{"gene":"EPPIN","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2001,"finding":"EPPIN (epididymal protease inhibitor) was cloned and identified as a novel cysteine-rich protein on human chromosome 20q12-13.2, containing both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences, expressed specifically in testis and epididymis and localized on ejaculated spermatozoa.","method":"cDNA cloning, Northern blot, RT-PCR, Western blot, immunohistochemistry","journal":"Gene","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods in foundational discovery paper","pmids":["11404006"],"is_preprint":false},{"year":2004,"finding":"EPPIN exhibits dose- and time-dependent antibacterial activity against E. coli that requires intact disulfide bonds; it permeabilizes both outer and inner bacterial membranes causing morphological alterations, but does not disrupt eukaryotic membranes.","method":"Colony-forming unit antibacterial assay, membrane permeabilization assay, scanning electron microscopy, erythrocyte hemolytic assay, reduction/alkylation of cysteines","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mechanistic dissection (disulfide bond requirement, membrane permeabilization)","pmids":["15229136"],"is_preprint":false},{"year":2004,"finding":"EPPIN binds semenogelin I (SEMG1) on the surface of ejaculated spermatozoa; the C-terminal fragment EPPIN(75-133) binds the SEMG1(164-283) fragment containing Cys239, and reduction/carboxymethylation of Cys239 blocks binding, indicating a disulfide bond is involved in the interaction.","method":"Immunoprecipitation, co-localization, far-Western blot, saturation binding with 125I-Sg, recombinant fragment mapping, chemical modification","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 1-2 — six orthogonal experimental approaches including radiolabeled binding and mutagenic chemical modification","pmids":["15590901"],"is_preprint":false},{"year":2004,"finding":"Immunization of male monkeys with recombinant EPPIN produced high-titer antibodies, caused complete infertility in all high-titer animals, and fertility was reversible upon cessation of immunization, demonstrating EPPIN's essential role in primate reproduction.","method":"Active immunization in Macaca radiata, fertility testing, antibody titer measurement","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — in vivo loss-of-function via immunization with defined fertility phenotype, replicated across 7/9 animals","pmids":["15539605"],"is_preprint":false},{"year":2006,"finding":"EPPIN modulates PSA (prostate-specific antigen) enzyme activity toward semenogelin digestion; when EPPIN is bound to SEMG1, PSA-mediated digestion is inhibited, and antibodies to the EPPIN C-terminus block this modulation. Human spermatozoa have a receptor for EPPIN.","method":"In vitro PSA digestion assay of SEMG1 with/without EPPIN, antibody blocking experiments, virtual structural modeling","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro enzymatic assay with antibody blocking, single lab","pmids":["16423450"],"is_preprint":false},{"year":2007,"finding":"Native EPPIN from seminal plasma and spermatozoa forms an EPPIN protein complex (EPC) containing lactotransferrin (LTF), clusterin (CLU), and semenogelin (SEMG1); these components co-localize on the sperm tail from the time spermatozoa leave the seminiferous tubule.","method":"Column chromatography isolation, 2D SDS-PAGE, mass spectrometry, Western blot, immunoprecipitation with anti-CLU and anti-LTF, immunofluorescence localization","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 — reciprocal immunoprecipitation combined with MS identification and co-localization","pmids":["17567961"],"is_preprint":false},{"year":2008,"finding":"The Kunitz domain of EPPIN is responsible for elastase inhibition, while neither the Kunitz nor WAP domain alone fully recapitulates EPPIN's antibacterial activity; both domains contribute to bacterial killing partly by permeabilizing the bacterial inner membrane and uncoupling respiratory electron transport.","method":"Domain expression in E. coli, protease inhibition assay (elastase, trypsin, chymotrypsin), colony forming unit assay, XTT respiratory assay","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 — domain dissection with multiple in vitro functional assays","pmids":["18331357"],"is_preprint":false},{"year":2011,"finding":"EPPIN's functions include modulating PSA enzyme activity, providing antimicrobial protection, and binding SEMG1 to inhibit sperm motility; EPPIN is secreted by Sertoli cells and epididymal epithelial cells, exists predominantly as a dimer, and forms a native complex with LTF and clusterin on the sperm surface.","method":"Review of experimental data including immunoprecipitation, functional assays, secretion studies","journal":"Biochemical Society transactions","confidence":"Medium","confidence_rationale":"Tier 3 — synthesis/review paper citing original experimental findings from same lab","pmids":["21936831"],"is_preprint":false},{"year":2012,"finding":"EPPIN (acting as the semenogelin receptor) binding by SEMG1 or anti-EPPIN antibodies targeting a 21 amino acid C-terminal region causes loss of intracellular calcium in spermatozoa, leading to loss of progressive and then complete motility; thimerosal rescues motility implicating internal calcium stores, and ammonium chloride rescues SEMG1-induced motility inhibition, indicating SEMG1 also decreases intracellular pH.","method":"Fluo-4 intracellular calcium measurement, sperm motility assay, pharmacological rescue with thimerosal and ammonium chloride, antibody blocking with defined epitope","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (calcium imaging, motility, pharmacological rescue) with mechanistic dissection","pmids":["22075473"],"is_preprint":false},{"year":2012,"finding":"EPPIN's C-terminal sequence C102-P133 contains the major binding site for SEMG1, and residues Cys102, Tyr107, and Phe117 are required for SEMG1 binding; residues Tyr107 and Phe117 are also required for interaction with lactotransferrin (LTF).","method":"Recombinant fragment binding assays, site-directed mutagenesis of key residues, functional motility inhibition assays","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with binding assays identifying specific residues required for interaction","pmids":["22699487"],"is_preprint":false},{"year":2009,"finding":"Anti-EPPIN antibodies inhibit the calcium ionophore A23187-induced acrosome reaction in human spermatozoa in a dose-dependent manner and reduce A23187-induced elevation of intracellular calcium, without affecting tyrosine phosphorylation; EPPIN relocates from acrosome and tail to equatorial segment and tail after the acrosome reaction.","method":"Immunofluorescence, sperm motility assay, Fluo-3 calcium measurement, anti-EPPIN antibody blocking, capacitation assay","journal":"Human reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — functional blocking with antibody, calcium imaging, single lab","pmids":["19801569"],"is_preprint":false},{"year":2012,"finding":"EPPIN expression in the rat