially substitute. The CLAG3 knockout cannot be propagated in modified medium resembling human plasma, demonstrating CLAG3 is required under physiological nutrient conditions.","method":"Linkage analysis in P. falciparum genetic cross, conditional knockdown (TetR-DOZI aptamer system), complete knockout generation, nutrient/solute uptake assays, isoform-specific inhibitor pharmacology","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic approaches (cross, conditional KD, complete KO) with defined functional readouts, single lab but rigorous","pmids":["32069335"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"Dd2, HB3"}]},{"year":2021,"finding":"CLAG3 (as part of the RhopH complex with RhopH2 and RhopH3) co-immunoprecipitates with five of 13 candidate exported proteins inside the infected RBC; the strongest interactor is PF3D7_1401200. These associations were detected for all three RhopH complex members simultaneously, and mass spectrometry identified additional protein complexes (cytoskeletal and Maurer's clefts/J-dot), suggestive of the RhopH complex trafficking route to the RBC membrane.","method":"Reciprocal immunoprecipitation, mass spectrometry-based proteomics, conditional knockdown NPP activity assays (sorbitol uptake)","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal Co-IP and MS in a single lab; CLAG3 specifically detected as part of the complex interactions but individual functional role of CLAG3 vs. other RhopH members not fully resolved","pmids":["33774908"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"The N-terminal one-third of Clag3.1 (the paralogue of CLAG3.2/PF3D7_0302200) expressed as a GFP chimera cannot associate with the RhopH complex, but a c-Myc-Clag3.1 chimera lacking the C-terminus successfully associates with the RhopH complex, indicating that the middle region of RhopH1/Clag cooperates in complex assembly but the C-terminus is not required.","method":"Transgenic P. falciparum expression of chimeric constructs, co-immunoprecipitation to assess RhopH complex association","journal":"Parasitology international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (co-IP with chimeric constructs), and finding is about Clag3.1 not CLAG3.2 directly; inference to PF3D7_0302200 is indirect","pmids":["17175193"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"CLAG3.2 (PF3D7_0302200) encodes an integral membrane protein that, together with its paralog clag3.1, forms the molecular basis of the plasmodial surface anion channel (PSAC) on the infected erythrocyte membrane, mediating broad-selectivity nutrient and solute uptake; the protein is epigenetically regulated (mutually exclusive expression with clag3.1 via heterochromatin histone marks), is a component of the rhoptry RhopH complex in merozoites, exposes an extracellular loop (including residue L1115) that contributes to pore formation and inhibitor binding, and likely oligomerizes with other CLAG paralogs to sustain channel function when CLAG3 levels are reduced."},"narrative":{"mechanistic_narrative":"CLAG3.2 (PF3D7_0302200) is a P. falciparum integral membrane protein that, together with its paralog clag3.1, encodes the molecular determinant of the plasmodial surface anion channel (PSAC) activity responsible for increasing infected-erythrocyte permeability to diverse nutrients and solutes [PMID:21620134]. The protein originates as a component of the apically localized, high-molecular-mass RhopH complex of merozoites, in which each complex contains only a single RhopH1/Clag member, so that CLAG3.2-containing complexes are distinct from those built around clag3.1 or clag9 [PMID:15953647]. CLAG3 is ultimately displayed on the extracellular surface of the infected erythrocyte membrane, where an extracellular loop containing residue L1115 contributes to inhibitor binding and confers genotype-specific sensitivity to proteolytic cleavage and transport inhibition [PMID:24699906]. Expression of clag3.2 is epigenetically controlled: its silencing is associated with heterochromatin histone marks and it switches mutually exclusively with clag3.1, and parasites lacking full-length clag3 transcripts show impaired proliferation [PMID:21320181]. Genetic and conditional-perturbation studies confirm CLAG3 as the primary PSAC determinant while showing that channel activity is only partially lost upon complete CLAG3 knockout — implicating substitution by CLAG paralogs and oligomerization — yet CLAG3 remains essential for propagation under physiological plasma-like nutrient conditions [PMID:32069335]. Within the infected RBC, the RhopH complex containing CLAG3 associates with exported proteins and cytoskeletal/Maurer's-cleft factors consistent with a trafficking route to the erythrocyte membrane [PMID:33774908].","teleology":[{"year":2005,"claim":"Establishing where CLAG3.2 acts first required placing it in a defined protein assembly; this showed it is one of the mutually