{"gene":"ACRBP","run_date":"2026-06-09T22:02:39","timeline":{"discoveries":[{"year":1994,"finding":"SP32 (ACRBP) was purified from porcine sperm and identified as a binding protein specific for 55-, 53-, and 49-kDa forms of proacrosin and an acrosin intermediate, but not for the 43-kDa acrosin intermediate or 35-kDa mature acrosin. SP32 significantly accelerated autoactivation of proacrosin at basic pH in vitro and shifted the maturation pathway to accumulate the 49-kDa intermediate instead of the 43-kDa intermediate, suggesting it interacts with both amino- and carboxyl-terminal sequences of the 53-kDa proacrosin. SP32 is produced as a 61-kDa precursor with a signal peptide, and the carboxyl-terminal half corresponds to the mature protein.","method":"Protein purification, SDS-PAGE, in vitro binding assays, in vitro proacrosin autoactivation assay, cDNA cloning and sequence analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro biochemical reconstitution of binding and enzymatic activity, protein purification to homogeneity, cDNA-based structural analysis","pmids":["8144514"],"is_preprint":false},{"year":2005,"finding":"SP32 (ACRBP) in pig sperm undergoes tyrosine phosphorylation specifically during capacitation, as demonstrated by mass spectrometry identification, immunoprecipitation with anti-phosphotyrosine and anti-sp32 antibodies, and indirect immunofluorescence. After ionophore-induced acrosome reaction, anti-sp32 and anti-phosphotyrosine labeling on the acrosome disappeared, linking tyrosine-phosphorylated sp32 to capacitation-associated events.","method":"2D Western blot under non-reducing/reducing conditions, mass spectrometry/MS identification, immunoprecipitation, indirect immunofluorescence","journal":"Journal of andrology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal immunoprecipitation with two antibodies, MS identification, immunofluorescence, single lab with multiple orthogonal methods","pmids":["15955892"],"is_preprint":false},{"year":2012,"finding":"ACRBP/sp32 normally undergoes proteolytic processing from a 58.5 kDa precursor to a 27.5 kDa mature form in mouse sperm, and this processing does not occur in PCSK4 null mice, identifying ACRBP as a likely substrate (direct or indirect) of proprotein convertase PCSK4. In PCSK4 null mice, proacrosin failed to undergo autoactivation and sperm head/acrosome morphological defects were observed, supporting a role for mature ACRBP in regulating proacrosin conversion.","method":"2D differential in-gel electrophoresis, Western blot, immunolocalization in PCSK4 knockout mice","journal":"Molecular human reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with defined biochemical phenotype and multiple readouts, single lab; ACRBP as direct vs. indirect PCSK4 substrate not fully resolved","pmids":["22357636"],"is_preprint":false},{"year":2013,"finding":"In mouse, two forms of ACRBP are generated by alternative splicing: ACRBP-W (wild-type) processed to mature ACRBP-C, and ACRBP-V5 (intron 5-retaining variant). GST pull-down assays revealed that ACRBP-V5 and ACRBP-C bind different domains in the C-terminal region of proacrosin (pro-ACR). ACRBP-C significantly accelerated autoactivation of pro-ACR in vitro. ACRBP-W and ACRBP-V5 co-localize with pro-ACR in acrosomal granules of early round spermatids, while sperm acrosome contains only ACRBP-C.","method":"GST pull-down assay, in vitro proacrosin autoactivation assay, immunolocalization, RT-PCR, Western blot","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical reconstitution (GST pulldown, autoactivation assay) plus localization, single lab with multiple orthogonal methods","pmids":["23426433"],"is_preprint":false},{"year":2016,"finding":"ACRBP-null male mice lacking both ACRBP-W and ACRBP-V5 exhibit severely reduced fertility due to acrosome malformation; spermatids fail to form a large acrosomal granule, resulting in fragmented acrosome structure. Transgenic rescue with ACRBP-V5 alone restored acrosomal granule formation, demonstrating that ACRBP-V5 functions specifically in formation and configuration of the acrosomal granule during early spermiogenesis. Exogenously expressed ACRBP-W blocked proacrosin autoactivation in the acrosome, establishing its role in retaining proacrosin in an inactive state until acrosomal exocytosis.","method":"ACRBP knockout mice, transgenic rescue with ACRBP-V5, fertility assays, acrosome morphology analysis, proacrosin autoactivation assay in acrosome","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — genetic knockout plus isoform-specific transgenic rescue with defined phenotypic readouts and biochemical validation, single lab but multiple rigorous orthogonal approaches","pmids":["27303034"],"is_preprint":false},{"year":2018,"finding":"ACRBP-deficient mouse sperm showed markedly reduced numbers in the oviduct after