{"gene":"PRSS21","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2002,"finding":"PRSS21 (TESP5/testisin) is anchored to the sperm cell membrane via a glycosylphosphatidylinositol (GPI) anchor, as demonstrated by release from cell surface upon treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). The protein is selectively incorporated into lipid rafts (Triton X-100-insoluble microdomains) of sperm membranes, and exhibits serine protease enzymatic activity with substrate specificity distinct from acrosin and pancreatic trypsin.","method":"Recombinant expression in HEK293 cells, PI-PLC treatment, detergent fractionation, substrate specificity and inhibitor assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — reconstituted recombinant protein, PI-PLC release assay, enzymatic characterization with multiple substrates and inhibitors","pmids":["11861648"],"is_preprint":false},{"year":2000,"finding":"PRSS21 (testisin) gene encodes a serine protease with a C-terminal hydrophobic extension predicted to function as a GPI membrane anchor. The gene maps to human chromosome 16p13.3, spans ~4.5 kb, contains 6 exons and 5 introns, and produces two isoforms via alternative pre-mRNA splicing. The gene structure is similar to PRSS8 (prostasin), suggesting evolutionary gene duplication. A CpG island at the 5' region and transcription factor binding sites (Sp1, AP1, testis-specific elements) were identified in the promoter region.","method":"Genomic library screening, sequence analysis, Northern blot, radiation hybrid mapping, FISH, 5'-RACE","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 — direct genomic and molecular characterization with multiple methods","pmids":["11004480"],"is_preprint":false},{"year":1998,"finding":"PRSS21 (ESP-1) is a membrane-type serine protease containing a signal peptide, propeptide, active form sequence with a trypsin-type catalytic triad (His, Asp, Ser), and a C-terminal hydrophobic stretch consistent with membrane anchoring. It is expressed in eosinophils (but not neutrophils), and is most abundant in testis and prostate.","method":"cDNA cloning, sequence analysis, tissue distribution by Northern blot/RT-PCR","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 for sequence/domain architecture; Tier 3 for expression data; single lab","pmids":["9826525"],"is_preprint":false},{"year":1999,"finding":"The PRSS21 (esp-1) gene spans ~4.6 kb with 6 exons and 5 introns, maps to 16p13.3 by radiation hybrid and FISH analyses. A transcription initiation site was identified 106 nt upstream of the start codon. Dual-luciferase reporter analysis showed a GC-rich region (positions -106 to -189) containing an AP-1/Sp-1 binding site is responsible for minimum promoter activity in HeLa cells.","method":"BAC genomic cloning, radiation hybrid mapping, FISH, RNase protection, primer extension, dual-luciferase reporter assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 — reporter assay with defined promoter elements; multiple orthogonal methods","pmids":["10600542"],"is_preprint":false},{"year":2001,"finding":"Mouse Prss21 (testisin) is expressed exclusively in testis among adult tissues, with mRNA appearing at ~day 18 postnatally coinciding with the appearance of spermatids. Immunostaining localizes murine testisin to the cytoplasm and plasma membrane of round and elongating spermatids, demonstrating temporal and spatial regulation during spermatogenesis.","method":"Northern blot, immunohistochemistry, developmental expression analysis","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization by immunostaining with developmental time-course; single lab","pmids":["11231276"],"is_preprint":false},{"year":2003,"finding":"Rat Esp-1/Testisin is expressed predominantly in testis and localizes immunohistochemically to elongated spermatids (steps 12-19), distinct from mouse expression in round spermatids, suggesting species-specific roles in proteolytic events during spermatogenesis.","method":"RT-PCR, quantitative real-time PCR, immunohistochemistry","journal":"The journal of medical investigation : JMI","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization by immunostaining; single lab","pmids":["12630572"],"is_preprint":false},{"year":2005,"finding":"Hypermethylation of the 5' CpG island of PRSS21 (testisin) gene silences its expression in testicular germ cell tumors (100% of 8 tested) and tumor cell lines. Treatment with demethylating agents and/or histone deacetylase inhibitors reactivates PRSS21 expression, demonstrating that CpG methylation and histone deacetylation together regulate PRSS21 transcription. Stable re-expression of testisin in PRSS21-negative Tera-2 testicular cancer cells suppressed anchorage-dependent growth and tumor formation in SCID mice, indicating a tumor suppressor function.","method":"Bisulfite sequencing, demethylating agent treatment (5-aza-2'-deoxycytidine), HDAC inhibitor treatment, stable transfection, soft agar assay, SCID xenograft model","journal":"British journal of cancer","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including functional rescue and in vivo tumor assay; single lab with strong evidence","pmids":["15685234"],"is_preprint":false},{"year":2005,"finding":"Mouse PRSS21 (Prss21/testis serine protease-1) is anchored to plasma membranes via a GPI anchor analogous to other members of the chromosome 17A3.3 tryptase locus, and can be released from the cell surface by phosphatidylinositol-specific phospholipase C.","method":"PI-PLC cell surface release assay, biochemical fractionation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct biochemical demonstration; single lab, consistent with prior findings","pmids":["16143303"],"is_preprint":false},{"year":2008,"finding":"PRSS21-null mouse epididymal sperm show severely impaired