epididymis is androgen-dependent: surgical castration down-regulates EPPIN mRNA and protein in caput and cauda epididymis, and testosterone replacement reverses this effect.","method":"Surgical castration, testosterone replacement, RT-PCR, Western blot, immunolocalization","journal":"Molecular reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo loss-of-function (castration) with rescue (testosterone), single lab","pmids":["23070980"],"is_preprint":false},{"year":2017,"finding":"EPPIN inhibits LPS-induced NF-κB activation in monocytes by a mechanism involving accumulation of phosphorylated IκBα, suppresses LPS-induced chemokine production, and reduces neutrophil recruitment to the lung in vivo; EGF treatment upregulates EPPIN expression and secretion in monocytes.","method":"NF-κB reporter assay, Western blot for phospho-IκBα, cytokine/chemokine measurement, in vivo lung inflammation model with neutrophil recruitment assay, EGF stimulation","journal":"The European respiratory journal","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro mechanistic pathway (NF-κB/IκBα) plus in vivo confirmation, single lab","pmids":["28705940"],"is_preprint":false},{"year":2020,"finding":"In mice, EPPIN acts as a protein hub on sperm surface, binding seminal vesicle secreted proteins SVS2, SVS3A, SVS5, and SVS6 after exposure to seminal vesicle fluid; SVS2 binding was confirmed by in silico docking, far-Western blot, and co-localization on sperm head and flagellum.","method":"Immunoprecipitation from mouse spermatozoa + LC-MS/MS, Western blot, in silico protein-protein interaction, far-Western blot, immunofluorescence co-localization","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — MS-based interactome with orthogonal far-Western and co-localization confirmation","pmids":["32044375"],"is_preprint":false},{"year":2021,"finding":"Antibodies targeting the Kunitz domain epitope Ser103-Cys123 (S21C) reduce progressive motility, hyperactivated motility, and multiple kinematic parameters of mouse sperm and impair in vitro fertilization; antibodies targeting the WFDC domain epitope Gln20-Glu39 (Q20E) cause milder motility inhibition; neither affects capacitation-induced tyrosine phosphorylation.","method":"Antibody blocking with domain-specific epitope antibodies, CASA (computer-assisted sperm analysis), in vitro fertilization, capacitation tyrosine phosphorylation Western blot","journal":"Molecular human reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — domain-specific antibody functional dissection with multiple motility parameters and IVF readout","pmids":["34792600"],"is_preprint":false},{"year":2021,"finding":"siRNA-mediated knockdown of Eppin in mouse seminiferous tubules significantly reduces sperm motility and decreases T-type Ca2+ currents and mRNA expression of three T-type Ca2+ channel subtypes in spermatogenic cells, linking EPPIN to regulation of calcium channel activity.","method":"siRNA microinjection into seminiferous tubules, CASA, patch clamp recording of T-type Ca2+ currents, RT-PCR for channel subtype expression","journal":"Reproductive biology","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo knockdown with electrophysiology and motility readouts, single lab","pmids":["33607572"],"is_preprint":false},{"year":2023,"finding":"Computational modeling reveals EPPIN has a binding pocket formed between WFDC and Kunitz domains at the hinge region; residues Phe63 and Lys68 (WFDC), Asp71 (hinge), and Asn113, Asn114, Asn115 (Kunitz) are identified as hot spots for SEMG1, EP055, and EP012 binding; hydrophobic/hydrophilic residues allow plasma membrane anchoring orienting the binding pocket to solvent.","method":"Molecular dynamics simulation, normal mode analysis, computational docking, homology modeling","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 4 — computational prediction only, no experimental validation of individual residues","pmids":["37658081"],"is_preprint":false},{"year":2025,"finding":"SEMG1 inhibits sperm hyperactivation and directly suppresses CatSper calcium channel currents at physiologically relevant concentrations in an EPPIN-dependent manner; two functional domains of SEMG1 (Q32-V118 and R98-S220) mediate this inhibition, with R98-G375 retaining full EPPIN-binding capacity, while Q32-V118 does not require EPPIN binding to inhibit CatSper.","method":"Electrophysiological patch clamp of CatSper, CASA hyperactivation analysis, recombinant SEMG1 truncation fragments, NH4Cl alkalinization rescue, EPPIN-binding assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 — direct electrophysiology of CatSper with truncation mutant dissection, but preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.09.05.674523"],"is_preprint":true}],"current_model":"EPPIN is a cysteine-rich, dual-domain (Kunitz + WAP/WFDC) protease inhibitor secreted by Sertoli and epididymal epithelial cells onto the sperm surface, where it forms a protein complex (EPC) with lactotransferrin and clusterin; upon ejaculation, semenogelin-1 (SEMG1) binds the EPPIN C-terminus (via residues Cys102, Tyr107, Phe117) to inhibit progressive sperm motility by causing loss of intracellular calcium—likely through suppression of the CatSper calcium channel and disturbance of intracellular pH—while also modulating PSA-mediated SEMG1 digestion and providing antimicrobial protection via membrane permeabilization."},"narrative":{"teleology":[{"year":2001,"claim":"The identification of EPPIN as a novel dual-domain protease inhibitor specifically expressed in testis and epididymis and localized on ejaculated spermatozoa established the gene as a sperm-associated factor with potential reproductive function.","evidence":"cDNA cloning, Northern blot, RT-PCR, Western blot, and immunohistochemistry in human tissues","pmids":["11404006"],"confidence":"High","gaps":["No functional role demonstrated","Protein partners on sperm surface unknown","Mechanism of sperm surface attachment unknown"]},{"year":2004,"claim":"Demonstration that EPPIN kills bacteria by disulfide-dependent membrane permeabilization without damaging eukaryotic membranes established an innate antimicrobial function in the male reproductive tract.","evidence":"Colony-forming unit assays, membrane permeabilization assays, SEM, hemolytic assays, and cysteine reduction/alkylation in E. coli","pmids":["15229136"],"confidence":"High","gaps":["Range of susceptible pathogens not defined","In vivo antimicrobial contribution not tested","Which domain mediates antibacterial activity was unknown"]},{"year":2004,"claim":"The discovery that EPPIN binds SEMG1 on the sperm surface via its C-terminal region through a disulfide-dependent interaction, together with the finding that anti-EPPIN immunization causes reversible infertility in primates, established EPPIN–SEMG1 interaction as a functionally essential axis for male fertility.","evidence":"Immunoprecipitation, radiolabeled saturation binding, recombinant fragment mapping, and active immunization fertility trials in Macaca radiata","pmids":["15590901","15539605"],"confidence":"High","gaps":["How SEMG1 binding