mating and a marked reduction in ability to access unfertilized oocytes, despite normal sperm motility and head morphology in recovered oviductal sperm. This suggests male subfertility in ACRBP-null mice is attributable primarily to incompleteness of the acrosome reaction rather than impaired sperm migration.","method":"ACRBP knockout mice, in vivo sperm tracking in female reproductive tract post-mating, motility assessment, morphological analysis","journal":"The Journal of reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined in vivo phenotype in knockout model with specific cellular readouts, single lab","pmids":["30606959"],"is_preprint":false},{"year":2021,"finding":"In boar sperm, antibody blocking of ACRBP on the sperm surface reduced capacitation and spontaneous acrosome reaction, and decreased sperm-zona pellucida (ZP) binding. Anti-ACRBP antibodies on the sperm head also reduced the ability of sperm to undergo the acrosome reaction in response to solubilized ZP or SERCA inhibition, indicating ACRBP on the sperm surface participates in sperm-ZP binding and primes sperm for the acrosome reaction.","method":"Antibody inhibition during IVF, sperm-ZP binding assay, acrosome reaction assay with solubilized ZP and ionophore/SERCA inhibitor, immunofluorescence","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional antibody blocking with multiple downstream readouts in a defined porcine model, single lab","pmids":["34086710"],"is_preprint":false},{"year":2013,"finding":"sp32 tyrosine phosphorylation levels increase during proacrosin activation in pig sperm capacitation and acrosome reaction, correlating with conversion of 55 kDa proacrosin to 35 kDa active acrosin forms, as measured by Western blot in differently processed sperm populations.","method":"SDS-PAGE, Western blot of differently processed pig spermatozoa (fresh, capacitation, acrosome reaction, thawed)","journal":"Genetics and molecular research : GMR","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, correlative Western blot without direct manipulation of sp32 phosphorylation","pmids":["24391004"],"is_preprint":false},{"year":2015,"finding":"Expression level and tyrosine phosphorylation of sp32 differed across capacitated, frozen-thawed, and post-acrosomal reaction boar sperm, with sp32 expression higher in capacitated and post-acrosomal reaction sperm vs. fresh or frozen-thawed sperm, supporting a role for sp32 phosphorylation in activation of the proacrosin/acrosin system.","method":"SDS-PAGE, Western blot, Coomassie staining of acrosomal membrane proteins across sperm treatment groups","journal":"Genetics and molecular research : GMR","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, correlative Western blot, no direct manipulation","pmids":["25867384"],"is_preprint":false},{"year":2012,"finding":"Knockdown of OY-TES-1 (ACRBP) by RNAi in human bone marrow-derived mesenchymal stem cells caused cell growth inhibition, cell cycle arrest, apoptosis induction, and reduced migration ability, indicating ACRBP influences proliferation and migration in these cells.","method":"RNAi knockdown, cell viability assay, cell cycle analysis, apoptosis assay, migration assay","journal":"Cell biology international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, RNAi with cellular phenotype readouts but no pathway placement or molecular mechanism identified","pmids":["22651134"],"is_preprint":false},{"year":2015,"finding":"Knockdown of OY-TES-1 (ACRBP) by siRNA in hepatocellular carcinoma cell lines (HepG2 and BEL-7404) decreased cell growth, induced G0/G1 arrest and apoptosis, and reduced migration and invasion. This was accompanied by increased caspase-3 expression, decreased cyclin E, and decreased MMP2 and MMP9 protein levels.","method":"siRNA knockdown, cell proliferation assay, cell cycle analysis, apoptosis assay, migration/invasion assay, Western blot for caspase-3, cyclin E, MMP2, MMP9","journal":"International journal of clinical and experimental pathology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, siRNA with downstream protein markers but no direct mechanistic pathway placement for ACRBP itself","pmids":["26339343"],"is_preprint":false},{"year":2021,"finding":"Knockdown of CT23 (ACRBP) in BEL-7404 HCC cells altered expression of 1051 genes; functional analysis identified metallothionein 1 (MT1) as maximally enriched. Western blot and cell behavior assays confirmed CT23 modulates cell proliferation, migration, and apoptosis through regulation of MT1 expression in HCC cells.","method":"RNAi knockdown, microarray gene expression profiling, bioinformatic analysis, Western blot, cell proliferation/migration/apoptosis assays","journal":"Cell biology international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect mechanism via transcriptome analysis; direct mechanistic link