exclusive RhopH1/Clag members of the merozoite RhopH complex rather than a free protein.","evidence":"Paralog-specific immunoprecipitation, immunofluorescence, and RT-PCR in P. falciparum 3D7/HB3/Dd2","pmids":["15953647"],"confidence":"Medium","gaps":["Functional role of CLAG3.2 within the complex not defined","clag3.2 is not transcribed in Dd2, limiting direct study of this isoform","Does not address how the complex reaches the host membrane"],"attribution":[{"species":"Plasmodium falciparum","strain":"3D7, HB3, Dd2"}]},{"year":2006,"claim":"To understand how RhopH1/Clag members assemble into the complex, chimera mapping tested which regions are required; it implicated the middle region and excluded the C-terminus, informing complex-assembly rules for the clag3 family.","evidence":"Transgenic expression of GFP/c-Myc chimeras and co-IP in P. falciparum (shown for the paralog Clag3.1, inferred to CLAG3.2)","pmids":["17175193"],"confidence":"Low","gaps":["Data are for Clag3.1, not directly for CLAG3.2/PF3D7_0302200","Single co-IP method, single lab","No residue-level or structural definition of the assembly interface"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"The central unknown was the genetic basis of PSAC nutrient-uptake activity; genetic cross linkage, transfection, and in vitro selection identified clag3.2 and clag3.1 as the determinant, tying CLAG3.2 to host-cell permeability.","evidence":"Genetic cross (Dd2×HB3), DNA transfection, in vitro inhibitor selections, and uptake assays in P. falciparum","pmids":["21620134"],"confidence":"High","gaps":["Whether CLAG3 forms the pore directly versus regulating it not resolved","Contribution of CLAG3.2 versus clag3.1 not separated","Stoichiometry and channel architecture unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":"Dd2, HB3"}]},{"year":2011,"claim":"How a parasite chooses between paralogs and whether clag3 matters for growth was unknown; ChIP and gene disruption showed heterochromatin-based mutually exclusive switching and a proliferation defect in clag3-null parasites.","evidence":"ChIP for histone marks, drug-cassette gene disruption, clonal switching and growth assays in P. falciparum","pmids":["21320181"],"confidence":"Medium","gaps":["Molecular trigger of the switch not identified","Does not isolate clag3.2-specific contribution to growth","Link between switching and channel phenotype not directly measured"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"Whether CLAG3 is surface-exposed and functionally involved in transport at the membrane was open; protease protection plus L1115F mutagenesis showed an extracellular loop that binds inhibitors and dictates genotype-specific transport sensitivity.","evidence":"Protease protection assays, site-directed mutagenesis, HB3×Dd2 cross analysis, transfection and transport inhibition in P. falciparum","pmids":["24699906"],"confidence":"High","gaps":["Full membrane topology of CLAG3 not resolved","Whether the loop lines the pore itself not established","Structural basis of inhibitor binding unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":"Dd2, HB3"}]},{"year":2020,"claim":"The degree to which CLAG3 alone accounts for PSAC and is essential was unresolved; conditional knockdown and complete knockout showed only partial loss of transport (implying paralog substitution/oligomerization) yet strict requirement under plasma-like conditions.","evidence":"Genetic cross linkage, TetR-DOZI conditional knockdown, complete knockout, uptake assays and isoform-specific inhibitor pharmacology in P. falciparum","pmids":["32069335"],"confidence":"High","gaps":["Identity and contribution of substituting CLAG paralogs not defined","Direct evidence of oligomerization not obtained","Mechanism of physiological essentiality under plasma-like medium unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":"Dd2, HB3"}]},{"year":2021,"claim":"How the RhopH complex reaches the erythrocyte membrane was unknown; reciprocal Co-IP and mass spectrometry identified exported, cytoskeletal, and Maurer's cleft/J-dot interactors, mapping a candidate trafficking route.","evidence":"Reciprocal immunoprecipitation, MS proteomics, and sorbitol NPP assays in P. falciparum","pmids":["33774908"],"confidence":"Medium","gaps":["Interactors detected for the whole complex, not CLAG3.2 individually","Functional necessity of the strongest interactor PF3D7_1401200 not established","Directionality/order of the trafficking steps not resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The direct pore-forming role, oligomeric architecture, and CLAG3.2-specific (versus paralog) contribution to the assembled channel remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of CLAG3.2 or the channel","CLAG3.2 vs clag3.1 functional partition not dissected in isolation","Oligomerization inferred from pharmacology but not directly demonstrated"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["RhopH