regulates sperm motility was unknown","Specific EPPIN residues required for SEMG1 binding not yet mapped","Downstream signaling pathway in spermatozoa uncharacterized"]},{"year":2006,"claim":"Showing that EPPIN–SEMG1 binding inhibits PSA-mediated semenogelin digestion revealed EPPIN as a modulator of semen liquefaction, linking its sperm-surface function to the kinetics of motility acquisition after ejaculation.","evidence":"In vitro PSA digestion assays with and without EPPIN, antibody blocking experiments","pmids":["16423450"],"confidence":"Medium","gaps":["Physiological relevance of PSA modulation in vivo not confirmed","Whether EPPIN directly inhibits PSA protease activity or sterically shields SEMG1 not resolved"]},{"year":2007,"claim":"Isolation of the native EPPIN protein complex (EPC) containing lactotransferrin and clusterin from seminal plasma and sperm revealed that EPPIN functions within a multi-protein assembly on the sperm tail rather than as an isolated factor.","evidence":"Column chromatography, 2D SDS-PAGE, mass spectrometry, reciprocal immunoprecipitation, and immunofluorescence from human spermatozoa and seminal plasma","pmids":["17567961"],"confidence":"High","gaps":["Stoichiometry and assembly order of the EPC unknown","Functional contribution of LTF and CLU within the complex not tested","Mechanism of EPC attachment to sperm membrane uncharacterized"]},{"year":2008,"claim":"Domain dissection showed the Kunitz domain mediates elastase inhibition while both Kunitz and WAP domains are needed for full antibacterial activity, including bacterial inner membrane permeabilization and respiratory uncoupling, resolving domain-specific contributions to EPPIN's dual functions.","evidence":"Recombinant domain expression, protease inhibition assays, colony-forming unit assays, and XTT respiratory assays in E. coli","pmids":["18331357"],"confidence":"High","gaps":["No structural basis for how two domains cooperate in bacterial killing","Antibacterial activity against Gram-positive organisms not tested"]},{"year":2009,"claim":"Anti-EPPIN antibodies blocked the calcium ionophore-induced acrosome reaction and calcium influx, and EPPIN was shown to relocalize on the sperm head during acrosome reaction, extending EPPIN's role beyond motility to acrosomal exocytosis.","evidence":"Antibody blocking with immunofluorescence, Fluo-3 calcium imaging, and capacitation assays on human spermatozoa","pmids":["19801569"],"confidence":"Medium","gaps":["Whether EPPIN directly regulates acrosomal calcium channels or acts indirectly is unknown","Physiological significance of EPPIN relocalization not demonstrated"]},{"year":2012,"claim":"Mapping of the SEMG1-binding site to EPPIN residues Cys102, Tyr107, and Phe117, combined with showing that SEMG1 binding causes intracellular calcium loss and pH decrease leading to motility inhibition, provided the first integrated signaling mechanism for how the EPPIN–SEMG1 axis controls sperm motility.","evidence":"Site-directed mutagenesis and binding assays for residue mapping; Fluo-4 calcium imaging, motility analysis, and pharmacological rescue with thimerosal (calcium) and NH4Cl (pH) on human spermatozoa","pmids":["22699487","22075473"],"confidence":"High","gaps":["Identity of the calcium channel or transporter affected was unknown","Mechanism linking pH decrease to motility loss not elucidated","Whether EPPIN itself transduces the signal or acts solely as SEMG1 receptor unclear"]},{"year":2012,"claim":"Androgen-dependent regulation of EPPIN expression in the epididymis was established, connecting reproductive hormone signaling to EPPIN abundance on spermatozoa.","evidence":"Surgical castration, testosterone replacement, RT-PCR, and Western blot in rat epididymis","pmids":["23070980"],"confidence":"Medium","gaps":["Androgen response elements in the EPPIN promoter not identified","Whether androgen regulation occurs at transcriptional or post-transcriptional level not resolved"]},{"year":2017,"claim":"Discovery that EPPIN inhibits NF-κB activation by promoting phospho-IκBα accumulation and suppresses neutrophil recruitment in vivo extended EPPIN's function beyond reproduction to anti-inflammatory innate immunity.","evidence":"NF-κB reporter assays, phospho-IκBα Western blots in monocytes, and in vivo lung inflammation model with neutrophil counting","pmids":["28705940"],"confidence":"Medium","gaps":["Direct molecular target of EPPIN in the NF-κB pathway not identified","Whether this anti-inflammatory function operates in the male reproductive tract not shown","Mechanism of phospho-IκBα stabilization unclear"]},{"year":2020,"claim":"Identification of mouse seminal vesicle proteins SVS2, SVS3A, SVS5, and SVS6 as EPPIN-binding partners confirmed EPPIN's role as a protein hub on spermatozoa and extended the model to non-primate species where SEMG1 orthologs differ.","evidence":"Immunoprecipitation–LC-MS/MS from mouse spermatozoa, far-Western blot, and immunofluorescence co-localization","pmids":["32044375"],"confidence":"Medium","gaps":["Functional consequence of SVS binding on mouse sperm motility not tested","Whether SVS proteins mimic SEMG1 function in motility regulation unknown"]},{"year":2021,"claim":"Domain-specific antibody blocking showed the Kunitz domain epitope (Ser103-Cys123) is the primary functional surface for motility and fertilization, while siRNA knockdown of Eppin in seminiferous tubules reduced T-type Ca²⁺ channel currents, providing the first link between EPPIN and a specific calcium channel family in spermatogenic cells.","evidence":"Epitope-specific antibody blocking with CASA and IVF in mouse; siRNA knockdown with patch-clamp electrophysiology and RT-PCR in mouse spermatogenic cells","pmids":["34792600","33607572"],"confidence":"Medium","gaps":["Whether T-type Ca²⁺ channel regulation is direct or indirect not determined","T-type channels function in spermatogenic cells but CatSper dominates in mature sperm — channel identity on mature sperm unresolved","IVF effects not confirmed in vivo"]},{"year":null,"claim":"The identity of the calcium channel(s) on mature spermatozoa through which the EPPIN–SEMG1 axis suppresses motility remains to be definitively established, and no high-resolution structure of the EPPIN–SEMG1 complex or the EPPIN protein complex (EPC) exists.","evidence":"","pmids":[],"confidence":"High","gaps":["No crystal or cryo-EM structure of EPPIN or its complexes","Whether CatSper is the physiological target of the EPPIN–SEMG1 axis on mature sperm requires peer-reviewed confirmation","Mechanism of EPPIN membrane anchoring unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,4,8,9]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,6]},{"term_id":"GO:0090729","term_label":"toxin activity","supporting_discovery_ids":[1,6]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,5,7]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[3,8,14,15]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,6,12]}],"complexes":["EPPIN protein complex (EPC: EPPIN–LTF–CLU)"],"partners":["SEMG1","LTF","CLU","KLK3"],"other_free_text":[]},"mechanistic_narrative":"EPPIN