between ACRBP and MT1 not biochemically established","pmids":["33559934"],"is_preprint":false}],"current_model":"ACRBP (SP32/OY-TES-1/CT23) is an acrosomal matrix protein that exists as two alternatively spliced isoforms in mice (ACRBP-V5 and ACRBP-W/ACRBP-C): ACRBP-V5 is essential for acrosomal granule formation and packaging of proacrosin during early spermiogenesis, while ACRBP-C (the processed form of ACRBP-W) binds proacrosin via the C-terminal region to retain it in an inactive state and accelerates its autoactivation upon acrosomal exocytosis; the protein also undergoes tyrosine phosphorylation during sperm capacitation and, when present on the sperm surface, participates in zona pellucida binding and priming of the acrosome reaction."},"narrative":{"mechanistic_narrative":"ACRBP (SP32/OY-TES-1/CT23) is an acrosomal matrix protein that controls the packaging and timed activation of the sperm protease proacrosin during spermiogenesis and fertilization [PMID:8144514, PMID:27303034]. It was first purified as a proacrosin-binding protein that selectively recognizes the 55-/53-/49-kDa proacrosin and acrosin-intermediate forms and accelerates proacrosin autoactivation at basic pH in vitro, acting through both amino- and carboxyl-terminal sequences of proacrosin [PMID:8144514]. In mouse, alternative splicing generates two functionally distinct isoforms that bind separate domains of the proacrosin C-terminus: ACRBP-V5 drives formation and configuration of the large acrosomal granule during early spermiogenesis, while the processed ACRBP-C retains proacrosin in an inactive state and accelerates its autoactivation upon acrosomal exocytosis [PMID:23426433, PMID:27303034]. Genetic ablation of both isoforms causes acrosome malformation, fragmented acrosomes, and severe subfertility, with ACRBP-V5 transgenic rescue restoring acrosomal granule formation; the subfertility reflects an incomplete acrosome reaction rather than impaired sperm migration [PMID:27303034, PMID:30606959]. Maturation of ACRBP from precursor to mature form depends on the proprotein convertase PCSK4, and ACRBP undergoes tyrosine phosphorylation during capacitation, with surface ACRBP contributing to sperm–zona pellucida binding and priming of the acrosome reaction [PMID:22357636, PMID:15955892, PMID:34086710]. A separate body of low-confidence work links ACRBP/OY-TES-1/CT23 knockdown to proliferation, migration, and apoptosis phenotypes in mesenchymal stem cells and hepatocellular carcinoma lines, but this somatic role has not been mechanistically resolved in the available corpus.","teleology":[{"year":1994,"claim":"Established the founding biochemical activity of ACRBP: it is a dedicated proacrosin-binding protein that physically engages proacrosin and controls its maturation kinetics.","evidence":"Protein purification from porcine sperm, in vitro binding assays, and proacrosin autoactivation assays with cDNA-based structural analysis","pmids":["8144514"],"confidence":"High","gaps":["Did not define the structural basis of binding or distinguish whether acceleration occurs in vivo","Performed in porcine sperm without genetic confirmation of physiological role"]},{"year":2005,"claim":"Linked ACRBP to capacitation signaling by showing it is a capacitation-specific tyrosine phosphorylation target whose acrosomal labeling is lost upon the acrosome reaction.","evidence":"2D Western blot, mass spectrometry identification, reciprocal immunoprecipitation, and immunofluorescence in pig sperm","pmids":["15955892"],"confidence":"High","gaps":["Kinase responsible not identified","Functional consequence of phosphorylation for proacrosin binding not tested"]},{"year":2012,"claim":"Placed ACRBP maturation within a proteolytic processing pathway by identifying PCSK4 as required for conversion of the ACRBP precursor to its mature form.","evidence":"2D differential in-gel electrophoresis, Western blot, and immunolocalization in PCSK4-knockout mice","pmids":["22357636"],"confidence":"Medium","gaps":["Direct versus indirect PCSK4 substrate relationship unresolved","Did not isolate the contribution of ACRBP processing from other PCSK4 substrates in the phenotype"]},{"year":2013,"claim":"Resolved that ACRBP acts through two alternatively spliced isoforms binding distinct proacrosin domains, separating granule-formation from autoactivation-control functions.","evidence":"GST pull-down, in vitro proacrosin autoactivation assay, immunolocalization, RT-PCR, and Western blot in mouse","pmids":["23426433"],"confidence":"High","gaps":["Did not establish in vivo necessity of each isoform","Mechanism of differential domain selection not structurally defined"]},{"year":2016,"claim":"Provided genetic proof of isoform-specific function: ACRBP-V5 is required for acrosomal granule formation, while ACRBP-W retains proacrosin inactive until