ability to bind the zona pellucida (ZP) and undergo ZP-induced acrosome reaction in vitro, and severely impaired sperm-egg fusion in vitro. Normal male fertility in vivo is rescued by exposure of Prss21-null sperm to the uterine microenvironment or uterine fluids in vitro, demonstrating that uterine factors compensate for the loss of PRSS21 protease activity.","method":"Knockout mouse model, in vitro fertilization assays, ZP-binding assays, acrosome reaction measurement, uterine fluid treatment","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"High","confidence_rationale":"Tier 2 — clean KO with multiple defined cellular phenotypes; multiple orthogonal assays; single lab","pmids":["18754795"],"is_preprint":false},{"year":2009,"finding":"PRSS21-deficient (testisin knockout) mice display defective epididymal sperm maturation: spermatozoa show decreased motility, angulated/curled tails, fragile necks, and increased susceptibility to decapitation, with structural defects arising during transit from corpus to cauda epididymis. PRSS21-null sperm fail to swell under hypotonic conditions, indicating impaired osmotic stress responses essential for sperm maturation.","method":"PRSS21 knockout mouse model, histology, electron microscopy, sperm motility analysis, hypotonic swelling test","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 — clean KO with multiple defined morphological and functional phenotypes; multiple orthogonal methods","pmids":["19571264"],"is_preprint":false},{"year":2010,"finding":"Mice doubly deficient in ACR (acrosin) and PRSS21 are subfertile in vivo due to incomplete fertilization. Double-knockout epididymal sperm fail to undergo acrosomal exocytosis on the zona pellucida surface, fail to traverse the ZP, and are defective in penetration through cumulus matrix—demonstrating that the combined trypsin-like serine protease activity of ACR and PRSS21 is essential for sperm penetration through cumulus and ZP in vitro. Female reproductive tract partially compensates for this loss in vivo.","method":"Double knockout mouse model, in vitro fertilization, acrosome reaction assays, cumulus penetration assays, artificial uterine insemination","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 — double KO epistasis with multiple defined cellular phenotypes and genetic rescue experiments","pmids":["20484738"],"is_preprint":false},{"year":2018,"finding":"In stallion spermatozoa, testisin (PRSS21) surface expression increases significantly upon capacitation and further upon acrosome reaction, and localizes to the equatorial region of the sperm head. Testisin is also detected in epididymal luminal fluid and epididymal epithelial cells. Testisin forms multiprotein complexes and co-immunoprecipitates with zona pellucida-binding proteins including ZPBP, ZAN, acrosin, heat-shock proteins, and TCP1 complex components, indicating participation in the ZP-binding complex.","method":"Live cell immunofluorescence, flow cytometry, immunohistochemistry, Blue Native PAGE, co-immunoprecipitation, mass spectrometry","journal":"Andrology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including live imaging, co-IP, and MS; single lab","pmids":["30549223"],"is_preprint":false},{"year":2019,"finding":"PRSS21 (testisin) activity suppresses ovarian tumor metastasis, ascites accumulation, and intraperitoneal tumor seeding in a murine xenograft model. Mechanistically, catalytically active testisin activates protease-activated receptor-2 (PAR-2) on the cancer cell surface, which in turn suppresses the synthesis and secretion of pro-angiogenic angiopoietins ANG2 and ANGPTL4, thereby antagonizing vascular permeability and edema associated with metastasis.","method":"Murine xenograft model, catalytic mutant expression, gene profiling, PAR-2 mechanistic studies, ANG2/ANGPTL4 secretion assays","journal":"Journal of molecular medicine (Berlin, Germany)","confidence":"High","confidence_rationale":"Tier 2 — in vivo model with catalytic mutant controls and mechanistic pathway studies; multiple orthogonal methods","pmids":["30911775"],"is_preprint":false},{"year":2019,"finding":"Fluoride exposure downregulates PRSS21 expression (mRNA and protein) in rat epididymis and is associated with reduced sperm ability to penetrate the egg cumulus cell layer, linking PRSS21 epididymal expression to sperm fertilizing ability.","method":"In vivo fluoride exposure model, Western blot, immunofluorescence, sperm-egg binding assay","journal":"Journal of agricultural and food chemistry","confidence":"Low","confidence_rationale":"Tier 3 — correlative reduction in expression; no direct functional rescue or specific mechanistic link established","pmids":["31008594"],"is_preprint":false},{"year":2020,"finding":"Testisin (PRSS21) deficiency in mice leads to increased vascular permeability and hemorrhage during corpus luteal angiogenesis. In vitro, siRNA knockdown of testisin in microvascular endothelial cells impairs tubule-like formation on Matrigel and increases paracellular permeability to FITC-albumin. Loss of testisin decreases VE-cadherin expression and increases phospho(Tyr658)-VE-cadherin at adherens junctions, without affecting tight junction proteins (occludin, claudin-5, ZO-1), identifying testisin as a novel regulator of VE-cadherin-mediated endothelial adhesion during angiogenesis.","method":"Prss21 knockout mouse model, siRNA knockdown, Evans blue dye vascular permeability assay, live cell imaging on Matrigel, FITC-albumin transwell permeability assay, Western blot, immunofluorescence","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — in vivo KO and in vitro KD with multiple orthogonal functional readouts; single lab with strong mechanistic data","pmids":["32511276"],"is_preprint":false},{"year":2024,"finding":"SPEM2 interacts with