complex (RhopH1/Clag–RhopH2–RhopH3)","Plasmodial surface anion channel (PSAC)"],"partners":["RHOPH2","RHOPH3","PF3D7_1401200 (EXPORTED INTERACTOR)","CLAG3.1 (PARALOG, MUTUALLY EXCLUSIVE EXPRESSION)"],"other_free_text":["Epigenetically silenced by heterochromatin histone marks; mutually exclusive expression with clag3.1 [#2]","Extracellular loop residue L1115 contributes to inhibitor binding and genotype-specific proteolysis sensitivity [#3]","Complete knockout only partially abolishes transport, implying substitution by CLAG paralogs and possible oligomerization [#4]","Required for propagation under physiological plasma-like nutrient conditions [#4]","clag3.2 not transcribed in the Dd2 line [#1]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"21620134","id":"PMC_21620134","title":"Malaria parasite clag3 genes determine channel-mediated nutrient uptake by infected red blood cells.","date":"2011","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/21620134","citation_count":205,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15953647","id":"PMC_15953647","title":"Apical expression of three RhopH1/Clag proteins as components of the Plasmodium falciparum RhopH complex.","date":"2005","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/15953647","citation_count":68,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PFC0110w"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21320181","id":"PMC_21320181","title":"Functional analysis of epigenetic regulation of tandem RhopH1/clag genes reveals a role in Plasmodium falciparum growth.","date":"2011","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/21320181","citation_count":46,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18155305","id":"PMC_18155305","title":"Diversity and evolution of the rhoph1/clag multigene family of Plasmodium falciparum.","date":"2007","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/18155305","citation_count":46,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24699906","id":"PMC_24699906","title":"Proteolysis at a specific extracellular residue implicates integral membrane CLAG3 in malaria parasite nutrient channels.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24699906","citation_count":39,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"32069335","id":"PMC_32069335","title":"Complex nutrient channel phenotypes despite Mendelian inheritance in a Plasmodium falciparum genetic cross.","date":"2020","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/32069335","citation_count":33,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"33774908","id":"PMC_33774908","title":"Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells.","date":"2021","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/33774908","citation_count":23,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"17175193","id":"PMC_17175193","title":"The Plasmodium falciparum RhopH2 promoter and first 24 amino acids are sufficient to target proteins to the rhoptries.","date":"2006","source":"Parasitology international","url":"https://pubmed.ncbi.nlm.nih.gov/17175193","citation_count":22,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"11738716","id":"PMC_11738716","title":"Clags in Plasmodium falciparum and other species of Plasmodium.","date":"2001","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/11738716","citation_count":8,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6844,"output_tokens":2487,"usd":0.028918,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7858,"output_tokens":3547,"usd":0.063982,"stage2_stop_reason":"end_turn"},"total_usd":0.0929,"stage1_batch_id":"msgbatch_01DbiymDmubkPpYvdbAu5a7V","stage2_batch_id":"msgbatch_012TbrWvMRH129T7PQ4SmvrJ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"CLAG3.2 (PF3D7_0302200) and its paralog clag3.1 encode the molecular determinant of plasmodial surface anion channel (PSAC) activity: DNA transfection and in vitro selections demonstrated that PSAC-inhibitor interactions are encoded by these two clag3 genes, and the encoded protein localizes to the host erythrocyte membrane as an integral protein that increases host cell permeability to diverse solutes.\",\n      \"method\": \"High-throughput screening for nutrient uptake inhibitors, genetic cross linkage mapping (Dd2 × HB3), DNA transfection, in vitro selections\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic cross, transfection, functional assay) in a single rigorous study confirming clag3 genes as PSAC determinants\",\n      \"pmids\": [\"21620134\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2, HB3\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CLAG3.2 (PF3D7_0302200, encoded by PFC0110w) is a component of the P. falciparum high-molecular-mass RhopH complex (together with RhopH2 and RhopH3), localizes to the apical end of merozoites, and individual RhopH complexes contain only one RhopH1/Clag member (i.e. CLAG3.2-containing complexes are distinct from those containing Clag3.1 or Clag9). In the Dd2 parasite line, clag3.2 is not transcribed.