exocytosis.","evidence":"ACRBP-knockout mice with ACRBP-V5 transgenic rescue, fertility and acrosome morphology assays, and proacrosin autoactivation assays","pmids":["27303034"],"confidence":"High","gaps":["Did not define how ACRBP-V5 shapes granule architecture","Molecular trigger releasing proacrosin from ACRBP-W at exocytosis not identified"]},{"year":2018,"claim":"Attributed ACRBP-null subfertility specifically to an incomplete acrosome reaction rather than defective sperm transport.","evidence":"In vivo sperm tracking in the female reproductive tract, motility, and morphology analysis in knockout mice","pmids":["30606959"],"confidence":"Medium","gaps":["Did not quantify the acrosome reaction step that fails","Single-lab knockout phenotype without orthogonal model"]},{"year":2021,"claim":"Extended ACRBP function to the sperm surface, implicating it in zona pellucida binding and priming the acrosome reaction.","evidence":"Antibody inhibition during IVF, sperm-ZP binding and acrosome reaction assays with solubilized ZP and SERCA inhibition in boar sperm","pmids":["34086710"],"confidence":"Medium","gaps":["Surface ACRBP binding partner on the sperm or ZP not identified","Relationship between surface and matrix pools of ACRBP unclear"]},{"year":2021,"claim":"Reported a somatic role for ACRBP/OY-TES-1/CT23 in tumor and stem cell proliferation, migration, and apoptosis, including transcriptional regulation of MT1.","evidence":"RNAi/siRNA knockdown with proliferation, cycle, apoptosis, and migration assays plus microarray and Western blot in MSCs and HCC lines","pmids":["22651134","26339343","33559934"],"confidence":"Low","gaps":["Correlative knockdown phenotypes without a defined molecular mechanism for ACRBP","Direct biochemical link between ACRBP and MT1 not established","No reconciliation with the acrosomal proacrosin-binding function"]},{"year":null,"claim":"How tyrosine phosphorylation, PCSK4 processing, and the acrosome-reaction trigger are coordinated to time proacrosin release, and whether the somatic cancer-cell phenotypes reflect a genuine non-reproductive function, remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of the ACRBP-proacrosin complex","Mechanism of proacrosin release at exocytosis undefined","Somatic function lacks molecular mechanism"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3,4]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[3,4]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0,4]}],"complexes":[],"partners":["ACR","PCSK4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NEB7","full_name":"Acrosin-binding protein","aliases":["Acrosin-binding protein, 60 kDa form","Cancer/testis antigen 23","CT23","Cancer/testis antigen OY-TES-1","Proacrosin-binding protein sp32"],"length_aa":543,"mass_kda":61.4,"function":"Acrosomal protein that maintains proacrosin (pro-ACR) as an enzymatically inactive zymogen in the acrosome. Involved also in the acrosome formation","subcellular_location":"Secreted; Cytoplasmic vesicle, secretory vesicle, acrosome","url":"https://www.uniprot.org/uniprotkb/Q8NEB7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ACRBP","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/ACRBP","total_profiled":1310},"omim":[{"mim_id":"608352","title":"ACROSIN-BINDING PROTEIN; ACRBP","url":"https://www.omim.org/entry/608352"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"testis","ntpm":271.0}],"url":"https://www.proteinatlas.org/search/ACRBP"},"hgnc":{"alias_symbol":["SP32","OY-TES-1","CT23"],"prev_symbol":[]},"alphafold":{"accession":"Q8NEB7","domains":[{"cath_id":"-","chopping":"39-120","consensus_level":"high","plddt":89.697,"start":39,"end":120},{"cath_id":"-","chopping":"319-413","consensus_level":"medium","plddt":79.0998,"start":319,"end":413},{"cath_id":"-","chopping":"426-537","consensus_level":"high","plddt":83.0729,"start":426,"end":537}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NEB7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NEB7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NEB7-F1-predicted_aligned_error_v6.png","plddt_mean":63.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ACRBP","jax_strain_url":"https://www.jax.org/strain/search?query=ACRBP"},"sequence":{"accession":"Q8NEB7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NEB7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NEB7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NEB7"}},"corpus_meta":[{"pmid":"8144514","id":"PMC_8144514","title":"An 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international","url":"https://pubmed.ncbi.nlm.nih.gov/33559934","citation_count":3,"is_preprint":false},{"pmid":"35463688","id":"PMC_35463688","title":"Immunohistochemistry Study of OY-TES-1 Location in Fetal and Adult Human Tissues.","date":"2022","source":"Journal of healthcare