PRSS21 (and other sperm proteins ZPBP, PRSS54, PRSS55, ADAM2, ADAM3) and is required for their processing and maturation in epididymal sperm, placing PRSS21 downstream of SPEM2 in a regulatory pathway for sperm maturation.","method":"Co-immunoprecipitation, Western blot in Spem2 knockout mouse model","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP interaction with functional context from KO model; single lab","pmids":["38421455"],"is_preprint":false}],"current_model":"PRSS21 (testisin) is a GPI-anchored serine protease tethered to lipid rafts on the sperm and endothelial cell surface, where it mediates epididymal sperm maturation (regulating motility, morphology, osmotic responses, and ZP-binding/acrosome reaction), participates in a zona pellucida-binding multiprotein complex, activates PAR-2 to suppress pro-angiogenic angiopoietins (ANG2, ANGPTL4) thereby inhibiting tumor metastasis and ascites, and regulates endothelial VE-cadherin adherens junctions to maintain vascular integrity during angiogenesis; its gene is transcriptionally silenced by 5' CpG island hypermethylation and histone deacetylation in testicular germ cell tumors, contributing to its tumor suppressor function."},"narrative":{"teleology":[{"year":1998,"claim":"Identification of PRSS21 as a membrane-type serine protease with a catalytic triad and C-terminal hydrophobic anchor domain established it as a new trypsin-family member expressed predominantly in testis.","evidence":"cDNA cloning, sequence analysis, and tissue distribution by Northern blot/RT-PCR","pmids":["9826525"],"confidence":"Medium","gaps":["Enzymatic activity not directly demonstrated","Membrane anchoring mechanism not confirmed biochemically","Function unknown"]},{"year":2000,"claim":"Genomic characterization revealed the 6-exon gene structure, chromosomal location at 16p13.3, a 5′ CpG island, and structural similarity to PRSS8 (prostasin), suggesting evolutionary duplication and potential for epigenetic regulation.","evidence":"Genomic library screening, FISH, radiation hybrid mapping, 5′-RACE, promoter reporter assays in HeLa cells","pmids":["11004480","10600542"],"confidence":"High","gaps":["CpG island functional significance in disease not yet tested","Testis-specific transcription factor requirements undefined"]},{"year":2002,"claim":"Biochemical demonstration that PRSS21 is GPI-anchored, resides in lipid raft microdomains, and possesses serine protease activity with unique substrate specificity resolved its membrane tethering mechanism and confirmed enzymatic function.","evidence":"Recombinant expression in HEK293 cells, PI-PLC release, Triton X-100 detergent fractionation, substrate and inhibitor profiling","pmids":["11861648"],"confidence":"High","gaps":["Physiological substrates unidentified","Role of lipid raft localization in function unknown"]},{"year":2001,"claim":"Developmental expression analysis showed PRSS21 appears at postnatal day 18 coinciding with spermatid differentiation and localizes to round/elongating spermatid plasma membranes, placing it temporally and spatially in spermatogenesis.","evidence":"Northern blot, immunohistochemistry, developmental time-course in mouse testis","pmids":["11231276","12630572"],"confidence":"Medium","gaps":["Functional role during spermiogenesis not established","Species-specific differences in spermatid stage expression not mechanistically explained"]},{"year":2005,"claim":"Discovery that PRSS21 is epigenetically silenced by CpG hypermethylation and histone deacetylation in testicular germ cell tumors, and that re-expression suppresses tumorigenicity, established testisin as a tumor suppressor.","evidence":"Bisulfite sequencing, 5-aza-dC and HDAC inhibitor treatment, stable re-expression in Tera-2 cells, soft agar assay, SCID mouse xenograft","pmids":["15685234"],"confidence":"High","gaps":["Mechanism of tumor suppression at the molecular level unknown","Downstream targets of testisin protease activity in cancer cells unidentified"]},{"year":2008,"claim":"Prss21-knockout mice revealed that PRSS21 is required for zona pellucida binding, ZP-induced acrosome reaction, and sperm-egg fusion in vitro, but uterine factors compensate in vivo, defining a redundant reproductive safeguard.","evidence":"Knockout mouse, IVF assays, ZP-binding and acrosome reaction quantification, uterine fluid rescue","pmids":["18754795"],"confidence":"High","gaps":["Identity of compensatory uterine factors unknown","Direct proteolytic substrates mediating ZP binding unidentified"]},{"year":2009,"claim":"Extended KO phenotyping demonstrated that PRSS21 is required for epididymal sperm maturation including acquisition of motility, normal tail morphology, and osmotic stress response, pinpointing the corpus-to-cauda epididymal transit as the critical window.","evidence":"Prss21 KO mouse, electron microscopy, motility analysis, hypotonic swelling test","pmids":["19571264"],"confidence":"High","gaps":["Molecular targets of PRSS21 proteolysis during epididymal transit unknown","Whether PRSS21 acts cell-autonomously on sperm or via epididymal fluid not resolved"]},{"year":2010,"claim":"Double knockout of ACR (acrosin) and PRSS21 produced subfertility in vivo and complete failure of ZP penetration and cumulus traversal in vitro, establishing functional redundancy between these two trypsin-like proteases in fertilization.","evidence":"Acr/Prss21 double KO mouse, IVF, cumulus penetration assays, artificial insemination","pmids":["20484738"],"confidence":"High","gaps":["Whether ACR and PRSS21 share substrates or act on distinct targets unknown","Additional compensating proteases in vivo not identified"]},{"year":2018,"claim":"Identification of PRSS21 in a multiprotein ZP-binding complex (with ZPBP, ZAN, acrosin, HSPs, TCP1) on capacitated sperm provided