is a cysteine-rich, dual-domain (Kunitz + WAP/WFDC) protease inhibitor that functions as a sperm-surface protein hub essential for male fertility and innate antimicrobial defense in the reproductive tract. Secreted by Sertoli and epididymal epithelial cells, EPPIN assembles a native complex with lactotransferrin and clusterin on the sperm tail, and upon ejaculation binds semenogelin-1 (SEMG1) via C-terminal residues Cys102, Tyr107, and Phe117; this SEMG1 binding inhibits progressive motility through loss of intracellular calcium and decreased intracellular pH, and modulates PSA-mediated semenogelin digestion [PMID:22075473, PMID:22699487, PMID:16423450]. EPPIN also exhibits disulfide-dependent antibacterial activity by permeabilizing bacterial membranes and uncoupling respiratory electron transport, with both Kunitz and WAP domains contributing, and inhibits NF-κB signaling in monocytes to suppress neutrophil-driven inflammation [PMID:15229136, PMID:18331357, PMID:28705940]. Immunization of male primates with EPPIN produces reversible infertility, establishing it as a contraceptive target [PMID:15539605]."},"prefetch_data":{"uniprot":{"accession":"O95925","full_name":"Eppin","aliases":["Cancer/testis antigen 71","CT71","Epididymal protease inhibitor","Protease inhibitor WAP7","Serine protease inhibitor-like with Kunitz and WAP domains 1","WAP four-disulfide core domain protein 7"],"length_aa":133,"mass_kda":15.3,"function":"Serine protease inhibitor that plays an essential role in male reproduction and fertility. Modulates the hydrolysis of SEMG1 by KLK3/PSA (a serine protease), provides antimicrobial protection for spermatozoa in the ejaculate coagulum, and binds SEMG1 thereby inhibiting sperm motility","subcellular_location":"Secreted; Cell surface","url":"https://www.uniprot.org/uniprotkb/O95925/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPPIN","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EPPIN","total_profiled":1310},"omim":[{"mim_id":"619430","title":"SERINE PEPTIDASE INHIBITOR, KUNITZ-TYPE, 4; SPINT4","url":"https://www.omim.org/entry/619430"},{"mim_id":"613941","title":"SERINE PEPTIDASE INHIBITOR, KUNITZ-TYPE, 3; SPINT3","url":"https://www.omim.org/entry/613941"},{"mim_id":"609031","title":"EPIDIDYMAL PROTEASE INHIBITOR; EPPIN","url":"https://www.omim.org/entry/609031"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"epididymis","ntpm":309.3}],"url":"https://www.proteinatlas.org/search/EPPIN"},"hgnc":{"alias_symbol":["EPPIN1","EPPIN2","EPPIN3","dJ461P17.2","WAP7","WFDC7","CT71"],"prev_symbol":["SPINLW1"]},"alphafold":{"accession":"O95925","domains":[{"cath_id":"4.10.410.10","chopping":"21-127","consensus_level":"medium","plddt":91.7875,"start":21,"end":127}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95925","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95925-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95925-F1-predicted_aligned_error_v6.png","plddt_mean":89.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPPIN","jax_strain_url":"https://www.jax.org/strain/search?query=EPPIN"},"sequence":{"accession":"O95925","fasta_url":"https://rest.uniprot.org/uniprotkb/O95925.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95925/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95925"}},"corpus_meta":[{"pmid":"15539605","id":"PMC_15539605","title":"Reversible immunocontraception in male monkeys immunized with eppin.","date":"2004","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/15539605","citation_count":139,"is_preprint":false},{"pmid":"11404006","id":"PMC_11404006","title":"Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis.","date":"2001","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/11404006","citation_count":98,"is_preprint":false},{"pmid":"15229136","id":"PMC_15229136","title":"Antimicrobial activity of human EPPIN, an androgen-regulated, sperm-bound protein with a whey acidic protein motif.","date":"2004","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/15229136","citation_count":89,"is_preprint":false},{"pmid":"15590901","id":"PMC_15590901","title":"Association of eppin with semenogelin on human spermatozoa.","date":"2004","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/15590901","citation_count":68,"is_preprint":false},{"pmid":"17567961","id":"PMC_17567961","title":"Characterization of an eppin protein complex from human semen and spermatozoa.","date":"2007","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/17567961","citation_count":64,"is_preprint":false},{"pmid":"16423450","id":"PMC_16423450","title":"Eppin: an effective target for male contraception.","date":"2006","source":"Molecular and cellular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/16423450","citation_count":53,"is_preprint":false},{"pmid":"26593445","id":"PMC_26593445","title":"Non-hormonal male contraception: A review and development of an Eppin based contraceptive.","date":"2015","source":"Pharmacology & therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/26593445","citation_count":50,"is_preprint":false},{"pmid":"12909348","id":"PMC_12909348","title":"Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2.","date":"2003","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/12909348","citation_count":37,"is_preprint":false},{"pmid":"21525168","id":"PMC_21525168","title":"Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis.","date":"2011","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/21525168","citation_count":36,"is_preprint":false},{"pmid":"20488803","id":"PMC_20488803","title":"Variants of the EPPIN gene affect the risk of idiopathic male infertility in the Han-Chinese population.","date":"2010","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/20488803","citation_count":19,"is_preprint":false},{"pmid":"19801569","id":"PMC_19801569","title":"The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa.","date":"2009","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/19801569","citation_count":19,"is_preprint":false},{"pmid":"23070980","id":"PMC_23070980","title":"Epididymal protease inhibitor (EPPIN) is differentially expressed in the male rat reproductive tract and immunolocalized in maturing spermatozoa.","date":"2012","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/23070980","citation_count":18,"is_preprint":false},{"pmid":"21936831","id":"PMC_21936831","title":"Functional studies of eppin.","date":"2011","source":"Biochemical Society transactions","url":"https://pubmed.ncbi.nlm.nih.gov/21936831","citation_count":16,"is_preprint":false},{"pmid":"22075473","id":"PMC_22075473","title":"Loss of calcium in human spermatozoa via EPPIN, the semenogelin receptor.","date":"2012","source":"Biology of 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epididymal protease inhibitor and a target for male contraception.","date":"2007","source":"Society of Reproduction and Fertility supplement","url":"https://pubmed.ncbi.nlm.nih.gov/17566290","citation_count":14,"is_preprint":false},{"pmid":"34792600","id":"PMC_34792600","title":"Dissecting EPPIN protease inhibitor