engineering","url":"https://pubmed.ncbi.nlm.nih.gov/35463688","citation_count":2,"is_preprint":false},{"pmid":"38090303","id":"PMC_38090303","title":"Collaborative multidisciplinary management and expertise of cT2-3 locally advanced operable esophageal squamous cell carcinoma: a report of two cases.","date":"2023","source":"Journal of thoracic disease","url":"https://pubmed.ncbi.nlm.nih.gov/38090303","citation_count":2,"is_preprint":false},{"pmid":"21968305","id":"PMC_21968305","title":"[Construction of eukaryotic expression vector encoding ACRBP and its expression in hepatocarcinoma cells].","date":"2011","source":"Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/21968305","citation_count":1,"is_preprint":false},{"pmid":"41099151","id":"PMC_41099151","title":"Cancer-testis antigen ACRBP: Cytotoxic response to its HLA-A2 restricted peptide and immune features in ovarian cancer.","date":"2025","source":"Human vaccines & immunotherapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/41099151","citation_count":0,"is_preprint":false},{"pmid":"41428155","id":"PMC_41428155","title":"OY-TES-1 Splice Variant V5a in Glioma: A Driver of Malignancy and Potential Therapeutic Target.","date":"2025","source":"Current medical science","url":"https://pubmed.ncbi.nlm.nih.gov/41428155","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.08.15.608142","title":"Morpho-physiological and transcriptomic responses of field pennycress to waterlogging","date":"2024-08-16","source":"bioRxiv","url":"https://doi.org/10.1101/2024.08.15.608142","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":21899,"output_tokens":3417,"usd":0.058476,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10976,"output_tokens":2947,"usd":0.064278,"stage2_stop_reason":"end_turn"},"total_usd":0.122754,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1994,\n      \"finding\": \"SP32 (ACRBP) was purified from porcine sperm and identified as a binding protein specific for 55-, 53-, and 49-kDa forms of proacrosin and an acrosin intermediate, but not for the 43-kDa acrosin intermediate or 35-kDa mature acrosin. SP32 significantly accelerated autoactivation of proacrosin at basic pH in vitro and shifted the maturation pathway to accumulate the 49-kDa intermediate instead of the 43-kDa intermediate, suggesting it interacts with both amino- and carboxyl-terminal sequences of the 53-kDa proacrosin. SP32 is produced as a 61-kDa precursor with a signal peptide, and the carboxyl-terminal half corresponds to the mature protein.\",\n      \"method\": \"Protein purification, SDS-PAGE, in vitro binding assays, in vitro proacrosin autoactivation assay, cDNA cloning and sequence analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro biochemical reconstitution of binding and enzymatic activity, protein purification to homogeneity, cDNA-based structural analysis\",\n      \"pmids\": [\"8144514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SP32 (ACRBP) in pig sperm undergoes tyrosine phosphorylation specifically during capacitation, as demonstrated by mass spectrometry identification, immunoprecipitation with anti-phosphotyrosine and anti-sp32 antibodies, and indirect immunofluorescence. After ionophore-induced acrosome reaction, anti-sp32 and anti-phosphotyrosine labeling on the acrosome disappeared, linking tyrosine-phosphorylated sp32 to capacitation-associated events.\",\n      \"method\": \"2D Western blot under non-reducing/reducing conditions, mass spectrometry/MS identification, immunoprecipitation, indirect immunofluorescence\",\n      \"journal\": \"Journal of andrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal immunoprecipitation with two antibodies, MS identification, immunofluorescence, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"15955892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ACRBP/sp32 normally undergoes proteolytic processing from a 58.5 kDa precursor to a 27.5 kDa mature form in mouse sperm, and this processing does not occur in PCSK4 null mice, identifying ACRBP as a likely substrate (direct or indirect) of proprotein convertase PCSK4. In PCSK4 null mice, proacrosin failed to undergo autoactivation and sperm head/acrosome morphological defects were observed, supporting a role for mature ACRBP in regulating proacrosin conversion.\",\n      \"method\": \"2D differential in-gel electrophoresis, Western blot, immunolocalization in PCSK4 knockout mice\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with defined biochemical phenotype and multiple readouts, single lab; ACRBP as direct vs. indirect PCSK4 substrate not fully resolved\",\n      \"pmids\": [\"22357636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In mouse, two forms of ACRBP are generated by alternative splicing: ACRBP-W (wild-type) processed to mature ACRBP-C, and ACRBP-V5 (intron 5-retaining variant). GST pull-down assays revealed that ACRBP-V5 and ACRBP-C bind different domains in the C-terminal region of proacrosin (pro-ACR). ACRBP-C significantly accelerated autoactivation of pro-ACR in vitro. ACRBP-W and ACRBP-V5 co-localize with pro-ACR in acrosomal granules of early round spermatids, while sperm acrosome contains only ACRBP-C.