a molecular framework for how testisin contributes to zona recognition.","evidence":"Blue Native PAGE, co-immunoprecipitation, mass spectrometry, flow cytometry, and immunofluorescence in stallion sperm","pmids":["30549223"],"confidence":"High","gaps":["Whether PRSS21 proteolytically processes complex components or serves a structural role unknown","Whether this complex is conserved across species not established"]},{"year":2019,"claim":"Catalytically active testisin was shown to suppress ovarian tumor metastasis by activating PAR-2 on cancer cells, which in turn reduces secretion of pro-angiogenic ANG2 and ANGPTL4, providing a mechanistic basis for its tumor suppressor activity beyond testicular cancer.","evidence":"Murine xenograft with catalytic-dead mutant controls, gene profiling, PAR-2 signaling and angiopoietin secretion assays","pmids":["30911775"],"confidence":"High","gaps":["How PAR-2 activation leads to angiopoietin suppression mechanistically not fully delineated","Whether PAR-2 is a direct cleavage substrate of testisin not confirmed with purified components"]},{"year":2020,"claim":"PRSS21 was established as a regulator of endothelial barrier function: its loss decreases VE-cadherin and increases phospho-VE-cadherin at adherens junctions, increasing vascular permeability without affecting tight junctions, revealing a non-reproductive physiological role.","evidence":"Prss21 KO mouse corpus luteum analysis, siRNA in endothelial cells, Evans blue and FITC-albumin permeability assays, Matrigel angiogenesis assay","pmids":["32511276"],"confidence":"High","gaps":["Whether PRSS21 directly cleaves VE-cadherin or acts indirectly unknown","Which kinase pathway mediates VE-cadherin Tyr658 phosphorylation upon PRSS21 loss not identified"]},{"year":2024,"claim":"Placement of PRSS21 downstream of SPEM2 in a sperm maturation regulatory pathway showed that SPEM2 is required for PRSS21 processing and maturation, identifying an upstream regulator.","evidence":"Co-immunoprecipitation and Western blot in Spem2 KO mouse epididymal sperm","pmids":["38421455"],"confidence":"Medium","gaps":["Whether SPEM2 directly processes PRSS21 or acts as a chaperone unknown","Reciprocal validation and stoichiometry not established"]},{"year":null,"claim":"The direct physiological substrates of PRSS21 protease activity — in both reproductive and vascular contexts — remain unidentified, which is the central open question for understanding its mechanism of action.","evidence":"","pmids":[],"confidence":"High","gaps":["No physiological substrate identified by cleavage-site mapping or reconstitution","Structural basis of substrate specificity undetermined","Whether PAR-2 and VE-cadherin are direct or indirect targets not resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,8,10,12]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4,7,11]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[8,9,10,11]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,12]}],"complexes":["zona pellucida-binding complex"],"partners":["PAR2","ACR","ZPBP","ZAN","SPEM2","ADAM2","ADAM3"],"other_free_text":[]},"mechanistic_narrative":"PRSS21 (testisin) is a GPI-anchored trypsin-like serine protease that functions in sperm maturation, fertilization competence, tumor suppression, and endothelial barrier integrity. In the male reproductive tract, PRSS21 localizes to spermatid and sperm plasma membranes within lipid rafts and participates in a zona pellucida-binding multiprotein complex; Prss21-knockout sperm exhibit impaired motility, morphological defects, defective osmotic responses, and failure to bind the ZP or undergo acrosome reaction, establishing PRSS21 as essential for epididymal sperm maturation and fertilization in vitro [PMID:11861648, PMID:19571264, PMID:18754795, PMID:30549223]. In endothelial cells, PRSS21 maintains VE-cadherin adherens junctions and vascular integrity during angiogenesis, as its loss increases paracellular permeability and hemorrhage [PMID:32511276]. PRSS21 also acts as a tumor suppressor: its gene is silenced by 5′ CpG island hypermethylation in testicular germ cell tumors, re-expression suppresses tumor growth, and catalytically active testisin inhibits ovarian cancer metastasis by activating PAR-2 to downregulate pro-angiogenic angiopoietins ANG2 and ANGPTL4 [PMID:15685234, PMID:30911775]."},"prefetch_data":{"uniprot":{"accession":"Q9Y6M0","full_name":"Testisin","aliases":["Eosinophil serine protease 1","ESP-1","Serine protease 21"],"length_aa":314,"mass_kda":34.9,"function":"Could regulate proteolytic events associated with testicular germ cell maturation","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y6M0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PRSS21","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PRSS21","total_profiled":1310},"omim":[{"mim_id":"609341","title":"TRYPTASE, GAMMA-1; TPSG1","url":"https://www.omim.org/entry/609341"},{"mim_id":"608159","title":"PROTEASE, SERINE, 21; PRSS21","url":"https://www.omim.org/entry/608159"},{"mim_id":"608018","title":"PROTEASE, SERINE, 27; PRSS27","url":"https://www.omim.org/entry/608018"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Mid piece","reliability":"Approved"},{"location":"Principal piece","reliability":"Approved"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"testis","ntpm":81.8}],"url":"https://www.proteinatlas.org/search/PRSS21"},"hgnc":{"alias_symbol":["ESP-1","TEST1"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y6M0","domains":[{"cath_id":"2.40.10.10","chopping":"46-289","consensus_level":"medium","plddt":94.0377,"start":46,"end":289}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6M0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6M0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6M0-F1-predicted_aligned_error_v6.png","plddt_mean":84.