domains in sperm motility and fertilizing ability: repercussions for male contraceptive development.","date":"2021","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/34792600","citation_count":11,"is_preprint":false},{"pmid":"32044375","id":"PMC_32044375","title":"Epididymal protease inhibitor (EPPIN) is a protein hub for seminal vesicle-secreted protein SVS2 binding in mouse spermatozoa.","date":"2020","source":"Molecular and cellular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/32044375","citation_count":11,"is_preprint":false},{"pmid":"20158997","id":"PMC_20158997","title":"Variants in the Eppin gene show association with semen quality in Han-Chinese population.","date":"2009","source":"Reproductive biomedicine online","url":"https://pubmed.ncbi.nlm.nih.gov/20158997","citation_count":9,"is_preprint":false},{"pmid":"37658081","id":"PMC_37658081","title":"Interactions of the male contraceptive target EPPIN with semenogelin-1 and small organic ligands.","date":"2023","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/37658081","citation_count":8,"is_preprint":false},{"pmid":"25864521","id":"PMC_25864521","title":"Enhanced Suppression of Fertility Can be Achieved by Priming with FSHR and Eppin and Further Boosting with Their B-cell Epitope Peptides.","date":"2015","source":"American journal of reproductive immunology (New York, N.Y. : 1989)","url":"https://pubmed.ncbi.nlm.nih.gov/25864521","citation_count":7,"is_preprint":false},{"pmid":"28705940","id":"PMC_28705940","title":"Characterisation of eppin function: expression and activity in the lung.","date":"2017","source":"The European respiratory journal","url":"https://pubmed.ncbi.nlm.nih.gov/28705940","citation_count":5,"is_preprint":false},{"pmid":"21817855","id":"PMC_21817855","title":"A highly specific antibody response after protein prime-peptide boost immunization with Eppin/B-cell epitope in mice.","date":"2011","source":"Human vaccines","url":"https://pubmed.ncbi.nlm.nih.gov/21817855","citation_count":3,"is_preprint":false},{"pmid":"18466682","id":"PMC_18466682","title":"Purification and characterization of biologically active recombinant human Eppin expressed in Escherichia coli.","date":"2008","source":"Chinese medical journal","url":"https://pubmed.ncbi.nlm.nih.gov/18466682","citation_count":3,"is_preprint":false},{"pmid":"33607572","id":"PMC_33607572","title":"siRNA-mediated Eppin testicular silencing causes changes in sperm motility and calcium currents in mice.","date":"2021","source":"Reproductive biology","url":"https://pubmed.ncbi.nlm.nih.gov/33607572","citation_count":2,"is_preprint":false},{"pmid":"25424926","id":"PMC_25424926","title":"Nasal immunization using a mimovirus vaccine based on the Eppin B-cell epitope induced suppressed fertility in mice.","date":"2014","source":"Human vaccines & immunotherapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/25424926","citation_count":2,"is_preprint":false},{"pmid":"21461566","id":"PMC_21461566","title":"Distribution of Eppin in mouse and human testis.","date":"2010","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/21461566","citation_count":1,"is_preprint":false},{"pmid":"25619219","id":"PMC_25619219","title":"[The association study between Eppin gene polymorphisms and idiopathic male infertility].","date":"2014","source":"Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]","url":"https://pubmed.ncbi.nlm.nih.gov/25619219","citation_count":1,"is_preprint":false},{"pmid":"21892195","id":"PMC_21892195","title":"Preparation and immunogenicity of tag-free recombinant human eppin.","date":"2011","source":"Asian journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/21892195","citation_count":0,"is_preprint":false},{"pmid":"17345778","id":"PMC_17345778","title":"[Advances in the studies of epididymal protease inhibitor--Eppin].","date":"2007","source":"Zhonghua nan ke xue = National journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/17345778","citation_count":0,"is_preprint":false},{"pmid":"30324162","id":"PMC_30324162","title":"The Expressional Pattern of Epididymal Protease Inhibitor (EPPIN) in the Male Syrian Hamsters.","date":"2018","source":"Development & reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/30324162","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.05.674523","title":"Semenogelin-1 Inhibition of Mouse Sperm Hyperactivation Reveals Two Functional Domains Modulating CatSper Channel","date":"2025-09-10","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.05.674523","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16359,"output_tokens":4163,"usd":0.055761},"stage2":{"model":"claude-opus-4-6","input_tokens":7649,"output_tokens":3169,"usd":0.176205},"total_usd":0.231966,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"EPPIN (epididymal protease inhibitor) was cloned and identified as a novel cysteine-rich protein on human chromosome 20q12-13.2, containing both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences, expressed specifically in testis and epididymis and localized on ejaculated spermatozoa.\",\n      \"method\": \"cDNA cloning, Northern blot, RT-PCR, Western blot, immunohistochemistry\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in foundational discovery paper\",\n      \"pmids\": [\"11404006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EPPIN exhibits dose- and time-dependent antibacterial activity against E. coli that requires intact disulfide bonds; it permeabilizes both outer and inner bacterial membranes causing morphological alterations, but does not disrupt eukaryotic membranes.\",\n      \"method\": \"Colony-forming unit antibacterial assay, membrane permeabilization assay, scanning electron microscopy, erythrocyte hemolytic assay, reduction/alkylation of cysteines\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mechanistic dissection (disulfide bond requirement, membrane permeabilization)\",\n      \"pmids\": [\"15229136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EPPIN binds semenogelin I (SEMG1) on the surface of ejaculated spermatozoa; the C-terminal fragment EPPIN(75-133) binds the SEMG1(164-283) fragment containing Cys239, and reduction/carboxymethylation of Cys239 blocks binding, indicating a disulfide bond is involved in the interaction.\",\n      \"method\": \"Immunoprecipitation, co-localization, far-Western blot, saturation binding with 125I-Sg, recombinant fragment mapping, chemical modification\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — six orthogonal experimental approaches including radiolabeled binding and mutagenic chemical modification\",\n      \"pmids\": [\"15590901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Immunization of male monkeys with recombinant EPPIN produced high-titer antibodies, caused complete infertility in all high-titer animals, and fertility was reversible upon cessation of immunization, demonstrating EPPIN's essential role in primate reproduction.