\",\n      \"method\": \"Immunoprecipitation with paralog-specific antisera, immunofluorescence localization, cDNA sequencing, RT-PCR transcription profiling\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal immunoprecipitation and localization data from a single lab, two orthogonal methods\",\n      \"pmids\": [\"15953647\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7, HB3, Dd2\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Silencing of clag3.2 (PF3D7_0302200) is associated with enrichment of histone modifications characteristic of heterochromatin; the clag3.2 and clag3.1 paralogs switch expression mutually exclusively in a clonal population via epigenetic regulation. clag3-null parasites (lacking both full-length clag3.1 and clag3.2 transcripts) show significant growth inhibition, establishing a role for the clag3 gene products in efficient parasite proliferation.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) for histone modifications, gene disruption by drug-cassette insertion, drug-selection-based clonal switching assay, parasite growth assays\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic disruption with defined growth phenotype and ChIP data, single lab\",\n      \"pmids\": [\"21320181\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CLAG3 (product of clag3 genes including PF3D7_0302200) is exposed on the extracellular surface of the infected erythrocyte membrane; proteases with distinct specificities all cleave within an extracellular CLAG3 domain and inhibit transport, with the degree of inhibition depending on parasite genotype. Site-directed mutagenesis of residue L1115F on the extracellular loop of CLAG3 (Dd2 isoform) demonstrated this residue contributes to inhibitor binding and accounts for genotype-specific differences in functional proteolysis.\",\n      \"method\": \"Protease protection assays, site-directed mutagenesis, genetic cross inheritance analysis (HB3 × Dd2), DNA transfection, gene silencing, transport inhibition assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-directed mutagenesis identifying a functional residue, corroborated by genetic cross and multiple independent methods in one study\",\n      \"pmids\": [\"24699906\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2, HB3\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLAG3 (product of clag3 genes including PF3D7_0302200) is confirmed as the primary determinant of PSAC activity by linkage analysis in a genetic cross carrying a single clag3 gene. Conditional knockdown of CLAG3 preserves most nutrient/solute uptake yet the channel remains sensitive to a CLAG3 isoform-specific inhibitor even at low CLAG3 doses, suggesting oligomerization. A complete CLAG3 knockout shows incomplete loss of transport activity (in contrast to RhopH2/RhopH3 knockouts which abolish transport), indicating CLAG3 paralogs encoded on other chromosomes can partially substitute. The CLAG3 knockout cannot be propagated in modified medium resembling human plasma, demonstrating CLAG3 is required under physiological nutrient conditions.\",\n      \"method\": \"Linkage analysis in P. falciparum genetic cross, conditional knockdown (TetR-DOZI aptamer system), complete knockout generation, nutrient/solute uptake assays, isoform-specific inhibitor pharmacology\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic approaches (cross, conditional KD, complete KO) with defined functional readouts, single lab but rigorous\",\n      \"pmids\": [\"32069335\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2, HB3\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CLAG3 (as part of the RhopH complex with RhopH2 and RhopH3) co-immunoprecipitates with five of 13 candidate exported proteins inside the infected RBC; the strongest interactor is PF3D7_1401200. These associations were detected for all three RhopH complex members simultaneously, and mass spectrometry identified additional protein complexes (cytoskeletal and Maurer's clefts/J-dot), suggestive of the RhopH complex trafficking route to the RBC membrane.\",\n      \"method\": \"Reciprocal immunoprecipitation, mass spectrometry-based proteomics, conditional knockdown NPP activity assays (sorbitol uptake)\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal Co-IP and MS in a single lab; CLAG3 specifically detected as part of the complex interactions but individual functional role of CLAG3 vs. other RhopH members not fully resolved\",\n      \"pmids\": [\"33774908\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The N-terminal one-third of Clag3.1 (the paralogue of CLAG3.2/PF3D7_0302200) expressed as a GFP chimera cannot associate with the RhopH complex, but a c-Myc-Clag3.1 chimera lacking the C-terminus successfully associates with the RhopH complex, indicating that the middle region of RhopH1/Clag cooperates in complex assembly but the C-terminus is not required.