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PRSS21","jax_strain_url":"https://www.jax.org/strain/search?query=PRSS21"},"sequence":{"accession":"Q9Y6M0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y6M0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y6M0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6M0"}},"corpus_meta":[{"pmid":"26861414","id":"PMC_26861414","title":"Maternal 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The protein is selectively incorporated into lipid rafts (Triton X-100-insoluble microdomains) of sperm membranes, and exhibits serine protease enzymatic activity with substrate specificity distinct from acrosin and pancreatic trypsin.\",\n      \"method\": \"Recombinant expression in HEK293 cells, PI-PLC treatment, detergent fractionation, substrate specificity and inhibitor assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted recombinant protein, PI-PLC release assay, enzymatic characterization with multiple substrates and inhibitors\",\n      \"pmids\": [\"11861648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PRSS21 (testisin) gene encodes a serine protease with a C-terminal hydrophobic extension predicted to function as a GPI membrane anchor. The gene maps to human chromosome 16p13.3, spans ~4.5 kb, contains 6 exons and 5 introns, and produces two isoforms via alternative pre-mRNA splicing. The gene structure is similar to PRSS8 (prostasin), suggesting evolutionary gene duplication. A CpG island at the 5' region and transcription factor binding sites (Sp1, AP1, testis-specific elements) were identified in the promoter region.\",\n      \"method\": \"Genomic library screening, sequence analysis, Northern blot, radiation hybrid mapping, FISH, 5'-RACE\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct genomic and molecular characterization with multiple methods\",\n      \"pmids\": [\"11004480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PRSS21 (ESP-1) is a membrane-type serine protease containing a signal peptide, propeptide, active form sequence with a trypsin-type catalytic triad (His, Asp, Ser), and a C-terminal hydrophobic stretch consistent with membrane anchoring. It is expressed in eosinophils (but not neutrophils), and is most abundant in testis and prostate.\",\n      \"method\": \"cDNA cloning, sequence analysis, tissue distribution by Northern blot/RT-PCR\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 for sequence/domain architecture; Tier 3 for expression data; single lab\",\n      \"pmids\": [\"9826525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The PRSS21 (esp-1) gene spans ~4.6 kb with 6 exons and 5 introns, maps to 16p13.3 by radiation hybrid and FISH analyses. A transcription initiation site was identified 106 nt upstream of the start codon. Dual-luciferase reporter analysis showed a GC-rich region (positions -106 to -189) containing an AP-1/Sp-1 binding site is responsible for minimum promoter activity in HeLa cells.\",\n      \"method\": \"BAC genomic cloning, radiation hybrid mapping, FISH, RNase protection, primer extension, dual-luciferase reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reporter assay with defined promoter elements; multiple orthogonal methods\",\n      \"pmids\": [\"10600542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mouse Prss21 (testisin) is expressed exclusively in testis among adult tissues, with mRNA appearing at ~day 18 postnatally coinciding with the appearance of spermatids. Immunostaining localizes murine testisin to the cytoplasm and plasma membrane of round and elongating spermatids, demonstrating temporal and spatial regulation during spermatogenesis.\",\n      \"method\": \"Northern blot, immunohistochemistry, developmental expression analysis\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization by immunostaining with developmental time-course; single lab\",\n      \"pmids\": [\"11231276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Rat Esp-1/Testisin is expressed predominantly in testis and localizes immunohistochemically to elongated spermatids (steps 12-19), distinct from mouse expression in round spermatids, suggesting species-specific roles in proteolytic events during spermatogenesis.\",\n      \"method\": \"RT-PCR, quantitative real-time PCR, immunohistochemistry\",\n      \"journal\": \"The journal of medical investigation : JMI\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization by immunostaining; single lab\",\n      \"pmids\": [\"12630572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Hypermethylation of the 5' CpG island of PRSS21 (testisin) gene silences its expression in testicular germ cell tumors (100% of 8 tested) and tumor cell lines. Treatment with demethylating agents and/or histone deacetylase inhibitors reactivates PRSS21 expression, demonstrating that CpG methylation and histone deacetylation together regulate PRSS21 transcription. Stable re-expression of testisin in PRSS21-negative Tera-2 testicular cancer cells suppressed anchorage-dependent growth and tumor formation in SCID mice, indicating a tumor suppressor function.\",\n      \"method\": \"Bisulfite sequencing, demethylating agent treatment (5-aza-2'-deoxycytidine), HDAC inhibitor treatment, stable transfection, soft agar assay, SCID xenograft model\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including functional rescue and in vivo tumor assay; single lab with strong evidence\",\n      \"pmids\": [\"15685234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mouse PRSS21 (Prss21/testis serine protease-1) is anchored to plasma membranes via a GPI anchor analogous to other members of the chromosome 17A3.3 tryptase locus, and can be released from the cell surface by phosphatidylinositol-specific phospholipase C.