\",\n      \"method\": \"Active immunization in Macaca radiata, fertility testing, antibody titer measurement\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo loss-of-function via immunization with defined fertility phenotype, replicated across 7/9 animals\",\n      \"pmids\": [\"15539605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EPPIN modulates PSA (prostate-specific antigen) enzyme activity toward semenogelin digestion; when EPPIN is bound to SEMG1, PSA-mediated digestion is inhibited, and antibodies to the EPPIN C-terminus block this modulation. Human spermatozoa have a receptor for EPPIN.\",\n      \"method\": \"In vitro PSA digestion assay of SEMG1 with/without EPPIN, antibody blocking experiments, virtual structural modeling\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro enzymatic assay with antibody blocking, single lab\",\n      \"pmids\": [\"16423450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Native EPPIN from seminal plasma and spermatozoa forms an EPPIN protein complex (EPC) containing lactotransferrin (LTF), clusterin (CLU), and semenogelin (SEMG1); these components co-localize on the sperm tail from the time spermatozoa leave the seminiferous tubule.\",\n      \"method\": \"Column chromatography isolation, 2D SDS-PAGE, mass spectrometry, Western blot, immunoprecipitation with anti-CLU and anti-LTF, immunofluorescence localization\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal immunoprecipitation combined with MS identification and co-localization\",\n      \"pmids\": [\"17567961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The Kunitz domain of EPPIN is responsible for elastase inhibition, while neither the Kunitz nor WAP domain alone fully recapitulates EPPIN's antibacterial activity; both domains contribute to bacterial killing partly by permeabilizing the bacterial inner membrane and uncoupling respiratory electron transport.\",\n      \"method\": \"Domain expression in E. coli, protease inhibition assay (elastase, trypsin, chymotrypsin), colony forming unit assay, XTT respiratory assay\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain dissection with multiple in vitro functional assays\",\n      \"pmids\": [\"18331357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EPPIN's functions include modulating PSA enzyme activity, providing antimicrobial protection, and binding SEMG1 to inhibit sperm motility; EPPIN is secreted by Sertoli cells and epididymal epithelial cells, exists predominantly as a dimer, and forms a native complex with LTF and clusterin on the sperm surface.\",\n      \"method\": \"Review of experimental data including immunoprecipitation, functional assays, secretion studies\",\n      \"journal\": \"Biochemical Society transactions\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — synthesis/review paper citing original experimental findings from same lab\",\n      \"pmids\": [\"21936831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EPPIN (acting as the semenogelin receptor) binding by SEMG1 or anti-EPPIN antibodies targeting a 21 amino acid C-terminal region causes loss of intracellular calcium in spermatozoa, leading to loss of progressive and then complete motility; thimerosal rescues motility implicating internal calcium stores, and ammonium chloride rescues SEMG1-induced motility inhibition, indicating SEMG1 also decreases intracellular pH.\",\n      \"method\": \"Fluo-4 intracellular calcium measurement, sperm motility assay, pharmacological rescue with thimerosal and ammonium chloride, antibody blocking with defined epitope\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (calcium imaging, motility, pharmacological rescue) with mechanistic dissection\",\n      \"pmids\": [\"22075473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EPPIN's C-terminal sequence C102-P133 contains the major binding site for SEMG1, and residues Cys102, Tyr107, and Phe117 are required for SEMG1 binding; residues Tyr107 and Phe117 are also required for interaction with lactotransferrin (LTF).\",\n      \"method\": \"Recombinant fragment binding assays, site-directed mutagenesis of key residues, functional motility inhibition assays\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with binding assays identifying specific residues required for interaction\",\n      \"pmids\": [\"22699487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Anti-EPPIN antibodies inhibit the calcium ionophore A23187-induced acrosome reaction in human spermatozoa in a dose-dependent manner and reduce A23187-induced elevation of intracellular calcium, without affecting tyrosine phosphorylation; EPPIN relocates from acrosome and tail to equatorial segment and tail after the acrosome reaction.\",\n      \"method\": \"Immunofluorescence, sperm motility assay, Fluo-3 calcium measurement, anti-EPPIN antibody blocking, capacitation assay\",\n      \"journal\": \"Human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional blocking with antibody, calcium imaging, single lab\",\n      \"pmids\": [\"19801569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EPPIN expression in the rat epididymis is androgen-dependent: surgical castration down-regulates EPPIN mRNA and protein in caput and cauda epididymis, and testosterone replacement reverses this effect.\",\n      \"method\": \"Surgical castration, testosterone replacement, RT-PCR, Western blot, immunolocalization\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo loss-of-function (castration) with rescue (testosterone), single lab\",\n      \"pmids\": [\"23070980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EPPIN inhibits LPS-induced NF-κB activation in monocytes by a mechanism involving accumulation of phosphorylated IκBα, suppresses LPS-induced chemokine production, and reduces neutrophil recruitment to the lung in vivo; EGF treatment upregulates EPPIN expression and secretion in monocytes.\",\n      \"method\": \"NF-κB reporter assay, Western blot for phospho-IκBα, cytokine/chemokine measurement, in vivo lung inflammation model with neutrophil recruitment assay, EGF stimulation\",\n      \"journal\": \"The European respiratory journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro mechanistic pathway (NF-κB/IκBα) plus in vivo confirmation, single lab\",\n      \"pmids\": [\"28705940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In mice, EPPIN acts as a protein hub on sperm surface, binding seminal vesicle secreted proteins SVS2, SVS3A, SVS5, and SVS6 after exposure to seminal vesicle fluid; SVS2 binding was confirmed by in silico docking, far-Western blot, and co-localization on sperm head and flagellum.\",\n      \"method\": \"Immunoprecipitation from mouse spermatozoa + LC-MS/MS, Western blot, in silico protein-protein interaction, far-Western blot, immunofluorescence co-localization\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS-based interactome with orthogonal far-Western and co-localization confirmation\",\n      \"pmids\": [\"32044375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Antibodies targeting the Kunitz domain epitope Ser103-Cys123 (S21C) reduce progressive motility, hyperactivated motility, and multiple kinematic parameters of mouse sperm and impair in vitro fertilization; antibodies targeting the WFDC domain epitope Gln20-Glu39 (Q20E) cause milder motility inhibition; neither affects capacitation-induced tyrosine phosphorylation.