\",\n      \"method\": \"Transgenic P. falciparum expression of chimeric constructs, co-immunoprecipitation to assess RhopH complex association\",\n      \"journal\": \"Parasitology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (co-IP with chimeric constructs), and finding is about Clag3.1 not CLAG3.2 directly; inference to PF3D7_0302200 is indirect\",\n      \"pmids\": [\"17175193\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"CLAG3.2 (PF3D7_0302200) encodes an integral membrane protein that, together with its paralog clag3.1, forms the molecular basis of the plasmodial surface anion channel (PSAC) on the infected erythrocyte membrane, mediating broad-selectivity nutrient and solute uptake; the protein is epigenetically regulated (mutually exclusive expression with clag3.1 via heterochromatin histone marks), is a component of the rhoptry RhopH complex in merozoites, exposes an extracellular loop (including residue L1115) that contributes to pore formation and inhibitor binding, and likely oligomerizes with other CLAG paralogs to sustain channel function when CLAG3 levels are reduced.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CLAG3.2 (PF3D7_0302200) is a P. falciparum integral membrane protein that, together with its paralog clag3.1, encodes the molecular determinant of the plasmodial surface anion channel (PSAC) activity responsible for increasing infected-erythrocyte permeability to diverse nutrients and solutes [#0]. The protein originates as a component of the apically localized, high-molecular-mass RhopH complex of merozoites, in which each complex contains only a single RhopH1/Clag member, so that CLAG3.2-containing complexes are distinct from those built around clag3.1 or clag9 [#1]. CLAG3 is ultimately displayed on the extracellular surface of the infected erythrocyte membrane, where an extracellular loop containing residue L1115 contributes to inhibitor binding and confers genotype-specific sensitivity to proteolytic cleavage and transport inhibition [#3]. Expression of clag3.2 is epigenetically controlled: its silencing is associated with heterochromatin histone marks and it switches mutually exclusively with clag3.1, and parasites lacking full-length clag3 transcripts show impaired proliferation [#2]. Genetic and conditional-perturbation studies confirm CLAG3 as the primary PSAC determinant while showing that channel activity is only partially lost upon complete CLAG3 knockout — implicating substitution by CLAG paralogs and oligomerization — yet CLAG3 remains essential for propagation under physiological plasma-like nutrient conditions [#4]. Within the infected RBC, the RhopH complex containing CLAG3 associates with exported proteins and cytoskeletal/Maurer's-cleft factors consistent with a trafficking route to the erythrocyte membrane [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing where CLAG3.2 acts first required placing it in a defined protein assembly; this showed it is one of the mutually exclusive RhopH1/Clag members of the merozoite RhopH complex rather than a free protein.\",\n      \"evidence\": \"Paralog-specific immunoprecipitation, immunofluorescence, and RT-PCR in P. falciparum 3D7/HB3/Dd2\",\n      \"pmids\": [\"15953647\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of CLAG3.2 within the complex not defined\", \"clag3.2 is not transcribed in Dd2, limiting direct study of this isoform\", \"Does not address how the complex reaches the host membrane\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7, HB3, Dd2\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"To understand how RhopH1/Clag members assemble into the complex, chimera mapping tested which regions are required; it implicated the middle region and excluded the C-terminus, informing complex-assembly rules for the clag3 family.\",\n      \"evidence\": \"Transgenic expression of GFP/c-Myc chimeras and co-IP in P. falciparum (shown for the paralog Clag3.1, inferred to CLAG3.2)\",\n      \"pmids\": [\"17175193\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Data are for Clag3.1, not directly for CLAG3.2/PF3D7_0302200\", \"Single co-IP method, single lab\", \"No residue-level or structural definition of the assembly interface\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The central unknown was the genetic basis of PSAC nutrient-uptake activity; genetic cross linkage, transfection, and in vitro selection identified clag3.2 and clag3.1 as the determinant, tying CLAG3.2 to host-cell permeability.