\",\n      \"method\": \"PI-PLC cell surface release assay, biochemical fractionation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical demonstration; single lab, consistent with prior findings\",\n      \"pmids\": [\"16143303\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PRSS21-null mouse epididymal sperm show severely impaired ability to bind the zona pellucida (ZP) and undergo ZP-induced acrosome reaction in vitro, and severely impaired sperm-egg fusion in vitro. Normal male fertility in vivo is rescued by exposure of Prss21-null sperm to the uterine microenvironment or uterine fluids in vitro, demonstrating that uterine factors compensate for the loss of PRSS21 protease activity.\",\n      \"method\": \"Knockout mouse model, in vitro fertilization assays, ZP-binding assays, acrosome reaction measurement, uterine fluid treatment\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple defined cellular phenotypes; multiple orthogonal assays; single lab\",\n      \"pmids\": [\"18754795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PRSS21-deficient (testisin knockout) mice display defective epididymal sperm maturation: spermatozoa show decreased motility, angulated/curled tails, fragile necks, and increased susceptibility to decapitation, with structural defects arising during transit from corpus to cauda epididymis. PRSS21-null sperm fail to swell under hypotonic conditions, indicating impaired osmotic stress responses essential for sperm maturation.\",\n      \"method\": \"PRSS21 knockout mouse model, histology, electron microscopy, sperm motility analysis, hypotonic swelling test\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple defined morphological and functional phenotypes; multiple orthogonal methods\",\n      \"pmids\": [\"19571264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mice doubly deficient in ACR (acrosin) and PRSS21 are subfertile in vivo due to incomplete fertilization. Double-knockout epididymal sperm fail to undergo acrosomal exocytosis on the zona pellucida surface, fail to traverse the ZP, and are defective in penetration through cumulus matrix—demonstrating that the combined trypsin-like serine protease activity of ACR and PRSS21 is essential for sperm penetration through cumulus and ZP in vitro. Female reproductive tract partially compensates for this loss in vivo.\",\n      \"method\": \"Double knockout mouse model, in vitro fertilization, acrosome reaction assays, cumulus penetration assays, artificial uterine insemination\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — double KO epistasis with multiple defined cellular phenotypes and genetic rescue experiments\",\n      \"pmids\": [\"20484738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In stallion spermatozoa, testisin (PRSS21) surface expression increases significantly upon capacitation and further upon acrosome reaction, and localizes to the equatorial region of the sperm head. Testisin is also detected in epididymal luminal fluid and epididymal epithelial cells. Testisin forms multiprotein complexes and co-immunoprecipitates with zona pellucida-binding proteins including ZPBP, ZAN, acrosin, heat-shock proteins, and TCP1 complex components, indicating participation in the ZP-binding complex.\",\n      \"method\": \"Live cell immunofluorescence, flow cytometry, immunohistochemistry, Blue Native PAGE, co-immunoprecipitation, mass spectrometry\",\n      \"journal\": \"Andrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including live imaging, co-IP, and MS; single lab\",\n      \"pmids\": [\"30549223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PRSS21 (testisin) activity suppresses ovarian tumor metastasis, ascites accumulation, and intraperitoneal tumor seeding in a murine xenograft model. Mechanistically, catalytically active testisin activates protease-activated receptor-2 (PAR-2) on the cancer cell surface, which in turn suppresses the synthesis and secretion of pro-angiogenic angiopoietins ANG2 and ANGPTL4, thereby antagonizing vascular permeability and edema associated with metastasis.\",\n      \"method\": \"Murine xenograft model, catalytic mutant expression, gene profiling, PAR-2 mechanistic studies, ANG2/ANGPTL4 secretion assays\",\n      \"journal\": \"Journal of molecular medicine (Berlin, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo model with catalytic mutant controls and mechanistic pathway studies; multiple orthogonal methods\",\n      \"pmids\": [\"30911775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Fluoride exposure downregulates PRSS21 expression (mRNA and protein) in rat epididymis and is associated with reduced sperm ability to penetrate the egg cumulus cell layer, linking PRSS21 epididymal expression to sperm fertilizing ability.\",\n      \"method\": \"In vivo fluoride exposure model, Western blot, immunofluorescence, sperm-egg binding assay\",\n      \"journal\": \"Journal of agricultural and food chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — correlative reduction in expression; no direct functional rescue or specific mechanistic link established\",\n      \"pmids\": [\"31008594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Testisin (PRSS21) deficiency in mice leads to increased vascular permeability and hemorrhage during corpus luteal angiogenesis. In vitro, siRNA knockdown of testisin in microvascular endothelial cells impairs tubule-like formation on Matrigel and increases paracellular permeability to FITC-albumin. Loss of testisin decreases VE-cadherin expression and increases phospho(Tyr658)-VE-cadherin at adherens junctions, without affecting tight junction proteins (occludin, claudin-5, ZO-1), identifying testisin as a novel regulator of VE-cadherin-mediated endothelial adhesion during angiogenesis.