\",\n      \"method\": \"Antibody blocking with domain-specific epitope antibodies, CASA (computer-assisted sperm analysis), in vitro fertilization, capacitation tyrosine phosphorylation Western blot\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific antibody functional dissection with multiple motility parameters and IVF readout\",\n      \"pmids\": [\"34792600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"siRNA-mediated knockdown of Eppin in mouse seminiferous tubules significantly reduces sperm motility and decreases T-type Ca2+ currents and mRNA expression of three T-type Ca2+ channel subtypes in spermatogenic cells, linking EPPIN to regulation of calcium channel activity.\",\n      \"method\": \"siRNA microinjection into seminiferous tubules, CASA, patch clamp recording of T-type Ca2+ currents, RT-PCR for channel subtype expression\",\n      \"journal\": \"Reproductive biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo knockdown with electrophysiology and motility readouts, single lab\",\n      \"pmids\": [\"33607572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Computational modeling reveals EPPIN has a binding pocket formed between WFDC and Kunitz domains at the hinge region; residues Phe63 and Lys68 (WFDC), Asp71 (hinge), and Asn113, Asn114, Asn115 (Kunitz) are identified as hot spots for SEMG1, EP055, and EP012 binding; hydrophobic/hydrophilic residues allow plasma membrane anchoring orienting the binding pocket to solvent.\",\n      \"method\": \"Molecular dynamics simulation, normal mode analysis, computational docking, homology modeling\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 — computational prediction only, no experimental validation of individual residues\",\n      \"pmids\": [\"37658081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SEMG1 inhibits sperm hyperactivation and directly suppresses CatSper calcium channel currents at physiologically relevant concentrations in an EPPIN-dependent manner; two functional domains of SEMG1 (Q32-V118 and R98-S220) mediate this inhibition, with R98-G375 retaining full EPPIN-binding capacity, while Q32-V118 does not require EPPIN binding to inhibit CatSper.\",\n      \"method\": \"Electrophysiological patch clamp of CatSper, CASA hyperactivation analysis, recombinant SEMG1 truncation fragments, NH4Cl alkalinization rescue, EPPIN-binding assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — direct electrophysiology of CatSper with truncation mutant dissection, but preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.09.05.674523\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"EPPIN is a cysteine-rich, dual-domain (Kunitz + WAP/WFDC) protease inhibitor secreted by Sertoli and epididymal epithelial cells onto the sperm surface, where it forms a protein complex (EPC) with lactotransferrin and clusterin; upon ejaculation, semenogelin-1 (SEMG1) binds the EPPIN C-terminus (via residues Cys102, Tyr107, Phe117) to inhibit progressive sperm motility by causing loss of intracellular calcium—likely through suppression of the CatSper calcium channel and disturbance of intracellular pH—while also modulating PSA-mediated SEMG1 digestion and providing antimicrobial protection via membrane permeabilization.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EPPIN is a cysteine-rich, dual-domain (Kunitz + WAP/WFDC) protease inhibitor that functions as a sperm-surface protein hub essential for male fertility and innate antimicrobial defense in the reproductive tract. Secreted by Sertoli and epididymal epithelial cells, EPPIN assembles a native complex with lactotransferrin and clusterin on the sperm tail, and upon ejaculation binds semenogelin-1 (SEMG1) via C-terminal residues Cys102, Tyr107, and Phe117; this SEMG1 binding inhibits progressive motility through loss of intracellular calcium and decreased intracellular pH, and modulates PSA-mediated semenogelin digestion [PMID:22075473, PMID:22699487, PMID:16423450]. EPPIN also exhibits disulfide-dependent antibacterial activity by permeabilizing bacterial membranes and uncoupling respiratory electron transport, with both Kunitz and WAP domains contributing, and inhibits NF-κB signaling in monocytes to suppress neutrophil-driven inflammation [PMID:15229136, PMID:18331357, PMID:28705940]. Immunization of male primates with EPPIN produces reversible infertility, establishing it as a contraceptive target [PMID:15539605].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"The identification of EPPIN as a novel dual-domain protease inhibitor specifically expressed in testis and epididymis and localized on ejaculated spermatozoa established the gene as a sperm-associated factor with potential reproductive function.\",\n      \"evidence\": \"cDNA cloning, Northern blot, RT-PCR, Western blot, and immunohistochemistry in human tissues\",\n      \"pmids\": [\"11404006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No functional role demonstrated\", \"Protein partners on sperm surface unknown\", \"Mechanism of sperm surface attachment unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstration that EPPIN kills bacteria by disulfide-dependent membrane permeabilization without damaging eukaryotic membranes established an innate antimicrobial function in the male reproductive tract.\",\n      \"evidence\": \"Colony-forming unit assays, membrane permeabilization assays, SEM, hemolytic assays, and cysteine reduction/alkylation in E. coli\",\n      \"pmids\": [\"15229136\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Range of susceptible pathogens not defined\", \"In vivo antimicrobial contribution not tested\", \"Which domain mediates antibacterial activity was unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The discovery that EPPIN binds SEMG1 on the sperm surface via its C-terminal region through a disulfide-dependent interaction, together with the finding that anti-EPPIN immunization causes reversible infertility in primates, established EPPIN–SEMG1 interaction as a functionally essential axis for male fertility.\",\n      \"evidence\": \"Immunoprecipitation, radiolabeled saturation binding, recombinant fragment mapping, and active immunization fertility trials in Macaca radiata\",\n      \"pmids\": [\"15590901\", \"15539605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How SEMG1 binding regulates sperm motility was unknown\", \"Specific EPPIN residues required for SEMG1 binding not yet mapped\", \"Downstream signaling pathway in spermatozoa uncharacterized\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showing that EPPIN–SEMG1 binding inhibits PSA-mediated semenogelin digestion revealed EPPIN as a modulator of semen liquefaction, linking its sperm-surface function to the kinetics of motility acquisition after ejaculation.