\",\n      \"method\": \"GST pull-down assay, in vitro proacrosin autoactivation assay, immunolocalization, RT-PCR, Western blot\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical reconstitution (GST pulldown, autoactivation assay) plus localization, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"23426433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ACRBP-null male mice lacking both ACRBP-W and ACRBP-V5 exhibit severely reduced fertility due to acrosome malformation; spermatids fail to form a large acrosomal granule, resulting in fragmented acrosome structure. Transgenic rescue with ACRBP-V5 alone restored acrosomal granule formation, demonstrating that ACRBP-V5 functions specifically in formation and configuration of the acrosomal granule during early spermiogenesis. Exogenously expressed ACRBP-W blocked proacrosin autoactivation in the acrosome, establishing its role in retaining proacrosin in an inactive state until acrosomal exocytosis.\",\n      \"method\": \"ACRBP knockout mice, transgenic rescue with ACRBP-V5, fertility assays, acrosome morphology analysis, proacrosin autoactivation assay in acrosome\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — genetic knockout plus isoform-specific transgenic rescue with defined phenotypic readouts and biochemical validation, single lab but multiple rigorous orthogonal approaches\",\n      \"pmids\": [\"27303034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ACRBP-deficient mouse sperm showed markedly reduced numbers in the oviduct after mating and a marked reduction in ability to access unfertilized oocytes, despite normal sperm motility and head morphology in recovered oviductal sperm. This suggests male subfertility in ACRBP-null mice is attributable primarily to incompleteness of the acrosome reaction rather than impaired sperm migration.\",\n      \"method\": \"ACRBP knockout mice, in vivo sperm tracking in female reproductive tract post-mating, motility assessment, morphological analysis\",\n      \"journal\": \"The Journal of reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined in vivo phenotype in knockout model with specific cellular readouts, single lab\",\n      \"pmids\": [\"30606959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In boar sperm, antibody blocking of ACRBP on the sperm surface reduced capacitation and spontaneous acrosome reaction, and decreased sperm-zona pellucida (ZP) binding. Anti-ACRBP antibodies on the sperm head also reduced the ability of sperm to undergo the acrosome reaction in response to solubilized ZP or SERCA inhibition, indicating ACRBP on the sperm surface participates in sperm-ZP binding and primes sperm for the acrosome reaction.\",\n      \"method\": \"Antibody inhibition during IVF, sperm-ZP binding assay, acrosome reaction assay with solubilized ZP and ionophore/SERCA inhibitor, immunofluorescence\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional antibody blocking with multiple downstream readouts in a defined porcine model, single lab\",\n      \"pmids\": [\"34086710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"sp32 tyrosine phosphorylation levels increase during proacrosin activation in pig sperm capacitation and acrosome reaction, correlating with conversion of 55 kDa proacrosin to 35 kDa active acrosin forms, as measured by Western blot in differently processed sperm populations.\",\n      \"method\": \"SDS-PAGE, Western blot of differently processed pig spermatozoa (fresh, capacitation, acrosome reaction, thawed)\",\n      \"journal\": \"Genetics and molecular research : GMR\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, correlative Western blot without direct manipulation of sp32 phosphorylation\",\n      \"pmids\": [\"24391004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Expression level and tyrosine phosphorylation of sp32 differed across capacitated, frozen-thawed, and post-acrosomal reaction boar sperm, with sp32 expression higher in capacitated and post-acrosomal reaction sperm vs. fresh or frozen-thawed sperm, supporting a role for sp32 phosphorylation in activation of the proacrosin/acrosin system.\",\n      \"method\": \"SDS-PAGE, Western blot, Coomassie staining of acrosomal membrane proteins across sperm treatment groups\",\n      \"journal\": \"Genetics and molecular research : GMR\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, correlative Western blot, no direct manipulation\",\n      \"pmids\": [\"25867384\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Knockdown of OY-TES-1 (ACRBP) by RNAi in human bone marrow-derived mesenchymal stem cells caused cell growth inhibition, cell cycle arrest, apoptosis induction, and reduced migration ability, indicating ACRBP influences proliferation and migration in these cells.