\",\n      \"method\": \"Prss21 knockout mouse model, siRNA knockdown, Evans blue dye vascular permeability assay, live cell imaging on Matrigel, FITC-albumin transwell permeability assay, Western blot, immunofluorescence\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO and in vitro KD with multiple orthogonal functional readouts; single lab with strong mechanistic data\",\n      \"pmids\": [\"32511276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPEM2 interacts with PRSS21 (and other sperm proteins ZPBP, PRSS54, PRSS55, ADAM2, ADAM3) and is required for their processing and maturation in epididymal sperm, placing PRSS21 downstream of SPEM2 in a regulatory pathway for sperm maturation.\",\n      \"method\": \"Co-immunoprecipitation, Western blot in Spem2 knockout mouse model\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP interaction with functional context from KO model; single lab\",\n      \"pmids\": [\"38421455\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRSS21 (testisin) is a GPI-anchored serine protease tethered to lipid rafts on the sperm and endothelial cell surface, where it mediates epididymal sperm maturation (regulating motility, morphology, osmotic responses, and ZP-binding/acrosome reaction), participates in a zona pellucida-binding multiprotein complex, activates PAR-2 to suppress pro-angiogenic angiopoietins (ANG2, ANGPTL4) thereby inhibiting tumor metastasis and ascites, and regulates endothelial VE-cadherin adherens junctions to maintain vascular integrity during angiogenesis; its gene is transcriptionally silenced by 5' CpG island hypermethylation and histone deacetylation in testicular germ cell tumors, contributing to its tumor suppressor function.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PRSS21 (testisin) is a GPI-anchored trypsin-like serine protease that functions in sperm maturation, fertilization competence, tumor suppression, and endothelial barrier integrity. In the male reproductive tract, PRSS21 localizes to spermatid and sperm plasma membranes within lipid rafts and participates in a zona pellucida-binding multiprotein complex; Prss21-knockout sperm exhibit impaired motility, morphological defects, defective osmotic responses, and failure to bind the ZP or undergo acrosome reaction, establishing PRSS21 as essential for epididymal sperm maturation and fertilization in vitro [PMID:11861648, PMID:19571264, PMID:18754795, PMID:30549223]. In endothelial cells, PRSS21 maintains VE-cadherin adherens junctions and vascular integrity during angiogenesis, as its loss increases paracellular permeability and hemorrhage [PMID:32511276]. PRSS21 also acts as a tumor suppressor: its gene is silenced by 5′ CpG island hypermethylation in testicular germ cell tumors, re-expression suppresses tumor growth, and catalytically active testisin inhibits ovarian cancer metastasis by activating PAR-2 to downregulate pro-angiogenic angiopoietins ANG2 and ANGPTL4 [PMID:15685234, PMID:30911775].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of PRSS21 as a membrane-type serine protease with a catalytic triad and C-terminal hydrophobic anchor domain established it as a new trypsin-family member expressed predominantly in testis.\",\n      \"evidence\": \"cDNA cloning, sequence analysis, and tissue distribution by Northern blot/RT-PCR\",\n      \"pmids\": [\"9826525\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Enzymatic activity not directly demonstrated\", \"Membrane anchoring mechanism not confirmed biochemically\", \"Function unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Genomic characterization revealed the 6-exon gene structure, chromosomal location at 16p13.3, a 5′ CpG island, and structural similarity to PRSS8 (prostasin), suggesting evolutionary duplication and potential for epigenetic regulation.\",\n      \"evidence\": \"Genomic library screening, FISH, radiation hybrid mapping, 5′-RACE, promoter reporter assays in HeLa cells\",\n      \"pmids\": [\"11004480\", \"10600542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CpG island functional significance in disease not yet tested\", \"Testis-specific transcription factor requirements undefined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Biochemical demonstration that PRSS21 is GPI-anchored, resides in lipid raft microdomains, and possesses serine protease activity with unique substrate specificity resolved its membrane tethering mechanism and confirmed enzymatic function.\",\n      \"evidence\": \"Recombinant expression in HEK293 cells, PI-PLC release, Triton X-100 detergent fractionation, substrate and inhibitor profiling\",\n      \"pmids\": [\"11861648\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates unidentified\", \"Role of lipid raft localization in function unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Developmental expression analysis showed PRSS21 appears at postnatal day 18 coinciding with spermatid differentiation and localizes to round/elongating spermatid plasma membranes, placing it temporally and spatially in spermatogenesis.\",\n      \"evidence\": \"Northern blot, immunohistochemistry, developmental time-course in mouse testis\",\n      \"pmids\": [\"11231276\", \"12630572\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role during spermiogenesis not established\", \"Species-specific differences in spermatid stage expression not mechanistically explained\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Discovery that PRSS21 is epigenetically silenced by CpG hypermethylation and histone deacetylation in testicular germ cell tumors, and that re-expression suppresses tumorigenicity, established testisin as a tumor suppressor.