\",\n      \"evidence\": \"In vitro PSA digestion assays with and without EPPIN, antibody blocking experiments\",\n      \"pmids\": [\"16423450\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of PSA modulation in vivo not confirmed\", \"Whether EPPIN directly inhibits PSA protease activity or sterically shields SEMG1 not resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Isolation of the native EPPIN protein complex (EPC) containing lactotransferrin and clusterin from seminal plasma and sperm revealed that EPPIN functions within a multi-protein assembly on the sperm tail rather than as an isolated factor.\",\n      \"evidence\": \"Column chromatography, 2D SDS-PAGE, mass spectrometry, reciprocal immunoprecipitation, and immunofluorescence from human spermatozoa and seminal plasma\",\n      \"pmids\": [\"17567961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order of the EPC unknown\", \"Functional contribution of LTF and CLU within the complex not tested\", \"Mechanism of EPC attachment to sperm membrane uncharacterized\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Domain dissection showed the Kunitz domain mediates elastase inhibition while both Kunitz and WAP domains are needed for full antibacterial activity, including bacterial inner membrane permeabilization and respiratory uncoupling, resolving domain-specific contributions to EPPIN's dual functions.\",\n      \"evidence\": \"Recombinant domain expression, protease inhibition assays, colony-forming unit assays, and XTT respiratory assays in E. coli\",\n      \"pmids\": [\"18331357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural basis for how two domains cooperate in bacterial killing\", \"Antibacterial activity against Gram-positive organisms not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Anti-EPPIN antibodies blocked the calcium ionophore-induced acrosome reaction and calcium influx, and EPPIN was shown to relocalize on the sperm head during acrosome reaction, extending EPPIN's role beyond motility to acrosomal exocytosis.\",\n      \"evidence\": \"Antibody blocking with immunofluorescence, Fluo-3 calcium imaging, and capacitation assays on human spermatozoa\",\n      \"pmids\": [\"19801569\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether EPPIN directly regulates acrosomal calcium channels or acts indirectly is unknown\", \"Physiological significance of EPPIN relocalization not demonstrated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Mapping of the SEMG1-binding site to EPPIN residues Cys102, Tyr107, and Phe117, combined with showing that SEMG1 binding causes intracellular calcium loss and pH decrease leading to motility inhibition, provided the first integrated signaling mechanism for how the EPPIN–SEMG1 axis controls sperm motility.\",\n      \"evidence\": \"Site-directed mutagenesis and binding assays for residue mapping; Fluo-4 calcium imaging, motility analysis, and pharmacological rescue with thimerosal (calcium) and NH4Cl (pH) on human spermatozoa\",\n      \"pmids\": [\"22699487\", \"22075473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the calcium channel or transporter affected was unknown\", \"Mechanism linking pH decrease to motility loss not elucidated\", \"Whether EPPIN itself transduces the signal or acts solely as SEMG1 receptor unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Androgen-dependent regulation of EPPIN expression in the epididymis was established, connecting reproductive hormone signaling to EPPIN abundance on spermatozoa.\",\n      \"evidence\": \"Surgical castration, testosterone replacement, RT-PCR, and Western blot in rat epididymis\",\n      \"pmids\": [\"23070980\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Androgen response elements in the EPPIN promoter not identified\", \"Whether androgen regulation occurs at transcriptional or post-transcriptional level not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Discovery that EPPIN inhibits NF-κB activation by promoting phospho-IκBα accumulation and suppresses neutrophil recruitment in vivo extended EPPIN's function beyond reproduction to anti-inflammatory innate immunity.\",\n      \"evidence\": \"NF-κB reporter assays, phospho-IκBα Western blots in monocytes, and in vivo lung inflammation model with neutrophil counting\",\n      \"pmids\": [\"28705940\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular target of EPPIN in the NF-κB pathway not identified\", \"Whether this anti-inflammatory function operates in the male reproductive tract not shown\", \"Mechanism of phospho-IκBα stabilization unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identification of mouse seminal vesicle proteins SVS2, SVS3A, SVS5, and SVS6 as EPPIN-binding partners confirmed EPPIN's role as a protein hub on spermatozoa and extended the model to non-primate species where SEMG1 orthologs differ.\",\n      \"evidence\": \"Immunoprecipitation–LC-MS/MS from mouse spermatozoa, far-Western blot, and immunofluorescence co-localization\",\n      \"pmids\": [\"32044375\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of SVS binding on mouse sperm motility not tested\", \"Whether SVS proteins mimic SEMG1 function in motility regulation unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Domain-specific antibody blocking showed the Kunitz domain epitope (Ser103-Cys123) is the primary functional surface for motility and fertilization, while siRNA knockdown of Eppin in seminiferous tubules reduced T-type Ca²⁺ channel currents, providing the first link between EPPIN and a specific calcium channel family in spermatogenic cells.\",\n      \"evidence\": \"Epitope-specific antibody blocking with CASA and IVF in mouse; siRNA knockdown with patch-clamp electrophysiology and RT-PCR in mouse spermatogenic cells\",\n      \"pmids\": [\"34792600\", \"33607572\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether T-type Ca²⁺ channel regulation is direct or indirect not determined\", \"T-type channels function in spermatogenic cells but CatSper dominates in mature sperm — channel identity on mature sperm unresolved\", \"IVF effects not confirmed in vivo\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of the calcium channel(s) on mature spermatozoa through which the EPPIN–SEMG1 axis suppresses motility remains to be definitively established, and no high-resolution structure of the EPPIN–SEMG1 complex or the EPPIN protein complex (EPC) exists.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal or cryo-EM structure of EPPIN or its complexes\", \"Whether CatSper is the physiological target of the EPPIN–SEMG1 axis on mature sperm requires peer-reviewed confirmation\", \"Mechanism of EPPIN membrane anchoring unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 4, 8, 9]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0090729\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 5, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [3, 8, 14, 15]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 6, 12]}\n    ],\n    \"complexes\": [\n      \"EPPIN protein complex (EPC: EPPIN–LTF–CLU)\"\n    ],\n    \"partners\": [\n      \"SEMG1\",\n      \"LTF\",\n      \"CLU\",\n      \"KLK3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}