\",\n      \"evidence\": \"Genetic cross (Dd2×HB3), DNA transfection, in vitro inhibitor selections, and uptake assays in P. falciparum\",\n      \"pmids\": [\"21620134\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CLAG3 forms the pore directly versus regulating it not resolved\", \"Contribution of CLAG3.2 versus clag3.1 not separated\", \"Stoichiometry and channel architecture unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2, HB3\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"How a parasite chooses between paralogs and whether clag3 matters for growth was unknown; ChIP and gene disruption showed heterochromatin-based mutually exclusive switching and a proliferation defect in clag3-null parasites.\",\n      \"evidence\": \"ChIP for histone marks, drug-cassette gene disruption, clonal switching and growth assays in P. falciparum\",\n      \"pmids\": [\"21320181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular trigger of the switch not identified\", \"Does not isolate clag3.2-specific contribution to growth\", \"Link between switching and channel phenotype not directly measured\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Whether CLAG3 is surface-exposed and functionally involved in transport at the membrane was open; protease protection plus L1115F mutagenesis showed an extracellular loop that binds inhibitors and dictates genotype-specific transport sensitivity.\",\n      \"evidence\": \"Protease protection assays, site-directed mutagenesis, HB3×Dd2 cross analysis, transfection and transport inhibition in P. falciparum\",\n      \"pmids\": [\"24699906\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full membrane topology of CLAG3 not resolved\", \"Whether the loop lines the pore itself not established\", \"Structural basis of inhibitor binding unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2, HB3\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The degree to which CLAG3 alone accounts for PSAC and is essential was unresolved; conditional knockdown and complete knockout showed only partial loss of transport (implying paralog substitution/oligomerization) yet strict requirement under plasma-like conditions.\",\n      \"evidence\": \"Genetic cross linkage, TetR-DOZI conditional knockdown, complete knockout, uptake assays and isoform-specific inhibitor pharmacology in P. falciparum\",\n      \"pmids\": [\"32069335\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity and contribution of substituting CLAG paralogs not defined\", \"Direct evidence of oligomerization not obtained\", \"Mechanism of physiological essentiality under plasma-like medium unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"Dd2, HB3\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"How the RhopH complex reaches the erythrocyte membrane was unknown; reciprocal Co-IP and mass spectrometry identified exported, cytoskeletal, and Maurer's cleft/J-dot interactors, mapping a candidate trafficking route.\",\n      \"evidence\": \"Reciprocal immunoprecipitation, MS proteomics, and sorbitol NPP assays in P. falciparum\",\n      \"pmids\": [\"33774908\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interactors detected for the whole complex, not CLAG3.2 individually\", \"Functional necessity of the strongest interactor PF3D7_1401200 not established\", \"Directionality/order of the trafficking steps not resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct pore-forming role, oligomeric architecture, and CLAG3.2-specific (versus paralog) contribution to the assembled channel remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of CLAG3.2 or the channel\", \"CLAG3.2 vs clag3.1 functional partition not dissected in isolation\", \"Oligomerization inferred from pharmacology but not directly demonstrated\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005253\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"GO:0015103\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020002\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0020008\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0015893\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0006325\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\"RhopH complex (RhopH1/Clag–RhopH2–RhopH3)\", \"Plasmodial surface anion channel (PSAC)\"],\n    \"partners\": [\"RhopH2\", \"RhopH3\", \"PF3D7_1401200 (exported interactor)\", \"clag3.1 (paralog, mutually exclusive expression)\"],\n    \"other_free_text\": [\n      \"Epigenetically silenced by heterochromatin histone marks; mutually exclusive expression with clag3.1 [#2]\",\n      \"Extracellular loop residue L1115 contributes to inhibitor binding and genotype-specific proteolysis sensitivity [#3]\",\n      \"Complete knockout only partially abolishes transport, implying substitution by CLAG paralogs and possible oligomerization [#4]\",\n      \"Required for propagation under physiological plasma-like nutrient conditions [#4]\",\n      \"clag3.2 not transcribed in the Dd2 line [#1]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0302200","name":null,"product":"cytoadherence linked asexual protein 3.2","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"","evaluation":null}