\",\n      \"evidence\": \"Bisulfite sequencing, 5-aza-dC and HDAC inhibitor treatment, stable re-expression in Tera-2 cells, soft agar assay, SCID mouse xenograft\",\n      \"pmids\": [\"15685234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of tumor suppression at the molecular level unknown\", \"Downstream targets of testisin protease activity in cancer cells unidentified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Prss21-knockout mice revealed that PRSS21 is required for zona pellucida binding, ZP-induced acrosome reaction, and sperm-egg fusion in vitro, but uterine factors compensate in vivo, defining a redundant reproductive safeguard.\",\n      \"evidence\": \"Knockout mouse, IVF assays, ZP-binding and acrosome reaction quantification, uterine fluid rescue\",\n      \"pmids\": [\"18754795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of compensatory uterine factors unknown\", \"Direct proteolytic substrates mediating ZP binding unidentified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended KO phenotyping demonstrated that PRSS21 is required for epididymal sperm maturation including acquisition of motility, normal tail morphology, and osmotic stress response, pinpointing the corpus-to-cauda epididymal transit as the critical window.\",\n      \"evidence\": \"Prss21 KO mouse, electron microscopy, motility analysis, hypotonic swelling test\",\n      \"pmids\": [\"19571264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular targets of PRSS21 proteolysis during epididymal transit unknown\", \"Whether PRSS21 acts cell-autonomously on sperm or via epididymal fluid not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Double knockout of ACR (acrosin) and PRSS21 produced subfertility in vivo and complete failure of ZP penetration and cumulus traversal in vitro, establishing functional redundancy between these two trypsin-like proteases in fertilization.\",\n      \"evidence\": \"Acr/Prss21 double KO mouse, IVF, cumulus penetration assays, artificial insemination\",\n      \"pmids\": [\"20484738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ACR and PRSS21 share substrates or act on distinct targets unknown\", \"Additional compensating proteases in vivo not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identification of PRSS21 in a multiprotein ZP-binding complex (with ZPBP, ZAN, acrosin, HSPs, TCP1) on capacitated sperm provided a molecular framework for how testisin contributes to zona recognition.\",\n      \"evidence\": \"Blue Native PAGE, co-immunoprecipitation, mass spectrometry, flow cytometry, and immunofluorescence in stallion sperm\",\n      \"pmids\": [\"30549223\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PRSS21 proteolytically processes complex components or serves a structural role unknown\", \"Whether this complex is conserved across species not established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Catalytically active testisin was shown to suppress ovarian tumor metastasis by activating PAR-2 on cancer cells, which in turn reduces secretion of pro-angiogenic ANG2 and ANGPTL4, providing a mechanistic basis for its tumor suppressor activity beyond testicular cancer.\",\n      \"evidence\": \"Murine xenograft with catalytic-dead mutant controls, gene profiling, PAR-2 signaling and angiopoietin secretion assays\",\n      \"pmids\": [\"30911775\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PAR-2 activation leads to angiopoietin suppression mechanistically not fully delineated\", \"Whether PAR-2 is a direct cleavage substrate of testisin not confirmed with purified components\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"PRSS21 was established as a regulator of endothelial barrier function: its loss decreases VE-cadherin and increases phospho-VE-cadherin at adherens junctions, increasing vascular permeability without affecting tight junctions, revealing a non-reproductive physiological role.\",\n      \"evidence\": \"Prss21 KO mouse corpus luteum analysis, siRNA in endothelial cells, Evans blue and FITC-albumin permeability assays, Matrigel angiogenesis assay\",\n      \"pmids\": [\"32511276\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PRSS21 directly cleaves VE-cadherin or acts indirectly unknown\", \"Which kinase pathway mediates VE-cadherin Tyr658 phosphorylation upon PRSS21 loss not identified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placement of PRSS21 downstream of SPEM2 in a sperm maturation regulatory pathway showed that SPEM2 is required for PRSS21 processing and maturation, identifying an upstream regulator.\",\n      \"evidence\": \"Co-immunoprecipitation and Western blot in Spem2 KO mouse epididymal sperm\",\n      \"pmids\": [\"38421455\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SPEM2 directly processes PRSS21 or acts as a chaperone unknown\", \"Reciprocal validation and stoichiometry not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct physiological substrates of PRSS21 protease activity — in both reproductive and vascular contexts — remain unidentified, which is the central open question for understanding its mechanism of action.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological substrate identified by cleavage-site mapping or reconstitution\", \"Structural basis of substrate specificity undetermined\", \"Whether PAR-2 and VE-cadherin are direct or indirect targets not resolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 8, 10, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4, 7, 11]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [8, 9, 10, 11]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 12]}\n    ],\n    \"complexes\": [\n      \"zona pellucida-binding complex\"\n    ],\n    \"partners\": [\n      \"PAR2\",\n      \"ACR\",\n      \"ZPBP\",\n      \"ZAN\",\n      \"SPEM2\",\n      \"ADAM2\",\n      \"ADAM3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}