\",\n      \"method\": \"RNAi knockdown, cell viability assay, cell cycle analysis, apoptosis assay, migration assay\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, RNAi with cellular phenotype readouts but no pathway placement or molecular mechanism identified\",\n      \"pmids\": [\"22651134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Knockdown of OY-TES-1 (ACRBP) by siRNA in hepatocellular carcinoma cell lines (HepG2 and BEL-7404) decreased cell growth, induced G0/G1 arrest and apoptosis, and reduced migration and invasion. This was accompanied by increased caspase-3 expression, decreased cyclin E, and decreased MMP2 and MMP9 protein levels.\",\n      \"method\": \"siRNA knockdown, cell proliferation assay, cell cycle analysis, apoptosis assay, migration/invasion assay, Western blot for caspase-3, cyclin E, MMP2, MMP9\",\n      \"journal\": \"International journal of clinical and experimental pathology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, siRNA with downstream protein markers but no direct mechanistic pathway placement for ACRBP itself\",\n      \"pmids\": [\"26339343\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Knockdown of CT23 (ACRBP) in BEL-7404 HCC cells altered expression of 1051 genes; functional analysis identified metallothionein 1 (MT1) as maximally enriched. Western blot and cell behavior assays confirmed CT23 modulates cell proliferation, migration, and apoptosis through regulation of MT1 expression in HCC cells.\",\n      \"method\": \"RNAi knockdown, microarray gene expression profiling, bioinformatic analysis, Western blot, cell proliferation/migration/apoptosis assays\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect mechanism via transcriptome analysis; direct mechanistic link between ACRBP and MT1 not biochemically established\",\n      \"pmids\": [\"33559934\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ACRBP (SP32/OY-TES-1/CT23) is an acrosomal matrix protein that exists as two alternatively spliced isoforms in mice (ACRBP-V5 and ACRBP-W/ACRBP-C): ACRBP-V5 is essential for acrosomal granule formation and packaging of proacrosin during early spermiogenesis, while ACRBP-C (the processed form of ACRBP-W) binds proacrosin via the C-terminal region to retain it in an inactive state and accelerates its autoactivation upon acrosomal exocytosis; the protein also undergoes tyrosine phosphorylation during sperm capacitation and, when present on the sperm surface, participates in zona pellucida binding and priming of the acrosome reaction.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ACRBP (SP32/OY-TES-1/CT23) is an acrosomal matrix protein that controls the packaging and timed activation of the sperm protease proacrosin during spermiogenesis and fertilization [#0, #4]. It was first purified as a proacrosin-binding protein that selectively recognizes the 55-/53-/49-kDa proacrosin and acrosin-intermediate forms and accelerates proacrosin autoactivation at basic pH in vitro, acting through both amino- and carboxyl-terminal sequences of proacrosin [#0]. In mouse, alternative splicing generates two functionally distinct isoforms that bind separate domains of the proacrosin C-terminus: ACRBP-V5 drives formation and configuration of the large acrosomal granule during early spermiogenesis, while the processed ACRBP-C retains proacrosin in an inactive state and accelerates its autoactivation upon acrosomal exocytosis [#3, #4]. Genetic ablation of both isoforms causes acrosome malformation, fragmented acrosomes, and severe subfertility, with ACRBP-V5 transgenic rescue restoring acrosomal granule formation; the subfertility reflects an incomplete acrosome reaction rather than impaired sperm migration [#4, #5]. Maturation of ACRBP from precursor to mature form depends on the proprotein convertase PCSK4, and ACRBP undergoes tyrosine phosphorylation during capacitation, with surface ACRBP contributing to sperm–zona pellucida binding and priming of the acrosome reaction [#2, #1, #6]. A separate body of low-confidence work links ACRBP/OY-TES-1/CT23 knockdown to proliferation, migration, and apoptosis phenotypes in mesenchymal stem cells and hepatocellular carcinoma lines, but this somatic role has not been mechanistically resolved in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established the founding biochemical activity of ACRBP: it is a dedicated proacrosin-binding protein that physically engages proacrosin and controls its maturation kinetics.\",\n      \"evidence\": \"Protein purification from porcine sperm, in vitro binding assays, and proacrosin autoactivation assays with cDNA-based structural analysis\",\n      \"pmids\": [\"8144514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural basis of binding or distinguish whether acceleration occurs in vivo\", \"Performed in porcine sperm without genetic confirmation of physiological role\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked ACRBP to capacitation signaling by showing it is a capacitation-specific tyrosine phosphorylation target whose acrosomal labeling is lost upon the acrosome reaction.\",\n      \"evidence\": \"2D Western blot, mass spectrometry identification, reciprocal immunoprecipitation, and immunofluorescence in pig sperm\",\n      \"pmids\": [\"15955892\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible not identified\", \"Functional consequence of phosphorylation for proacrosin binding not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed ACRBP maturation within a proteolytic processing pathway by identifying PCSK4 as required for conversion of the ACRBP precursor to its mature form.\",\n      \"evidence\": \"2D differential in-gel electrophoresis, Western blot, and immunolocalization in PCSK4-knockout mice\",\n      \"pmids\": [\"22357636\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct versus indirect PCSK4 substrate relationship unresolved\", \"Did not isolate the contribution of ACRBP processing from other PCSK4 substrates in the phenotype\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved that ACRBP acts through two alternatively spliced isoforms binding distinct proacrosin domains, separating granule-formation from autoactivation-control functions.\",\n      \"evidence\": \"GST pull-down, in vitro proacrosin autoactivation assay, immunolocalization, RT-PCR, and Western blot in mouse\",\n      \"pmids\": [\"23426433\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish in vivo necessity of each isoform\", \"Mechanism of differential domain selection not structurally defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided genetic proof of isoform-specific function: ACRBP-V5 is required for acrosomal granule formation, while ACRBP-W retains proacrosin inactive until exocytosis.\",\n      \"evidence\": \"ACRBP-knockout mice with ACRBP-V5 transgenic rescue, fertility and acrosome morphology assays, and proacrosin autoactivation assays\",\n      \"pmids\": [\"27303034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how ACRBP-V5 shapes granule architecture\", \"Molecular trigger releasing proacrosin from ACRBP-W at exocytosis not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Attributed ACRBP-null subfertility specifically to an incomplete acrosome reaction rather than defective sperm transport.\",\n      \"evidence\": \"In vivo sperm tracking in the female reproductive tract, motility, and morphology analysis in knockout mice\",\n      \"pmids\": [\"30606959\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not quantify the acrosome reaction step that fails\", \"Single-lab knockout phenotype without orthogonal model\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended ACRBP function to the sperm surface, implicating it in zona pellucida binding and priming the acrosome reaction.\",\n      \"evidence\": \"Antibody inhibition during IVF, sperm-ZP binding and acrosome reaction assays with solubilized ZP and SERCA inhibition in boar sperm\",\n      \"pmids\": [\"34086710\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Surface ACRBP binding partner on the sperm or ZP not identified\", \"Relationship between surface and matrix pools of ACRBP unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Reported a somatic role for ACRBP/OY-TES-1/CT23 in tumor and stem cell proliferation, migration, and apoptosis, including transcriptional regulation of MT1.\",\n      \"evidence\": \"RNAi/siRNA knockdown with proliferation, cycle, apoptosis, and migration assays plus microarray and Western blot in MSCs and HCC lines\",\n      \"pmids\": [\"22651134\", \"26339343\", \"33559934\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Correlative knockdown phenotypes without a defined molecular mechanism for ACRBP\", \"Direct biochemical link between ACRBP and MT1 not established\", \"No reconciliation with the acrosomal proacrosin-binding function\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How tyrosine phosphorylation, PCSK4 processing, and the acrosome-reaction trigger are coordinated to time proacrosin release, and whether the somatic cancer-cell phenotypes reflect a genuine non-reproductive function, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of the ACRBP-proacrosin complex\", \"Mechanism of proacrosin release at exocytosis undefined\", \"Somatic function lacks molecular mechanism\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ACR\", \"PCSK4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}